Application of PGRMC1 in treatment of hepatocellular carcinoma

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of PGRMC1 in treatment of hepatocellular carcinoma. Specifically, the invention relates to application of PGRMC1, a PGRMC1 truncated body or an accelerant thereof in preparation of products for prevention, treatment and / or prognosis of HCC. The invention discloses that the liver specific gene PGRMC1 can obviously block the formation and development of HCC for the first time. Meanwhile, the applicant finds that a transmembrane structural domain and an N-terminal structural domain of the PGRMC1 are very important in inhibition of c-Myc translation and c-Myc induced liver tumor formation. Therefore, delivery of PGRMC1 or PGRMC11-47aa can inhibit the progress of HCC and prolong the lifetime of a receptor.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the use of PGRMC1 in the treatment of hepatocellular carcinoma. Background Art

[0002] Hepatocellular carcinoma (HCC) accounts for approximately 90% of all primary liver cancers and is the fourth leading cause of cancer-related deaths globally. There are approximately 800,000 new cases worldwide each year. Clinically, comprehensive treatment is mainly carried out through various means such as surgery, chemotherapy, and radiotherapy, and the prognosis of patients varies.

[0003] HCC is highly heterogeneous in many aspects, such as different etiologies and various molecular subgroups based on HCC omics analysis. Among these HCC molecular subgroups, some subgroups characterized by high levels of liver metabolism-related genes and / or preserved liver function have relatively better prognosis for patients. Liver-specific genes (LSGs) are a group of genes related to liver metabolic functions and are expressed at higher levels in the liver than in other organs. LSGs are markers of liver terminal differentiation and are involved in liver metabolism. In fact, LSGs are usually dysregulated in HCC tumors, and the role of LSGs in regulating HCC initiation and progression has rarely been explored.

[0004] Progesterone receptor membrane component 1 (PGRMC1) was initially discovered as a non-classical progesterone-binding protein that mediates the anti-apoptotic function of progesterone. PGRMC1 is a single-pass transmembrane protein with a carboxyl-terminal cytochrome b5 heme-binding domain. Therefore, PGRMC1 can bind heme and regulate CYP450 enzymes. In addition to its physiological functions, the role of PGRMC1 in tumors is also unclear.

[0005] Based on this, there is an urgent need to screen for key genes in the occurrence and development of HCC and their potential as drugs in the treatment of HCC. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the purpose of the present disclosure is to provide a key gene in the occurrence and development of HCC, that is, the use of PGRMC1 in the treatment of hepatocellular carcinoma.

[0007] To achieve the above purpose, the present disclosure adopts the following specific solutions:

[0008] In one aspect, the present disclosure provides the use of a PGRMC1 protein or a truncated form thereof, a nucleic acid molecule encoding the PGRMC1 protein or a truncated form thereof, or a promoter thereof in the preparation of a product for preventing, treating, and / or prognosticating HCC.

[0009] On the other hand, the present disclosure provides a PGRMC1 truncated form, wherein the truncated form is selected from:

[0010] (a) a polypeptide having the amino acid sequence shown in SEQ ID NO.6; or

[0011] (b) a polypeptide comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology with the amino acid sequence shown in SEQ ID NO.6; or

[0012] (c) a protein or polypeptide derived by inserting, substituting, or deleting one or more amino acids in the amino acid sequence of (a) or (b).

[0013] On the other hand, the present disclosure provides a nucleic acid molecule encoding the aforementioned PGRMC1 truncated form.

[0014] On the other hand, the present disclosure provides a pharmaceutical composition comprising:

[0015] (a) a prophylactically or therapeutically effective amount of PGRMC1, a PGRMC1 truncated form, or a promoter thereof;

[0016] (b) a pharmaceutically or immunologically acceptable carrier or excipient;

[0017] (c) optionally, one or more second active components for preventing and / or treating cancer.

[0018] On the other hand, the present disclosure provides an expression cassette comprising a polynucleotide sequence, wherein the polynucleotide sequence is operably linked from 5' to 3':

[0019] (a) a promoter sequence;

[0020] (b) a sequence encoding PGRMC1, a PGRMC1 truncated form, or a functional fragment or variant thereof;

[0021] Optionally, a sequence encoding an HA-tag polypeptide is further included between (a) and (b);

[0022] Preferably, the promoter is a modified TBG promoter, and its nucleotide sequence is shown in SEQ ID NO.8;

[0023] Preferably, the nucleic acid molecule encoding PGRMC1 comprises the nucleotide sequence shown in SEQ ID NO.2;

[0024] Preferably, the nucleic acid molecule encoding the PGRMC1 truncation variant comprises the nucleotide sequence as shown in SEQ ID NO.7;

[0025] Preferably, the expression cassette further comprises an expression control element, which is operably linked to the polynucleotide molecule;

[0026] Preferably, the expression cassette further comprises an enhancer, which is selected from the ApoE HCR enhancer, the CRMSBS2 enhancer, the TTRm enhancer, and the CMV enhancer;

[0027] Preferably, the expression cassette further comprises an intron, which is selected from the α1-antitrypsin intron, the β-globin intron 2, the SV40 intron, and the minute virus of mice intron;

[0028] Preferably, the expression cassette further comprises a polyA signal, which is at least one of the bovine growth hormone poly A (BGHpoly A), the short poly A, the SV40 polyA, and the human β-globin poly A.

[0029] On the other hand, the present disclosure provides an expression vector, which comprises the aforementioned expression cassette;

[0030] Preferably, the expression vector is selected from plasmids, cosmids, viral vectors, or RNA vectors;

[0031] Preferably, the viral vector is selected from retroviruses, parvoviruses, coronaviruses, negative-strand RNA viruses, rhabdoviruses, paramyxoviruses, positive-strand RNA viruses, or double-stranded DNA viruses;

[0032] Preferably, the double-stranded DNA virus is selected from adenoviruses, herpesviruses, poxviruses, noroviruses, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, baculoviruses, or hepatitis viruses;

[0033] Preferably, the retrovirus is selected from mammalian C-type, B-type viruses, D-type viruses, the HTLV-BLV group, lentiviruses, or foamy viruses;

[0034] Preferably, the expression vector is an adenovirus.

[0035] On the other hand, the present disclosure provides a cell, which comprises the aforementioned expression cassette or the aforementioned expression vector.

[0036] On the other hand, the present disclosure provides a method for screening a drug for treating HCC by promoting PGRMC1 expression, which comprises:

[0037] (A) Treating model cells, tissues or model animals with a candidate substance;

[0038] (B) Detecting changes in the expression level of PGRMC1 in the model cells, tissues or model animals, or changes in the level of the nucleic acid molecule encoding the aforementioned protein; and

[0039] (C) If the expression level of PGRMC1 or the level of the nucleic acid molecule encoding the aforementioned protein is higher than that before treatment with the candidate substance or higher than that in the normal control, it indicates that the candidate substance has the effect of treating HCC by promoting the expression of PGRMC1.

[0040] The beneficial effects of this application are at least as follows:

[0041] This application discloses for the first time that the liver-specific gene PGRMC1 can significantly block the formation and development of HCC. At the same time, the applicant found that the transmembrane domain and N-terminal domain of PGRMC1 are both important in inhibiting c-Myc translation and c-Myc-induced liver tumor formation. Therefore, delivering PGRMC1 or PGRMC1 1-47aa can both inhibit the progression of HCC and prolong the survival period of the recipient. Brief Description of the Drawings

[0042] Figure 1 Hepatocellular carcinoma (HCC) is characterized by heterogeneous expression of liver-specific genes and is associated with the c-Myc signaling pathway. (A) In cohorts 1-3, HCC patients were clustered according to the expression of liver-specific genes in non-tumor and tumor tissues. NT, non-tumor; T, tumor. (B) HCC patients were divided into low, medium, and high LSG expression groups according to the LSG level in tumor tissues. The number of cases is shown. The AFP level and TNM staging status of each patient were marked. LSGs, liver-specific genes. (C) Kaplan-Meier analysis of recurrence time and overall survival for three groups of HCC cases in cohorts 1-3. A log-rank test was performed. (D) Gene Set Enrichment Analysis (GSEA) was performed on HCC patients with high LSG levels and low LSG levels in tumors in cohorts 1-3. Venn diagram analysis revealed 34 features in the high LSG level group that were frequently enriched in three independent cohorts. The enrichment score was calculated using the weighted Kolmogorov-Smirnov (K-S) statistic, which is a two-tailed test. (E) c-Myc activation status in low, medium, and high LSG expression HCC subgroups in cohorts 1-3. (F) LSG expression status in c-Myc activation strong, medium, and weak HCC subgroups in cohorts 1-3. (E, F) The number of cases in each group or subgroup is shown as indicated. E-F, a two-sided chi-square test was performed. A chi-square test was performed.

[0043] Figure 2Liver-specific gene PGRMC1 significantly inhibits c-Myc-induced liver cancer tumorigenesis. (A) Venn diagram analysis of c-Myc- and survival-related LSGs in cohorts 1-2. c-Myc-related genes were screened by comparing the tumor tissue mRNA profiles between the strong and weak c-Myc activation subgroups. Genes with P < 0.001 and |fold change| > 2 were c-Myc-related genes. Survival-related LSGs were screened by Kaplan-Meier survival analysis, with the median of each LSG as the boundary. Genes with P < 0.05 were survival-related genes. Forty c-Myc / survival-related LSGs were identified and shown by the P values from c-Myc-related gene analysis. TPM refers to transcripts per million, averaged from three transcriptomics datasets (HPA, GTEx, and FANTOM5). (B) Expression levels of the top 5 c-Myc / survival-related LSGs in different c-Myc activation groups in cohort 1-3, and their hazard ratios for survival in the low LSG group versus the high LSG group. (Cohort 1, strong n = 67, medium n = 62, weak n = 47; cohort 2, strong n = 109, medium n = 133, weak n = 129; cohort 3, strong n = 66, medium n = 75, weak n = 30) Left: Violin plots show the distribution of each group of data, highlighting the median and quartiles. Right: Hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated to assess the risk. Data are presented as the median and range. (C) Overall survival analysis was performed using the Myc / Mcl1-induced HDTV mouse model to screen LSGs with the ability to inhibit c-Myc-mediated HCC development. Five to six mice were used in each group, i.e., 6 mice in the control, ABAT, FMO4, and PGRMC1 groups; 5 mice in the HAGH and SLC10A1 groups. (D) Tumor-bearing mice (including those that died due to tumor burden) in the five LSG groups and the control group of the Myc / Mcl1 mouse model. (E) Occurrence of liver cancer in the Myc / Mcl1-induced HCC mouse model with or without PGRMC1 overexpression. 7 mice in the control group and 6 mice in the PGRMC1 group. (F) Occurrence of liver cancer in the c-Myc-induced HCC mouse model with or without PGRMC1 overexpression. 8 mice in the control group and 6 mice in the PGRMC1 group. (E, F) Representative images are shown. Tumor-bearing mice, liver / body ratios, numbers of liver tumors, and tumor diameters were also quantified and compared between the two groups. A two-sided Student's t-test was performed. (G) Overall procedure of the Myc / Mcl1 / luciferase-induced HCC mouse model treated with AAV8 particles. Representative images of in vivo bioluminescence imaging in mice treated with AAV8.GFP or AAV8.PGRMC1 on day 28 after injection. (H) Average bioluminescence radiation at 14, 21, 24, and 28 days after injection of AAV8 particles (4 mice per group). Two-way ANOVA was performed.The overall survival of mice treated with AAV8.GFP or AAV8.PGRMC1 is shown and subjected to the Log-rank test.

[0044] Figure 3 The expression levels of PGRMC1 in tumors from different liver cancer patients are shown. Low-dose PGRMC1 still significantly inhibits c-Myc-induced liver cancer tumor formation. (A) A low dose of PGRMC1 was used in this model. This is 10ug PGRMC1, which is one-third of the original PGRMC1. There were 6 mice in the control group and 5 mice in the PGRMC1 group. Representative images are shown. The tumor-bearing mice, liver / body ratio, number of liver tumors, and tumor diameter between the two groups were also quantified and compared. (B) PGRMC1 levels in tumors from AFP-positive or AFP-negative HCC patients in cohorts 1-3. The number of cases is shown. (J) PGRMC1 levels in tumors of different tumor grades in cohort 2. The number of cases is shown.

[0045] Figure 4 PGRMC1 inhibits Myc-induced liver cancer, which can be reversed by silencing PERK. In the Myc / Mcl1-induced HCC mouse model, shRNAs were used to silence or not silencing Perk in the presence or absence of PGRMC1 to observe the development of liver cancer. Representative images are shown. The number of tumor-bearing mice, liver / body ratio, number of liver tumors, and tumor diameter between the two groups were also quantified and compared.

[0046] Figure 5 PGRMC1 interacts with PERK through its ER lumen region and dissociates BiP from PERK, activating PERK in an ER stress-independent manner. (A) The interaction between PGRMC1 and PERK in Huh7, Huh1, and 293T cells co-transfected with HA-PGRMC1 was detected by anti-HA IP. (B) Localization of the PERK region involved in PGRMC1 binding. Anti-Flag IP was performed in 293T and Huh7 cells co-transfected with HA-PGRMC1 and different PERK-flag vectors. C-perk, C-terminal region of perk; N-perk, N-segment region of PERK. (C) Localization of the PGRMC1 region involved in perk binding. Anti-HA IP was performed in 293T and Huh7 cells co-transfected with N-PERK-flag and different HA-PGRMC1 vectors. (D) When BiP-Flag was co-transfected with PGRMC1, anti-Flag IP was performed in 293T and Huh7 cells to detect the interaction between BiP and PERK. WT 、PGRMC1 Δ25-43 and PGRMC1 △1-24Anti-Flag IP was performed in 293T and Huh7 cells during co-transfection. (F) Exogenous c-Myc levels, p-eIF2α, and eIF2α levels in Huh7 cells transfected with PGRMC1 and with or without TUDCA and 4-PBA treatment. (G) Endogenous c-Myc protein levels, p-eIF2α, and eIF2α levels in Huh7 cells transfected with PGRMC1 and with or without TUDCA and 4-PBA treatment. (H) Schematic model of PGRMC1 activating PERK in a manner independent of ER stimulation. Representative immunoblot images in this figure are from the results of three or more independent experiments.

[0047] Figure 6 The N-terminal domain of PGRMC1 and endoplasmic reticulum localization are crucial for inhibiting c-Myc translation and c-Myc-induced liver tumorigenesis. (A) Confocal microscopy images of PGRMC1 and the ER marker molecule Calreticulin and their co-localization in Huh7 cells transfected with HA-Ctrl, HA-PGRMC1 WT 、HA-PGRMC1 Δ1-24 and HA-PGRMC1 Δ25-43 . (B) Boncat assay in Huh7 cells co-transfected with PGRMC1 WT or PGRMC1 Δ25-43 and MYC-HA. (C) Boncat assay in Huh7 cells transfected with PGRMC1 WT or PGRMC1 Δ25-43 . (D) Hepatocarcinogenesis in a Myc / Mcl1-induced HCC mouse model under overexpression conditions of PGRMC1 WT or PGRMC1 Δ25-43 . Representative images are shown. The tumor-bearing mice, liver / body ratio, number of liver tumors, and tumor diameter between the two groups were also quantified and compared. Six mice were used in each group. Student's t-test was used. (E) Boncat assay in Huh7 cells co-transfected with PGRMC1 WT or PGRMC1 Δ1-24 and MYC-HA. (F) Boncat assay in Huh7 cells transfected with PGRMC1-WT or PGRMC1-Δ1-24. (G) Hepatocarcinogenesis in a Myc / Mcl1-induced HCC mouse model under overexpression conditions of PGRMC1 WT or PGRMC1 Δ2-24 . Representative images are shown. The tumor-bearing mice, liver / body ratio, number of liver tumors, and tumor diameter between the two groups were also quantified and compared. 6 mice in the control group, 7 mice in the PGRMC1 group, PGRMC1 Δ2-24The group consisted of 8 animals. The two-tailed Student's t-test was used for D and G. The images in Figures A-C and E-F represent data from three or more independent experiments.

[0048] Figure 7 Micro-PGRMC1 (1-47aa) was able to interact with PERK, reduce the translation of c-Myc, and Myc / Mcl1-induced tumor formation. (A) Schematic diagram of micro-PGRMC1 (47aa), including the N-terminal region and transmembrane domain of PGRMC1. (B) HA-PGRMC1 WT and HA-PGRMC1 2-47aa Confocal microscopy images of co-localization with the ER marker Calretiredin and its co-localization in Huh7 cells. (C) Anti-HA IP was performed to detect the interaction between PGRMC1 and PERK, and N-PERK-flag in Huh7 cells transfected with HA-PGRMC1 WT 、HA-PGRMC1 2-47aa or PGRMC1 Δ25-43 。(D) Anti-Flag IP was used to detect the interaction between PERK and BiP in Huh7 cells transfected with BiP-flag and HA-PGRMC1 WT 、HA-PGRMC1 2-47aa or PGRMC1 Δ25-43 。(E) Boncat assay in Huh7 cells co-transfected with MYC-HA and PGRMC1 WT 、HA-PGRMC1 2-47aa or PGRMC1 Δ25-43 。The indicated proteins were detected. (F) Boncat assay in Huh7 cells transfected with PGRMC1 WT 、HA-PGRMC1 2-47aa or PGRMC1 Δ25-43 。(G) Hepatocarcinogenesis in a HCC mouse model with overexpression of PGRMC1 WT or PGRMC1 1-47aa induced by Myc / Mcl1. Representative images are shown. The tumor-bearing mice, liver / body ratio, number of liver tumors, and tumor diameter between the two groups were also quantified and compared. 6 mice were used in the control group, 7 in the PGRMC1 group, and 8 in the PGRMC1 1-47aa group. The two-tailed Student's t-test was used. (H) With AAV8.GFP or AAV8.PGRMC1 1-47aaGeneral procedure of the particle-treated Myc / Mcl1-induced in situ HCC mouse model. Five mice were used in each group. The total survival time of the mice treated with AAV8 was collected and compared. A log-rank t-test was performed. The images in Figures B-F represent data from three or more independent experiments. Detailed Description

[0049] I. Terms

[0050] To facilitate understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which this disclosure pertains.

[0051] As used herein, the articles "a" and "an" refer to one or more (i.e., at least one) of the grammatical objects to which the article refers. For example, "an element" means one element or more than one element.

[0052] The term "and / or" should be understood to mean either option or both options.

[0053] The term "comprising" or "including" generally means including the expressly specified features, but does not exclude other elements.

[0054] The term "about" generally means varying within a range of 0.5% - 10% above or below the specified value, e.g., within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0055] As used herein, the expressions "cell", "cell line", and "cell culture" are used interchangeably, and all such names include their progeny. Thus, the words "transformant" and "transformed cell" include the original test cells and the cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to deliberate or inadvertent mutations, all progeny may not be precisely identical in DNA content. Include mutant progeny having the same function or biological activity as screened in the original transformed cells. In cases where different names are meant, it is clear from the context.

[0056] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and this description includes the instances where the event or circumstance occurs or does not occur.

[0057] In the present application, the terms "protein variant" and "variant" generally refer to compounds having sequence homology with a native bioactive protein or polypeptide. The protein variants described in the present application may include proteins having an altered amino acid sequence by adding (including inserting), deleting, modifying, and / or substituting one or more amino acid residues, while retaining at least one biological activity of the parental sequence. For example, the variant may have at least about 0%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the parental protein. The variant may be naturally occurring or non-naturally occurring. Techniques known in the art can be used to generate non-naturally occurring variants. Protein variants may contain conservative or non-conservative amino acid substitutions, deletions, or additions.

[0058] In the present application, the term "truncated form" generally refers to anything less than the whole. In the present application, the "truncated form" may refer to a compound having an amino acid sequence less than the entire sequence of PGRMC1. For example, the truncated form has an amino acid sequence less than at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the parental protein.

[0059] The term "vector" generally refers to a vector that contains regulatory sequences necessary for the transcription and translation of one or more cloned nucleic acid molecules, and thus can transcribe and clone nucleic acid molecules. The vector may contain one or more regulatory sequences operably linked to the nucleic acid molecule, and this regulatory sequence can be selected according to the type of host cell used. Regulatory sequences include promoters, enhancers, and other expression control elements, such as polyadenylation (poly(A)+) sequences. Other vector components may include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more selectable genes, and transcription termination sequences.

[0060] The term "homology" generally refers to an amino acid sequence or nucleotide sequence that has a certain degree of homology with a compared amino acid sequence and a compared nucleotide sequence. The term "homology" can be equivalent to sequence "identity". Homologous sequences can include amino acid sequences that are at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the subject sequence. Generally, homologs will contain the same active sites, etc. as the subject amino acid sequence. Homology can be considered in terms of similarity (i.e., amino acid residues with similar chemical properties / functions), or homology can be expressed in terms of sequence identity. In this application, a sequence having a percent identity to any one of the SEQ ID NOs of the amino acid sequence or nucleotide sequence mentioned refers to a sequence having the said percent identity over the entire length of the SEQ ID NO mentioned.

[0061] To determine sequence identity, sequence alignment can be performed, which can be carried out in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art are able to determine the appropriate parameters for alignment, including any algorithms required to achieve optimal alignment in the full-length sequences being compared.

[0062] The term "expression vector" includes vectors as described above or known in the art, such as plasmids, minicircles, viral vectors, liposomes, etc., which contain a polynucleotide encoding a target gene product and are used to effect the expression of the gene product in a desired target cell. The expression vector also contains control elements operably linked to the coding region to facilitate the expression of the gene product in the target. The combination of control elements (e.g., promoters, enhancers, UTRs, miRNA targeting sequences, etc.) and one or more genes to which they are operably linked for expression is sometimes referred to as an "expression cassette". Many such control elements are known and available in the art, or can be readily constructed from components available in the art.

[0063] The terms "promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' of the promoter sequence. A promoter may be entirely derived from a native gene, or may be composed of different elements derived from different promoters found in nature, or may even contain synthetic DNA segments. Those skilled in the art will understand that different promoters may direct gene expression in different tissues or cell types, or at different developmental stages, or in response to different environmental or physiological conditions. A promoter that causes a gene to be expressed most of the time in most cell types is typically referred to as a "constitutive promoter". A promoter that causes a gene to be expressed in a specific cell type is typically referred to as a "cell-specific promoter" or "tissue-specific promoter". A promoter that causes a gene to be expressed at a specific stage of development or cell differentiation is typically referred to as a "development-specific promoter" or "cell differentiation-specific promoter". A promoter that is induced and causes gene expression after exposure or treatment of cells with an inducing agent, biomolecule, chemical, ligand, light, etc. is typically referred to as an "inducible promoter" or "regulatable promoter". It is further recognized that since the precise boundaries of regulatory sequences are not fully defined in most cases, DNA fragments of different lengths may have the same promoter activity.

[0064] A promoter sequence typically binds at its 3' end to the transcription start site and extends upstream (in the 5' direction) to include the minimum number of bases or elements required to initiate transcription at a detectable level above background. Within the promoter sequence will be found the transcription start site (usually defined, for example, by nuclease S1 mapping) and protein-binding domains (consensus sequences) responsible for the binding of RNA polymerase. In some aspects, promoters that can be used in conjunction with the present disclosure include tissue-specific promoters.

[0065] As used herein, an "enhancer" includes cis-acting elements that stimulate or inhibit the transcription of an adjacent gene. An enhancer that inhibits transcription is also referred to as a "silencer". An enhancer can act at distances of up to several thousand base pairs (kb) downstream of the coding sequence and transcription region in either orientation (i.e., it can be associated with the coding sequence).

[0066] As used herein, a "termination signal sequence" includes any genetic element that causes RNA polymerase to terminate transcription, such as a polyadenylation signal sequence.

[0067] As used herein, the terms "operably linked" or "functionally linked" refer to the juxtaposition of genetic elements such as promoters, enhancers, termination signal sequences, polyadenylation sequences, etc., wherein the elements are in a relationship that permits them to operate in an intended manner. For example, if a promoter aids in initiating transcription of a coding sequence, the promoter is operably linked to the coding region. There can be intervening residues inserted between the promoter and the coding region so long as this functional relationship is maintained.

[0068] The term "promoter" refers to a substance that can increase the level of PGRMC1 protein or promote its function. Promoters that can be used in the present disclosure include, but are not limited to: overexpression vectors from PGRMC1 or its truncated coding sequences, exogenous PGRMC1 or its truncated forms, naked DNA of PGRMC1 or its truncated coding sequences, liposome-encapsulated DNA of PGRMC1 or its truncated coding sequences, precursor proteins or conjugates or complexes that can be converted into PGRMC1 or its truncated forms in vivo. As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include their progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to deliberate or inadvertent mutations, all progeny may not be precisely identical in their DNA content. This includes mutant progeny having the same function or biological activity as screened in the original transformed cells. Where different names are intended, it will be clear from the context.

[0069] "Administering", "giving" and "treating", when applied to an animal, a human, an experimental subject, a cell, a tissue, an organ or a biological fluid, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid. "Administering", "giving" and "treating" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treating a cell includes the contact of a reagent with the cell, as well as the contact of a reagent with a fluid that is in contact with the cell. "Administering", "giving" and "treating" also mean treating a cell in vitro and ex vivo by a reagent, a diagnostic, a binding composition or by another cell. "Treating", when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

[0070] "Treatment" means administering a therapeutic agent, either internally or externally, to a patient having one or more disease symptoms, where the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the treated patient or population, whether by inducing regression of such symptoms or inhibiting their development to any clinically non-measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, as well as the ability of the drug to produce the desired effect in the patient. Whether a disease symptom has been alleviated can be evaluated by any clinical test method commonly used by a physician or other professional healthcare provider to assess the severity or progression of the symptom. Although the embodiments of the present disclosure (such as treatment methods or articles) may not be effective in alleviating the target disease symptoms present in every patient, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test method known in the art, such as the Student t-test, chi-square test, Mann-Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0071] As used throughout the specification and claims, the term "consisting essentially of" or variations thereof means including all of the recited elements or groups of elements, and optionally including other elements of like or different nature, which do not materially alter the basic or novel properties of the specified dosage regimen, method, or composition.

[0072] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested as any of the following: reduction in tumor volume, decrease in the number of tumor cells, decrease in the number of metastases, increase in life expectancy, or improvement in various physiological symptoms associated with cancer. The "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, antibodies (or antigen-binding portions thereof), and T cells of the present invention to prevent the emergence of tumors in the first place.

[0073] The terms "cancer" or "tumor" or "hyperproliferative disease" refer to the presence of cells having typical characteristics of cancer cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and certain characteristic morphological features. Cancer cells are usually in the form of tumors, but these cells can exist singly in an animal or can be non-tumorigenic cancer cells, such as leukemia cells. The term "cancer" includes pre-malignant cancers as well as malignant cancers. The term "pre-malignant lesion" as used herein refers to a lesion that, although not cancerous, has the potential to become cancerous. It also includes the terms "pre-malignant condition" or "potentially malignant condition". In particular, it denotes tissues showing benign morphological and / or histological alterations that have a greater than normal risk of malignant transformation; and denotes diseases or patient habits that do not necessarily alter the clinical presentation of the local tissue but are associated with a greater than normal risk of pre-cancerous lesions or cancer development (leukoplakia, erythroplakia, lichen planus erythematosus (lichenoid reaction) and any lesion or area showing cellular atypia or dysplasia on histological examination) in that tissue.

[0074] Cancer includes sarcomas and cancers of humans, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelioma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias such as acute lymphocytic leukemia and acute myelogenous leukemia (myeloblast, promyelocyte, myelomonocyte, monocyte, and erythroleukemia); chronic leukemias (chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström's macroglobulinemia, and heavy chain disease.

[0075] "Vector" is a composition of matter that contains a nucleic acid in isolation and can be used to deliver the isolated nucleic acid into a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0076] The terms "patient", "subject", "individual", etc. are used interchangeably herein and are intended to include living organisms (e.g., mammals) in which an immune response can be elicited. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species.

[0077] The term "kit" is any article (e.g., a package or container) that contains at least one reagent (e.g., a therapeutic agent, a probe, a small molecule, etc.) for specifically detecting and / or therapeutically affecting the expression of a biomarker described herein. The kit can be promoted, distributed, or sold as a whole for performing the methods described herein. The kit can contain one or more reagents that are essential for the expression of a composition used in the methods described herein. In certain embodiments, the kit can further contain reference standards, e.g., nucleic acids encoding proteins that do not affect or regulate a signaling pathway that controls immune response, cell growth, division, migration, survival, or apoptosis. Those skilled in the art can envision many such control proteins, including but not limited to: common molecular tags (e.g., green fluorescent protein and β-galactosidase); proteins that, by Gene Ontology reference, are not classified in any pathway that includes cell growth, division, migration, survival, or apoptosis; or ubiquitously expressed housekeeping proteins. The reagents in the kit can be provided in a single container or as a mixture of two or more reagents in a single container. Additionally, instructional materials describing the use of the compositions in the kit can be included.

[0078] The term "prognosis" refers to the act of predicting the course and outcome of a disease in advance. More specifically, the course after treatment may vary depending on the patient's physiological or environmental conditions, and it can be interpreted as all actions of predicting the course after treatment considering the patient's overall condition. For the present disclosure, prognosis prediction or diagnosis can be interpreted as predicting the disease-free survival rate or survival rate of a cancer patient by predicting the course and post-treatment situation of the cancer patient. For example, "good prognosis" indicates a high disease-free survival rate or survival rate of a cancer patient, which means that the cancer patient is more likely to be cured; "poor prognosis" indicates a low disease-free survival rate or survival rate of a cancer patient after radiotherapy, which means that the cancer of the cancer patient is likely to recur or the patient is likely to die from cancer.

[0079] The term "TNM staging" is the most commonly used tumor staging system internationally at present and is also the standard method for staging malignant tumors clinically. The TNM staging method was first proposed by the Frenchman Pierre Denoix between 1943 and 1952. Subsequently, the American Joint Committee on Cancer (AJCC) and the International Union Against Cancer (UICC) began to establish international staging criteria, and the first edition of the "Classification of Malignant Tumors INM" manual was officially published in 1968. Currently, the latest update is the eighth version. In the embodiments of the present disclosure, the meanings of the respective English and numerical codes in TNM are as follows: T (Tumor): the scope and size of the primary tumor, T0 indicates that there is no evidence of the presence of a primary tumor, T1-T4 indicate an increase in the size of the tumor in the primary focus and the impact on surrounding tissues, Tis indicates carcinoma in situ, without invasion, and Tx indicates that the condition of the primary tumor cannot be evaluated; N (Node): the spread of lymph nodes, N0 indicates that the lymph nodes are not affected, N1-N3 indicate an increase in the degree and scope of lymph node involvement in sequence, and Nx indicates that the condition of lymph node involvement cannot be evaluated; M (Metastasis): the presence or absence of metastasis, M0 indicates no metastasis, and M1 indicates distant metastasis.

[0080] II. Detailed Description of Specific Embodiments

[0081] In one aspect, the present disclosure provides the use of a PGRMC1 protein or a truncated form thereof, a nucleic acid molecule encoding the PGRMC1 protein or a truncated form thereof, or a promoter thereof in the preparation of a product for preventing, treating, and / or prognosticating HCC.

[0082] In some embodiments, the PGRMC1 protein is selected from:

[0083] (a) a polypeptide having the amino acid sequence shown in SEQ ID NO.1; or

[0084] (b) a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology with the amino acid sequence shown in SEQ ID NO.1; or

[0085] (c) a protein or polypeptide derived by inserting, substituting or deleting one or more amino acids in the amino acid sequence of (a) or (b); and / or

[0086] The nucleic acid molecule encoding the PGRMC1 protein is selected from:

[0087] (i) a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO.2; or

[0088] (ii) a nucleic acid molecule homologous to the nucleotide sequence shown in SEQ ID NO.2 or having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more homology;

[0089] (iii) a nucleic acid molecule derived by inserting, substituting or deleting one or more nucleotides in the nucleotide sequence of (i) or (ii).

[0090] In some embodiments, the amino acid sequence of the PGRMC1 protein is as shown in SEQ ID NO.1.

[0091] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the PGRMC1 protein is as shown in SEQ ID NO.2.

[0092] In some embodiments, the PGRMC1 truncation body comprises at least a transmembrane domain and an N-terminal domain.

[0093] In some embodiments, the PGRMC1 truncation body is selected from:

[0094] (a) a polypeptide having the amino acid sequence shown in SEQ ID NO.6; or

[0095] (b) a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology with the amino acid sequence shown in SEQ ID NO.6; or

[0096] (c) a protein or polypeptide derived by inserting, substituting or deleting one or more amino acids in the amino acid sequence of (a) or (b); and / or

[0097] The nucleic acid molecule encoding the PGRMC1 truncation body is selected from:

[0098] (i) a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO.7; or

[0099] (ii) a nucleic acid molecule that is homologous to the nucleotide sequence shown in SEQ ID NO.7 or has a homology of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more;

[0100] (iii) a nucleic acid molecule derived by inserting, substituting or deleting one or more nucleotides in the nucleotide sequence of (i) or (ii).

[0101] In some embodiments, the amino acid sequence of the PGRMC1 truncation is as shown in SEQ ID NO.6.

[0102] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the PGRMC1 truncation is as shown in SEQ ID NO.7.

[0103] In some embodiments, the promoter is selected from an overexpression vector of the PGRMC1 or its truncation coding sequence, exogenous PGRMC1 or its truncation, naked DNA of the PGRMC1 or its truncation coding sequence, liposome-encapsulated DNA of the PGRMC1 or its truncation coding sequence, a precursor protein or conjugate or complex capable of being converted into PGRMC1 or its truncation in vivo.

[0104] In some embodiments, the product is a drug or a kit. In some embodiments, the product further comprises a second active ingredient for preventing and / or treating cancer.

[0105] On the other hand, the present disclosure provides a PGRMC1 truncation, wherein the truncation is selected from:

[0106] (a) a polypeptide having the amino acid sequence shown in SEQ ID NO.6; or

[0107] (b) a polypeptide comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology with the amino acid sequence shown in SEQ ID NO.6; or

[0108] (c) a protein or polypeptide derived by inserting, substituting or deleting one or more amino acids in the amino acid sequence of (a) or (b).

[0109] In some embodiments, the amino acid sequence of the PGRMC1 truncation is as shown in SEQ ID NO.6.

[0110] On the other hand, the present disclosure provides a nucleic acid molecule encoding the aforementioned PGRMC1 truncation.

[0111] In some embodiments, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.7.

[0112] On the other hand, the present disclosure provides a pharmaceutical composition comprising:

[0113] (a) a prophylactically or therapeutically effective amount of PGRMC1, a truncated PGRMC1 or a promoter thereof;

[0114] (b) a pharmaceutically or immunologically acceptable carrier or excipient;

[0115] (c) optionally, one or more second active components for preventing and / or treating cancer.

[0116] On the other hand, the present disclosure provides an expression cassette comprising a polynucleotide sequence, wherein the polynucleotide sequence is operably linked from 5' to 3':

[0117] (a) a promoter sequence;

[0118] (b) a sequence encoding PGRMC1, a truncated PGRMC1 or a functional fragment or variant thereof;

[0119] Optionally, a sequence encoding an HA-tag polypeptide is further comprised between (a) and (b).

[0120] In some embodiments, the expression cassette comprises a sequence encoding an HA-tag polypeptide for cell experiments.

[0121] In some embodiments, the expression cassette does not comprise a sequence encoding an HA-tag polypeptide for animal experiments.

[0122] In some embodiments, the promoter is a modified TBG promoter, and its nucleotide sequence is as shown in SEQ ID NO.8.

[0123] In some embodiments, the nucleic acid molecule encoding PGRMC1 comprises the nucleotide sequence as shown in SEQ ID NO.2.

[0124] In some embodiments, the nucleic acid molecule encoding a truncated PGRMC1 comprises the nucleotide sequence as shown in SEQ ID NO.7.

[0125] In some embodiments, the expression cassette further comprises an expression control element, and the expression control element is operably linked to the polynucleotide molecule.

[0126] In some embodiments, the expression cassette further comprises an enhancer, and the enhancer is selected from ApoE HCR enhancer, CRMSBS2 enhancer, TTRm enhancer and CMV enhancer.

[0127] In some embodiments, the expression cassette further comprises an intron selected from the group consisting of the α1-antitrypsin intron, the β-globin intron 2, the SV40 intron, and the minute virus of mice intron.

[0128] In some embodiments, the expression cassette further comprises a polyA signal, which is at least one of bovine growth hormone polyA (BGH polyA), short polyA, SV40 polyA, and human β-globin polyA.

[0129] On the other hand, the present disclosure provides an expression vector comprising the aforementioned expression cassette.

[0130] In some embodiments, the expression vector is selected from the group consisting of plasmids, cosmids, viral vectors, or RNA vectors.

[0131] In some embodiments, the viral vector is selected from the group consisting of retroviruses, parvoviruses, coronaviruses, negative-strand RNA viruses, rhabdoviruses, paramyxoviruses, positive-strand RNA viruses, or double-stranded DNA viruses.

[0132] In some embodiments, the double-stranded DNA virus is selected from the group consisting of adenoviruses, herpesviruses, poxviruses, noroviruses, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, baculoviruses, or hepatitis viruses.

[0133] In some embodiments, the retrovirus is selected from the group consisting of mammalian C-type, B-type, D-type viruses, the HTLV-BLV group, lentiviruses, or foamy viruses.

[0134] In some embodiments, the expression vector is an adenovirus.

[0135] On the other hand, the present disclosure provides a cell comprising the aforementioned expression cassette or the aforementioned expression vector.

[0136] On the other hand, the present disclosure provides a method for screening a drug for treating HCC by promoting PGRMC1 expression, comprising:

[0137] (A) Treating a model cell, tissue, or model animal with a candidate substance;

[0138] (B) Detecting a change in the expression level of PGRMC1 in the model cell, tissue, or model animal, or a change in the level of a nucleic acid molecule encoding the aforementioned protein; and

[0139] (C) If the expression level of PGRMC1 or the level of the nucleic acid molecule encoding the aforementioned protein is higher than the level before treatment with the candidate substance or higher than the level in the normal control, it indicates that the candidate substance has the effect of treating HCC by promoting PGRMC1 expression.

[0140] In the following detailed description of the embodiments of the present disclosure, reference is made to the accompanying drawings. In the drawings, like reference numerals indicate like elements, and specific embodiments in which the present disclosure can be practiced are shown in an illustrative manner. These embodiments have been described in sufficient detail to enable those skilled in the art to practice the present disclosure. In other cases, well-known processes, structures, and technologies are not shown in detail so as not to obscure the understanding of this specification. Therefore, the following detailed description should not be construed as restrictive, and the technical solutions of the present disclosure are only defined by the appended claims.

[0141] Experimental materials and methods

[0142] Fluorescence co-localization

[0143] Cells with or without transfection were seeded on cover slips, and after 24 hours, the cells adhered to the wall. The medium was removed, the cells were washed once with PBS, fixed with pre-cooled methanol for 5 minutes, then permeabilized with 0.1% Triton X-100 for 10 minutes, washed once with PBST, and blocked with 3% BSA for 1 hour. The primary antibody was incubated at room temperature for 1 hour, washed three times with PBST, then the corresponding secondary antibody was incubated for 1 hour, and after washing three times with PBST, the cell nuclei were stained with DAPI in the mounting medium. Fluorescence images were acquired using a Zeiss LSM 880 AiryScan laser confocal microscope.

[0144] In vivo imaging of mice

[0145] In the in vivo bioluminescence imaging experiment of live mice, 3 weeks after the injection of Myc / Mcl1 / Luciferase HDTV, FVB / N mice were divided into two groups on average according to the bioluminescence signal, and 1x10 11 virus particles of AAV8-TBG-GFP or AAV8-TBG-PGRMC1 were injected via the tail vein respectively. Bioluminescence signals were collected at the set time points. The specific operation was to intraperitoneally inject the luciferase substrate into the mice. After 10 - 15 minutes, when the bioluminescence signal of the mice reached the maximum value, photos were taken and the data were saved, and at the same time, the survival data of the mice were collected. In order to observe the role of AAV8-TBG-PGRMC1(1 - 47aa) in inhibiting the progression of liver cancer, 3 weeks after the injection of Myc / Mcl1 / Luciferase HDTV, AAV8-TBG-GFP or AAV8-TBG-PGRMC1(1 - 47aa) (1x10 11 virus particles) were injected respectively, and the survival data of the mice were collected.

[0146] Sample data

[0147] The HCC datasets and clinical specimens are shown in Table 1 and Table 2.

[0148] Table 1. Information of the cohort samples

[0149]

[0150] Note: GSE14520 is the dataset number in the Gene Expression Omnibus (GEO) database; TCGA-LIHC are hepatocellular carcinoma patients in The Cancer Genome Atlas (TCGA) project; CHCC-HBV is the Chinese Hepatocellular Carcinoma Cohort Study - Hepatitis B virus.

[0151] Table 2. Overview of the clinical information of patients in HCC cohort 1 - 3

[0152]

[0153]

[0154]

[0155] Construct an expression plasmid

[0156] Design complementary pairing primers with AgeI and EcoRI restriction enzyme cleavage site sticky ends at the 5' end of the targeting sequence. After primer annealing and ligation, they are ligated to the linearized pLKO.1 vector to construct a knockdown plasmid.

[0157] (1) The reaction system for primer annealing and ligation is as follows:

[0158]

[0159] (2) Add each component to a PCR tube, centrifuge briefly to spin to the bottom of the tube, and place it in boiling water and cool to room temperature.

[0160] (3) Perform a double digestion reaction on the target vector, and the system is as follows:

[0161]

[0162] After mixing each component, let it stand in a 37°C constant temperature incubator for 3 - 5 hours for digestion.

[0163] (4) Perform nucleic acid electrophoresis on the digestion product, cut the gel and recover the linearized vector.

[0164] (5) Perform a ligation reaction on the linearized vector after gel cutting and recovery and the annealing and ligation product, and the system is as follows:

[0165]

[0166] (6) Add each component to a PCR tube, centrifuge briefly to pellet the components at the bottom of the tube, and incubate at 22 °C for 1 h for ligation.

[0167] (7) After the ligation reaction is completed, add the ligation product to 100 μL of DH5α competent cells and incubate on ice for 20 min.

[0168] (8) Heat shock at 42 °C for 1 min, incubate on ice for 2 min, add 800 μL of antibiotic-free LB medium, and recover at 37 °C in a shaker at 220 rpm for 1 h.

[0169] (9) After recovery, centrifuge at 4500 rpm at room temperature for 3 min, discard the supernatant, resuspend the pellet, spread on an ampicillin-resistant plate, and incubate overnight in a 37 °C constant temperature incubator.

[0170] (10) The next day, pick monoclonal colonies, culture the bacteria for 12 - 16 h, extract plasmid miniprep, and after verification by single digestion with XhoI, select the correct clones and send them to a sequencing company for sequencing.

[0171] Reverse Transcription and Real-Time Quantitative PCR

[0172] PrimeScript RT reagent Kit with gDNA Eraser from Takara Bio Inc., Japan and TB Premix Ex Taq reagent were used for mRNA reverse transcription and real-time quantitative PCR respectively. All operations were carried out on ice. TM RT reagent Kit with gDNA Eraser kit and TB Premix Ex Taq TM reagent.

[0173] (1) Remove genomic DNA and perform the reaction using the following system:

[0174]

[0175] (2) Add each component to a PCR tube, centrifuge briefly to pellet the components at the bottom of the tube, and react at 42 °C for 2 min to complete the removal of genomic DNA.

[0176] (3) For reverse transcription reaction, perform the reaction using the following system:

[0177]

[0178] (4) Add each component to a PCR tube, centrifuge briefly to pellet the components at the bottom of the tube, and perform the reverse transcription reaction according to the following program:

[0179] 37 °C, 15 min

[0180] 85 °C, 5 s

[0181] 4 °C, end

[0182] (5) After the reaction is completed, take out the reverse transcription reaction product and add dd H2O to dilute it 10 times for subsequent experiments.

[0183] (6) Prepare the following real-time quantitative PCR reaction mixture:

[0184]

[0185] (7) Mix the mixture well, aliquot it into a 96-well or 384-well fluorescence quantitative PCR plate, 7.5 μL per well, and add 2.5 μL of the diluted reverse transcription reaction product. Set up 3 replicate wells for each sample.

[0186] (8) Centrifuge the plate briefly and perform quantitative PCR reaction according to the following program:

[0187]

[0188]

[0189] End

[0190] (9) Copy the data for subsequent data processing.

[0191] High-pressure tail vein injection oncogene-induced tumorigenesis experiment

[0192] Filter normal saline with a 0.22-μm diameter filter membrane, dilute the plasmid in the filtered normal saline according to a ratio, and mix well. Anesthetize the experimental mice with isoflurane, preheat the mice's tails with about 55 °C hot water to dilate the veins, use a 5-mL syringe equipped with a 0.45-mm needle, aspirate 10% of the plasmid-normal saline mixture of the mouse body weight, and inject the mixture completely into the vein of the mouse's tail within 5 - 7 s to complete the high-pressure tail vein injection operation. Then, at the experimental time points, euthanize the mice, collect liver tissues, record the body weight, liver weight, number of tumor nodules and abnormalities of the mice, and take pictures to save the gross pictures of the mice and the front and back pictures of the livers.

[0193] Immunoblotting (Western blot, WB) experiment

[0194] (1) Preparation of protein samples (collection with 1X SDS loading buffer, taking a 6-well plate as an example)

[0195] Aspirate the culture medium, wash the cells with cold 1X PBS buffer, add 200 μL of 1X SDS loading buffer to each well, rotate the pipette tip in the same direction to aggregate the cells, transfer them to an Ep tube, and boil the samples at 95 °C for 15 min.

[0196] The 5X SDS loading buffer is prepared as follows:

[0197]

[0198] (2) Preparation of protein samples (collected with 1X lysis buffer, taking a 6-well plate as an example)

[0199] Aspirate the culture medium, wash the cells with cold 1X PBS buffer. Add 200 μL of 1X lysis buffer to each well, scrape the cells with an inverted pipette tip, transfer them to an Ep tube, vortex, and then transfer to a cold storage (4 °C) rotary drum for lysis for 30 min. Centrifuge at 12,000 rpm for 15 min at 4 °C. Transfer the supernatant to a new Ep tube, add 5X SDS loading buffer, and boil the sample at 95 °C for 15 min.

[0200] The 1X lysis buffer is prepared as follows:

[0201]

[0202] (3) SDS-PAGE electrophoresis

[0203] 1) Prepare 8% separating gel:

[0204]

[0205]

[0206] 2) Prepare 5% stacking gel:

[0207]

[0208] 3) Prepare 10X running buffer for electrophoresis: Weigh 60.4 g of Tris, 376 g of glycine, and 20 g of SDS, dissolve them in 2 L of double-distilled water. Dilute to 1X running buffer before use.

[0209] 4) Remove the prepared WB gel plate, pull out the comb, add 1X running buffer, load an appropriate volume of protein sample according to the experimental requirements, and the loading amount of protein marker is 2 μL. Apply a constant voltage of 70 - 80 V. After electrophoresis for about 30 min, when the sample enters the separating gel and the protein marker bands are separated, change to a constant voltage of 120 - 130 V and continue electrophoresis for about 30 - 60 min (adjust the electrophoresis time according to experimental requirements).

[0210] (4) Transfer membrane

[0211] 1) Prepare 10X trans buffer: Weigh 60.6 g of Tris and 288.2 g of glycine and dissolve them in 2 L of double-distilled water. When in use, dilute it to 1X trans buffer according to the ratio of 10X trans buffer: methanol: double-distilled water = 1:2:7.

[0212] 2) Prepare the transfer cassette, sponge sheets, and filter paper. Place them in the transfer cassette in sequence and pour 1X trans buffer into it to soak them. Remove the WB gel, remove the stacking gel part, retain the loading area, and place it on the filter paper. Soak the PVDF membrane in methanol for 3 - 5 s to "activate the membrane" until it becomes semi-transparent. After rinsing it in 1X trans buffer, cover it on the gel, and pay attention to expelling the air bubbles. Then close the transfer cassette, place it in the transfer tank, and supplement 1X trans buffer to submerge the transfer cassette. Place the transfer device in a foam box, perform ice bath, set a constant current of 260 mA, and transfer for 90 min.

[0213] (5) Blocking

[0214] 1) Prepare 10X TBS buffer: Weigh 175.2 g of NaCl and 48.4 g of Tris and dissolve them in 2 L of double-distilled water, and adjust the pH to 7.4.

[0215] 2) Prepare 1X TBST buffer: Take 50 mL of 10X TBS buffer, dilute it with 450 mL of double-distilled water to 1X, and add 250 μL of Tween-20 and mix well.

[0216] 3) Prepare 5% skim milk powder: Weigh 2.5 g of skim milk powder and dissolve it in 50 mL of 1X TBST buffer, and mix well thoroughly.

[0217] 4) After the transfer is completed, take out the PVDF membrane, quickly wash it twice with 1X TBST buffer, soak it in 5% skim milk powder, and block it at room temperature for 60 min on a horizontal shaker at 50 - 60 rpm.

[0218] (6) Incubation with primary antibody

[0219] 1) Prepare antibody dilution solution (5% BSA): Weigh 0.3 g of BSA and dissolve it in 6 mL of 1X TBST buffer, and mix well thoroughly.

[0220] 2) After blocking, quickly wash the membrane twice with 1X TBST buffer, cut the membrane according to the experimental requirements, dilute the antibody according to the antibody instruction manual, soak the membrane in the primary antibody dilution solution, and incubate it overnight at 60 rpm on a shaker at 4°C.

[0221] (7) Secondary antibody incubation

[0222] 1) Recover the primary antibody, quickly wash the membrane twice with 1X TBST buffer, and then wash the membrane 3 times at 80 rpm on a horizontal shaker for 12 minutes each time.

[0223] 2) Dilute the secondary antibody at a ratio of 1:10000, immerse the membrane in the secondary antibody dilution solution, and incubate it at 80 rpm on a horizontal shaker at room temperature for 1 hour.

[0224] (8) Darkroom development

[0225] 1) After the secondary antibody incubation, quickly wash the membrane twice with 1X TBST buffer, and then wash the membrane 3 times at 80 rpm on a horizontal shaker for 12 minutes each time.

[0226] 2) Prepare the developing solution, mix the ECL developing solution A and B at a ratio of 1:1, and bring it to the darkroom in the dark. Immerse the membrane in the developing solution for 5 - 10 seconds, lay it flat in the dark cassette, take out the film and press for development, and adjust the exposure time according to the strength of protein expression.

[0227] 3) After development, mark the developed film and scan and save it.

[0228] Statistical analysis

[0229] Statistical analysis was performed using Graphpad Prism 8 software. All errors were standard deviations (SD). Student's t-test and two-way ANOVA were used to analyze the data differences between groups. The Kaplan-Meier method in Graphpad Prism 8 software was used for the survival analysis of patients, and the statistical p-value was obtained through the Cox-Mantel log-rank test. All p-values were two-sided, and p < 0.05 was defined as a statistically significant difference.

[0230] Examples

[0231] Further understanding of the present disclosure can be obtained by referring to some specific examples given herein. These examples are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure in any way. Obviously, various modifications and changes can be made to the present disclosure without departing from the essence of the present disclosure. Therefore, these modifications and changes are also within the scope claimed in this application.

[0232] Example 1: The expression level of LSG is negatively correlated with the c-Myc signaling pathway

[0233] To evaluate the role of LSGs in HCC, the applicant analyzed the mRNA profiles of 759 LSGs in tumor and non-tumor tissues of 718 HCC patients in three independent cohorts (see Table 2). Compared with non-tumor liver tissues of HCC patients, the expression levels of these LSGs were mostly decreased in tumors, and samples in the three cohorts were classified into non-tumor and tumor tissues ( Figure 1 A). LSGs showed a heterogeneous expression pattern in HCC tissues. According to their expression levels in tumor tissues, HCC patients were divided into high, medium, and low LSG expression groups ( Figure 1 B). The levels of alpha-fetoprotein (AFP, a serum biomarker of HCC) in HCC patients in the high LSG group tended to be significantly decreased, and the tumor grade and tumor stage were also lower ( Figure 1 B). Correspondingly, in cohorts 1-3, high LSG patients had better overall survival than medium and low LSG patients ( Figure 1 C).

[0234] To detect the relevant molecular characteristics of patients in the high LSG group, gene set enrichment analysis was performed. Thirty-four co-enriched genes were generated in the three cohorts. Among them, hepatocyte metabolic function characteristics (n = 22) and HCC-related better prognosis characteristics (n = 9) were significantly enriched in the high LSG HCC subgroup ( Figure 1 D). In addition, the inhibition of three oncogenic signaling pathways, including two c-Myc signaling pathways, was also enhanced ( Figure 1 D).

[0235] MYC amplification is one of the most common genetic events in HCC. According to the defined c-Myc target genes used previously, HCC patients were subdivided into strong, medium, and weak c-Myc activation groups (Supplementary Fig.1B). Notably, HCC patients with weak c-Myc activation were enriched in the high LSG subgroup, while HCC patients with strong c-Myc activation were enriched in the low LSG subgroup ( Figure 1 E, P < 0.001 for each cohort). Conversely, high LSG patients were enriched in the HCC subgroup with weak c-Myc activation, while low LSG patients were enriched in the HCC subgroup with strong c-Myc activation ( Figure 1 F, P < 0.001 for each cohort).

[0236] Example 2: LSG PGRMC1 significantly inhibits c-Myc-induced HCC

[0237] The applicant screened LSGs that were negatively correlated with c-Myc activation and patient prognosis, and then evaluated their antitumor effects using a Myc / Mcl1-induced hydrodynamic tail vein injection HCC mouse model (HDTV). In cohort 1 and cohort 2, 40 LSGs showed differential expression (P<0.001) between the strong c-Myc activation subgroup and the weak c-Myc activation subgroup, and were associated with HCC prognosis (P<0.05)( Figure 2 A-B). Among them, the top 5 LSGs related to c-Myc (ABAT, FMO4, HAHG, PGRMC1, and SLC10A1) were further evaluated in cohort 3, and consistent data were obtained( Figure 2 B).

[0238] In the Myc / Mcl1-induced HDTV HCC model of 6-week-old ICR mice, the following plasmids were injected via the tail vein under high pressure: the control group was MYC / pT3EF1α, MCL1 / pT3EF1α, pT3EF1α, pCMV / SB; the experimental group was MYC / pT3EF1α, MCL1 / pT3EF1α, candidate gene / pT3EF1α, pCMV / SB. According to the abdominal size and mental state of the mice, euthanasia was performed when they were intolerant as their survival time; or the time of death due to tumor burden was observed. After all the mice in the control group were euthanized due to tumor burden, the remaining mice were euthanized, and the liver weight, body weight, number and size of nodules formed by HCC in the mice were recorded.

[0239] The results showed that 3 out of 5 LSGs significantly inhibited c-Myc-induced HCC formation (PGRMC1, FMO4, ABAT), prolonged the survival time of mice, and PGRMC1 had the strongest inhibitory ability( Figure 2 C-D). When all the mice (n = 6) in the control group ended their lives due to tumor burden, all the mice in the PGRMC1 group (n = 6) survived, and 60% of the mice (4 out of 6) did not form tumors( Figure 2 D).

[0240] In the Myc / Mcl1-induced HCC model of 6-week-old FVB mice, the ability of PGRMC1 to inhibit c-Myc-induced HCC was further confirmed. Control group treatment: c-Myc, Mcl1, pCMV / SB; overexpression group treatment: PGRMC1, c-Myc, Mcl1, pCMV / SB. The mice were sacrificed 5.5 weeks after injection. The liver tissues were dissected to observe the tumor formation. The liver weight, body weight, number and size of hepatocellular carcinoma nodules formed in the mice were recorded; and statistical comparative analysis was performed.

[0241] The results showed that at 5.5 weeks after HDTV, liver tumors appeared in all mice in the control group, while tumors appeared in only 42.9% of the mice in the PGRMC1 group. PGRMC1 also significantly reduced the Myc / Mcl1-mediated tumor burden, as evidenced by a significant decrease in the liver / volume ratio, tumor number, and tumor size ( Figure 2 E).

[0242] In addition, to avoid the potential influence of Mcl1 on FVB mice, the same experiment was conducted using a c-Myc-induced HCC mouse model alone. Treatment of the control group: c-Myc, pCMV / SB; treatment of the overexpression group: PGRMC1, c-Myc, pCMV / SB. Mice were sacrificed 9.5 weeks after injection. Liver tissues were dissected to observe tumor formation. The liver weight, body weight, number and size of hepatocellular carcinoma nodules formed in the mice were recorded; and statistical comparative analysis was performed.

[0243] The results showed that at 9.5 weeks after HDTV, liver tumors appeared in 87.5% of the mice in the control group, while only 16.7% (one-sixth) of the mice in the PGRMC1 group had a small tumor nodule ( Figure 2 F).

[0244] Therefore, in the three mouse models, PGRMC1 significantly reduced the c-Myc-mediated HCC tumor burden.

[0245] Furthermore, the applicant used the Myc / Mcl1 liver cancer mouse model and packaged AAV8.PGRMC1 to further study whether PGRMC1 could inhibit the tumor burden after the occurrence of liver cancer.

[0246] Experimental procedure:

[0247] a. Prepare high- and low-toxic adeno-associated viruses of the target gene.

[0248] The TBG promoter is used for liver-specific expression of foreign genes. Adeno-associated virus AAV8 can also specifically enable genes to be highly expressed in the liver. Construct a liver-specific driver TBG-driven PGRMC1 expression plasmid, and use TBG-GFP as a control. At the same time, transfect 293T cells with RepCap (AAV8 serotype-coated plasmid) and adeno-associated helper plasmid (Delta F6 helper plasmid) for adeno-associated virus coating. After collecting the virus, purify the virus by iodixanol gradient centrifugation, concentrate the virus using a centrifugal filter, and measure the titer by PCR for later use. Control the virus titer at 5x10 11 ~1x10 12 virus particles / ml.

[0249] b. Detect the role of AAV8-TBG-PGRMC1 virus in alleviating c-Myc-induced HCC.

[0250] Six-week-old FVB mice were used for construction. Plasmids c-Myc, Mcl1, Luciferase / pT3EF1α, and pCMV / SB were injected via the tail vein under high pressure. Three weeks later, at the early stage of tumorigenesis, 1x10 11 viral particles of adeno-associated virus PGRMC1 were injected via the tail vein. Two, three, 3.5, and four weeks after the injection, luciferin substrate was injected into the mice for in vivo imaging, tumor signals were recorded, and continuous survival analysis was performed. Thus, the possible role of PGRMC1 in the treatment of c-Myc-induced HCC was judged.

[0251] The results showed that AAV8-TBG-PGRMC1 significantly inhibited tumor growth, manifested as a decrease in liver luminescence ( Figure 2 G-H), and prolonged the overall survival of the mice. The median survival increased from 9 weeks to 14 weeks ( Figure 2 H). In summary, the liver-specific gene PGRMC1 significantly inhibited the formation and progression of c-Myc-induced HCC.

[0252] In addition, even when the dose of PGRMC1 in the Myc / Mcl1-induced HCC model was reduced to Figure 2 one-third of the PGRMC1 used in E and 2F, significant HCC inhibition was also obtained ( Figure 3 A).

[0253] In cohorts 1-3, HCC patients with lower PGRMC1 levels had higher serum AFP levels and poorer tumor differentiation. In contrast, the PGRMC1 levels in HCC patients with AFP>200 ng / ml were significantly lower than those in HCC patients with AFP≤200 ng / ml ( Figure 3 B), and gradually decreased from the well-differentiated state to the poorly-differentiated state in HCC tumors ( Figure 3 C). In summary, there is heterogeneous expression of LSG PGRMC1 in HCC tumor tissues, and HCC patients with low PGRMC1 levels have a poor prognosis.

[0254] Example 3: PGRMC1 inhibits the generation of c-Myc-induced tumors through the PERK pathway

[0255] PGRMC1 is mainly localized in the endoplasmic reticulum (ER), and the endoplasmic reticulum membrane protein PERK / phosphorylated eIF2α (p-eIF2α) axis is responsible for inhibiting protein synthesis. To detect whether PGRMC1 inhibits the generation of c-Myc-induced tumors through the PERK pathway, in this example, in FVB mice, a Myc / Mcl1 mouse orthotopic hepatocellular carcinoma model was established by high-pressure tail vein injection. Six-week-old mice were used, and the following groups of plasmids were injected via the tail vein under high pressure:

[0256] Group 1, c-Myc, Mcl1, pCMV / SB;

[0257] Group 2, PGRMC1, c-Myc, Mcl1, pCMV / SB;

[0258] Group 3, PGRMC1, shPERK#1, c-Myc, Mcl1, pCMV / SB;

[0259] Group 4, PGRMC1, shPERK#2, c-Myc, Mcl1, pCMV / SB.

[0260] The mice were sacrificed 30 days after injection. The liver tissues were dissected to observe the tumor formation. The liver weight, body weight, number and size of hepatocellular carcinoma nodules formed in the mice were recorded; and statistical comparative analysis was performed.

[0261] The results showed that overexpression of PGRMC1 significantly inhibited the generation of hepatocellular carcinoma induced by c-Myc / Mcl1. On the basis of overexpressing PGRMC1, knocking down PERK cancelled the tumor suppression mediated by PGRMC1. This could be seen from the tumor incidence, liver-to-body ratio, tumor number and tumor size ( Figure 4 ). It is speculated that PGRMC1 may interact with PERK through its ER lumen domain, resulting in a significant enhancement of the generation of hepatocellular carcinoma due to PERK activation.

[0262] Example 4: PGRMC1 interacts with PERK through its ER lumen domain, resulting in PERK activation

[0263] To study how PGRMC1 activates PERK, the applicant performed co-immunoprecipitation (co-IP) and detected the interaction between exogenous PGRMC1 and PERK as well as between endogenous PGRMC1 and PERK ( Figure 5 A). In this case, the interaction domains of PGRMC1 and PERK were mapped by a series of co-IP assays. As Figure 5 shown in B, PGRMC1 strongly interacted with the ER-lumen domain of PERK (N-PERK), but not strongly with the cytoplasmic domain (C-PERK). Meanwhile, when PGRMC1 lost its N-terminal region of 1-24aa (PGRMC1 Δ1-24 )(SEQ ID NO.3) or its transmembrane domain ( PGRMC1 Δ25-43 )(SEQ ID NO.5), the interaction between N-PERK and PGRMC1 was greatly reduced ( Figure 5 C). It shows that PGRMC1 interacts with PERK through its ER lumen domain.

[0264] PERK is normally present in an inactive form due to the binding of BiP that constitutes the PERK ER-lumen domain. Notably, PGRMC1 reduced the interaction between PERK and BiP in both the 293T cell line and the HCC cell line Huh7 ( Figure 5 D). In addition, PGRMC1 Δ25-43 or PGRMC1 Δ1-24 lacking the domain that interacts with PERK could not inhibit the interaction between PERK and BiP as PGRMC1WT did ( Figure 5 E). Then, the applicant tested whether PGRMC1-mediated PERK / p-eIF2α activation was dependent on endoplasmic reticulum stress. Two well-defined endoplasmic reticulum stress inhibitors, tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (4-PBA), were used. Both TUDCA and 4-PBA significantly reduced the level of p-eIF2α induced by endoplasmic reticulum stress. However, they did not reduce PGRMC1-mediated eIF2α phosphorylation or rescue the decrease in the level of exogenous c-Myc protein mediated by PGRMC1 ( Figure 5 F). In contrast, the decrease in endogenous c-Myc protein mediated by PGRMC1 was not rescued by these endoplasmic reticulum stress inhibitors either ( Figure 5 G).

[0265] In summary, the N-terminus of PGRMC1 and its transmembrane domain are important for the interaction between PGRMC1 and PERK in the endoplasmic reticulum lumen, thus separating PERK from BiP and activating PERK in a manner independent of endoplasmic reticulum stress. The action model is as shown in Figure 5 H.

[0266] Example 5: The ER localization of PGRMC1 and its N-terminus are both important in inhibiting c-Myc translation and c-Myc-induced liver tumorigenesis

[0267] Consistent with the model, immunofluorescence showed that the transmembrane domain of PGRMC1 was crucial for its endoplasmic reticulum localization ( Figure 6 A). PGRMC1 WT and PGRMC1 Δ1-24 were significantly co-localized with calreticulin, a marker protein of the endoplasmic reticulum, around the nucleus, while PGRMC1 Δ25-43 lost co-localization with calreticulin and was mainly located in the nucleus. At the same time, the N-terminus of PGRMC1 interacted with PERK in the endoplasmic lumen region, suggesting that the N-terminus of PGRMC1 was located in the endoplasmic lumen.

[0268] Then, the applicant tested the importance of the ER localization of PGRMC1 and its lumen domain in inhibiting c-Myc translation and c-Myc-induced HCC formation. As shown in Figure 6As shown in B-C, PGRMC1 without ER localization Δ25-43 no longer inhibits the protein translation of exogenous and endogenous c-Myc.

[0269] In FVB mice, a Myc / Mcl1 mouse orthotopic hepatocellular carcinoma model was established by high-pressure tail vein injection to detect whether the localization of PGRMC1 in the endoplasmic reticulum is necessary for inhibiting c-Myc-induced tumor formation. Six-week-old mice were used, and plasmids of the following groups were injected into the tail vein under high pressure:

[0270] Group 1: c-Myc, Mcl1, pCMV / SB;

[0271] Group 2: PGRMC1, c-Myc, Mcl1, pCMV / SB;

[0272] Group 3: PGRMC1 Δ25-43 , c-Myc, Mcl1, pCMV / SB.

[0273] The mice were sacrificed 5 weeks after injection. The liver tissues were dissected to observe the tumor formation. The liver weight, body weight, number and size of hepatocellular carcinoma nodules formed in the mice were recorded; and statistical comparative analysis was performed.

[0274] The results showed that in the HCC mouse model, PGRMC1 WT significantly inhibited liver tumor formation and tumor burden, while PGRMC1 Δ25-43 did not ( Figure 6 D). Among them, all the mice in the control group developed liver tumors, and the proportion of mice with liver tumors in the PGRMC1 group was 66.7%. And all the mice in the PGRMC1 Δ25-43 group developed tumors. At the same time, PGRMC1 Δ25-43 did not reduce the c-Myc-mediated tumor burden as PGRMC1 did, which was demonstrated by the liver / volume ratio, tumor number and tumor size.

[0275] In FVB mice, a Myc / Mcl1 mouse orthotopic hepatocellular carcinoma model was established by high-pressure tail vein injection to detect whether the luminal domain of the endoplasmic reticulum of PGRMC1 (i.e., the N-terminal domain) is necessary for inhibiting c-Myc-induced tumor formation. Six-week-old mice were used, and plasmids of the following groups were injected into the tail vein under high pressure:

[0276] Group 1, c-Myc, Mcl1, pCMV / SB;

[0277] Group 2, PGRMC1, c-Myc, Mcl1, pCMV / SB;

[0278] Group 3, PGRMC1 Δ2-24 , c-Myc, Mcl1, pCMV / SB.

[0279] The mice were sacrificed 30 days after injection. The liver tissues were dissected to observe the tumor formation. The liver weight, body weight, number and size of hepatocellular carcinoma nodules formed in the mice were recorded; and statistical comparative analysis was performed. It should be noted that PGRMC1 Δ2-24 No tag was added to the N-terminus of the vector, so the start codon methionine was retained; while for PGRMC1 Δ2-24 An HA tag was added before the gene sequence. To ensure that the gene starts translation from the methionine of the start codon before the HA tag, the start codon of the gene was not retained. The amino acid sequence of PGRMC1 Δ2-24 is shown in SEQ ID NO.4.

[0280] PGRMC1 Δ1-24 does not have the PGRMC1 ER lumen domain, and the results show that it also cannot inhibit the protein translation of exogenous and endogenous c-Myc ( Figure 6 E-F). In the orthotopic liver cancer mouse model, the tumor suppression effect of PGRMC1 Δ2-24 was also significantly weaker than that of PGRMC1 WT ( Figure 6 G). Compared with the PGRMC1 WT group, the tumor formation rate in the PGRMC1 Δ2-24 group increased, the liver / volume ratio increased, the number of tumors increased, and the tumor size increased.

[0281] In summary, the endoplasmic reticulum localization of PGRMC1 and its endoplasmic reticulum lumen domain are both important for inhibiting c-Myc translation and c-Myc-mediated tumor formation.

[0282] Example 6: mini PGRMC1 (PGRMC1 1-47aa ) is sufficient to activate PERK and inhibit Myc / Mcl 1-induced tumor formation.

[0283] To further clarify the role of PGRMC1 in the PERK / c-Myc axis, the applicant constructed a mini PGRMC1 that only contains its N-terminus and transmembrane domain, namely PGRMC1 1-47aa , and its amino acid sequence is shown in SEQ ID NO.6. ( Figure 7 A). Similar to PGRMC1 WT , PGRMC1 1-47aa colocalized with calreticulin ( Figure 7 B), and interacted with N-PERK ( Figure 7 C). At the same time, PGRMC1 1-47aa also inhibited the interaction between PERK and BiP ( Figure 7D), and significantly inhibited the exogenous c-Myc protein level and its protein translation ( Figure 7 E). At the same time, comparable data of endogenous c-Myc were also obtained ( Figure 7 F). PGRMC1 Δ25-43 was used as a negative control in Figure 7 C-7F.

[0284] In FVB mice, a Myc / Mcl1 mouse orthotopic hepatocellular carcinoma model was established by high-pressure tail vein injection. Six-week-old mice were used, and plasmids of the following groups were injected via the high-pressure tail vein:

[0285] Group 1: c-Myc, Mcl1, pCMV / SB;

[0286] Group 2: PGRMC1, c-Myc, Mcl1, pCMV / SB;

[0287] Group 3: PGRMC1 1-47aa , c-Myc, Mcl1, pCMV / SB.

[0288] The mice were sacrificed 30 days after injection. The liver tissues were dissected to observe the tumor formation. The liver weight, body weight, number and size of hepatocellular carcinoma nodules formed in the mice were recorded; and statistical comparative analysis was performed.

[0289] The results showed that PGRMC1 1-47aa significantly inhibited liver tumor formation and reduced the tumor burden, and the degree was similar to that of PGRMC1 WT ( Figure 7 G). All the mice in the control group developed liver tumors, while the tumor incidence rates of the PGRMC1 WT group and the PGRMC1 1-47aa group were 57.1% and 75% respectively. In addition, both PGRMC1 WT and PGRMC1 1-47aa could significantly reduce the tumor burden mediated by c-Myc, manifested as a decrease in the liver / volume ratio, tumor number and tumor size. There was no significant difference between the PGRMC1 WT group and the PGRMC1 1-47aa group.

[0290] PGRMC1 1-47aa could also play a role after the occurrence of liver cancer in the Myc / Mcl1 liver cancer mouse model. In this mouse model, 3 weeks after injecting the Myc / Mcl1 oncogene, the experimental procedure was referred to Example 3. When the tumors had developed, AAV particles were delivered via the tail vein ( Figure 7 H). Consistent with the function of wild-type PGRMC1, compared with the control group AAV8, PGRMC1 1-47aa significantly prolonged the overall survival of the mice (p = 0.009,Figure 7 H).

[0291] Overall, PGRMC1 1-47aa activates PERK and effectively inhibits Myc / Mcl1-induced tumor formation in vivo.

[0292] In summary, the liver-specific gene PGRMC1 can significantly block the formation and development of c-Myc-induced HCC. The transmembrane domain and N-terminal domain of PGRMC1 are both important in inhibiting c-Myc-induced liver tumor formation. Delivery of PGRMC1 or PGRMC1 1-47aa as a treatment for HCC has great potential in future biomedical development.

[0293] Sequence information

[0294]

[0295]

Claims

1. Use of PGRMC1 protein or its truncations, nucleic acid molecules encoding PGRMC1 protein or its truncations, or promoters thereof in the preparation of products for the prevention, treatment and / or prognosis of HCC.

2. The use according to claim 1, wherein The PGRMC1 protein is selected from: (a) a polypeptide having an amino acid sequence as shown in SEQ ID NO.1; or (b) comprising a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence shown in SEQ ID NO.1; or (c) a protein or polypeptide derived from the amino acid sequence of (a) or (b) by inserting, substituting or deleting one or more amino acids; and / or The nucleic acid molecule encoding the PGRMC1 protein is selected from: (i) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.2; or (ii) a nucleic acid molecule that is homologous to the nucleotide sequence shown in SEQ ID NO. 2 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more homology; (iii) A nucleic acid molecule derived from the nucleotide sequence of (i) or (ii) by inserting, substituting or deleting one or more nucleotides.

3. The use according to claim 1, wherein The PGRMC1 truncation contains at least the transmembrane domain and the N-terminal domain.

4. The use according to any one of claims 1 to 3, wherein The PGRMC1 truncate is selected from: (a) a polypeptide having an amino acid sequence as shown in SEQ ID NO.6; or (b) comprising a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence shown in SEQ ID NO.6; or (c) a protein or polypeptide derived from the amino acid sequence of (a) or (b) by inserting, substituting or deleting one or more amino acids; and / or The nucleic acid molecule encoding the PGRMC1 truncate is selected from: (i) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.7; or (ii) a nucleic acid molecule that is homologous to the nucleotide sequence shown in SEQ ID NO.7 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more homology; (iii) A nucleic acid molecule derived from the nucleotide sequence of (i) or (ii) by inserting, substituting or deleting one or more nucleotides.

5. The use according to any one of claims 1 to 4, wherein The promoter is selected from an overexpression vector of a PGRMC1 or truncate coding sequence, exogenous PGRMC1 or truncate thereof, naked DNA of a PGRMC1 or truncate coding sequence, liposome-encapsulated DNA of a PGRMC1 or truncate coding sequence, a precursor protein or conjugate or complex that can be converted into PGRMC1 or truncate thereof in vivo.

6. The use according to any one of claims 1 to 5, wherein The product is a medicine or a medicine kit; Preferably, the product further comprises a second active ingredient for preventing and / or treating cancer.

7. PGRMC1 truncations, wherein The truncated body is selected from: (a) a polypeptide having an amino acid sequence as shown in SEQ ID NO.6; or (b) comprising a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence shown in SEQ ID NO.6; or (c) A protein or polypeptide derived from the amino acid sequence of (a) or (b) by inserting, substituting or deleting one or more amino acids. A nucleic acid molecule encoding the PGRMC1 truncate according to claim 7 .

9. A pharmaceutical composition comprising: (a) a preventive or therapeutically effective amount of PGRMC1, PGRMC1 truncated form or a promoter thereof; (b) a pharmaceutically or immunologically acceptable carrier or excipient; (c) optionally, one or more second active ingredients for preventing and / or treating cancer.

10. An expression cassette comprising a polynucleotide sequence, wherein: The polynucleotide sequence is operably linked from 5' to 3' to: (a) Promoter sequence; (b) a sequence encoding PGRMC1, a PGRMC1 truncate or a functional fragment or variant thereof; Optionally, a sequence encoding an HA tag polypeptide is further included between (a) and (b); Preferably, the promoter is a modified TBG promoter, and its nucleotide sequence is shown in SEQ ID NO.8; Preferably, the nucleic acid molecule encoding PGRMC1 comprises the nucleotide sequence shown in SEQ ID NO.2; Preferably, the nucleic acid molecule encoding the PGRMC1 truncate comprises the nucleotide sequence shown in SEQ ID NO.7; Preferably, the expression cassette further comprises an expression control element, which is operably linked to the polynucleotide molecule; Preferably, the expression cassette further comprises an enhancer, and the enhancer is selected from the group consisting of ApoE HCR enhancer, CRMSBS2 enhancer, TTRm enhancer and CMV enhancer; Preferably, the expression cassette further comprises an intron, wherein the intron is selected from the group consisting of α1 antitrypsin intron, β-globin intron 2, SV40 intron, and minute virus of mice intron; Preferably, the expression cassette further comprises a polyA signal, and the polyA signal is at least one of bovine growth hormone poly A (BGH polyA), short poly A, SV40 polyA, and human β-globin poly A.

11. An expression vector comprising the expression cassette according to claim 10; Preferably, the expression vector is selected from a plasmid, a cosmid, a viral vector or an RNA vector; Preferably, the viral vector is selected from a retrovirus, a parvovirus, a coronavirus, a negative-strand RNA virus, a rhabdovirus, a paramyxovirus, a positive-strand RNA virus, or a double-stranded DNA virus; Preferably, the double-stranded DNA virus is selected from adenovirus, herpes virus, poxvirus, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, baculovirus or hepatitis virus; Preferably, the retrovirus is selected from mammalian C-type, B-type virus, D-type virus, HTLV-BLV collection, lentivirus or foamy virus; Preferably, the expression vector is an adenovirus.

12. A cell comprising the expression cassette of claim 10 or the expression vector of claim 11.

13. A method for screening a drug for treating HCC by promoting PGRMC1 expression, comprising: (A) treating model cells, tissues or model animals with a candidate substance; (B) detecting changes in the expression level of PGRMC1 in the model cells, tissues or model animals, or changes in the level of nucleic acid molecules encoding the aforementioned protein; and (C) If the expression level of PGRMC1 or the level of the nucleic acid molecule encoding the aforementioned protein is higher than the level before the candidate substance treatment or higher than the level in the normal control, it indicates that the candidate substance has the effect of treating HCC by promoting PGRMC1 expression.

Images