Application of SCRN1 inhibitors in the preparation of drugs for the treatment of hepatocellular carcinoma

Abstract

The present invention relates to the field of biomedicine and provides a new use of SCRN1, specifically the use of SCRN1 inhibitors in the preparation of drugs for treating hepatocellular carcinoma. The present invention further provides a recombinant vector for SCRN1 inhibitors and a pharmaceutical composition for treating hepatocellular carcinoma containing a SCRN1 inhibitor or its recombinant expression vector as an active ingredient. Experimental results show that SCRN1 expression is elevated in tumor tissues, which does not affect the normal proliferation of liver cancer cells, but can reduce the sensitivity of cells to sorafenib. ‑ / ‑ Tumor cells are more sensitive to sorafenib treatment, and patients with high SCRN1 expression are less likely to benefit from sorafenib treatment. Therefore, the present invention reveals a new use of the SCRN1 molecule in the classification and selection of treatment options for hepatocellular carcinoma, providing new ideas and approaches for the research, development and utilization of drugs targeting SCRN1.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to the use of SCRN1 as a molecular marker in the treatment of hepatocellular carcinoma, specifically to the use of SCRN1 inhibitors in the preparation of hepatocellular carcinoma therapeutic drugs, and further to the use of SCRN1 sgRNA and other inhibitors in the preparation of anti-hepatocellular carcinoma biotherapeutic drugs, especially as anti-tumor acquired drug resistance inhibitors. Background Art

[0002] Liver cancer (HCC) is one of the most common solid malignancies worldwide, ranking among the top three causes of mortality among malignant tumors. HCC can be divided into intrahepatic cholangiocarcinoma, hepatoblastoma, and hepatocellular carcinoma (HCC). HCC originates from hepatocytes and accounts for 75%-85% of primary liver cancers. Its etiology is complex and diverse, including hepatitis B / C virus infection, fatty liver disease, diabetes, aflatoxin infection, genomic alterations, and epigenetic modifications. In response to the complex pathogenic factors and disease progression, clinical treatment primarily involves surgical resection, tumor ablation, hepatic artery embolization, and systemic therapy. Early detection and timely treatment can prolong the survival of HCC patients. However, due to the complex etiology, insidious onset, and rapid progression of HCC, patients are often diagnosed in the middle and late stages of the disease. Therefore, systemic therapy is particularly important as the preferred treatment option for patients in the middle and late stages.

[0003] Sorafenib is an FDA-approved first-line systemic treatment for liver cancer. As a multi-target kinase inhibitor, sorafenib can block tumor cell proliferation by inhibiting the serine / threonine kinase (Raf) / mitogen-activated protein kinase (MAPK) pathway; it can also inhibit tumor angiogenesis by regulating platelet-derived growth factor receptor, FMS-like tyrosine kinase, hepatocyte factor receptor, and vascular endothelial growth factor receptor. In addition, inducing programmed cell death in tumor cells is also an important part of its anti-tumor effect. However, patients who are sensitive to sorafenib in the early stage of treatment usually develop acquired resistance within 6 months (Zhu YJ et al. New knowledge of the mechanisms of sorafenib resistance in liver cancer [J]. Acta Pharmacol Sin, 2017, 38 (5): 614-622.). Current research on the mechanisms of sorafenib resistance mainly includes drug transport and metabolism, cell proliferation and programmed cell death, epithelial-mesenchymal transition, tumor microenvironment, and gene modification. Previous studies have suggested that sorafenib induces cell apoptosis and autophagy by targeting kinase receptors on the cell membrane. However, in recent years, a growing number of studies have demonstrated that sorafenib primarily induces ferroptosis by inhibiting the glutamate-cysteine ​​transport system System xc on the cell membrane, and that this process is independent of classical kinase inhibition. Clinical data indicate that elevated expression of the iron metabolism-related gene MT1G is a biomarker of sorafenib resistance in hepatocellular carcinoma. Knockdown of MT1G promotes ferroptosis and restores sensitivity of resistant cells to sorafenib. In addition, decreased expression of epidermal growth factor receptor (EGFR) and activation of hepatocyte growth factor (HGF) can activate the AKT / ERK1 / 2-EGFR1 pathway, leading to decreased sensitivity of cells to sorafenib; increased methylation modification levels of the transporter protein SLC22 (Solute Carrier Family 22, SLC22) can lead to decreased expression and inhibit cellular uptake of sorafenib; continued use of sorafenib can lead to decreased tissue microvascular density and intracellular hypoxia, thereby promoting increased expression of hypoxia-inducible factor (HIF), which is conducive to the selection of drug-resistant cells that adapt to the hypoxic microenvironment.Although the mechanism of acquired resistance to sorafenib has been partially understood and studied, a large number of liver cancer patients still cannot benefit from it clinically, and it is impossible to predict the responsiveness of different individuals to treatment. Therefore, sorafenib can only effectively prolong the survival of liver cancer patients by about half a year.

[0004] Therefore, identifying and screening markers associated with sorafenib sensitivity in hepatocellular carcinoma and screening out the groups that benefit from sorafenib treatment are crucial to improving the efficacy of systemic treatment of advanced hepatocellular carcinoma. This is also likely to help discover potential targets for future targeted research and development of new drugs.

[0005] SCRN1 belongs to the secernin family, which consists of only three members (SCRN1-3). SCRN1 is the first discovered and most extensively studied molecule. SCRN1 is primarily expressed in neural tissue, with distribution also in organs such as the kidney, endometrium, and ovaries. Its expression is low in normal liver tissue. At the protein level, SCRN1 contains a C69 domain at its N-terminus and a coiled-coil domain at its C-terminus. The C-terminus is predicted to contain a FFAT-like motif.

[0006] In recent years, the function of SCRN1 in the nervous system has been gradually revealed: the continuously dynamic endoplasmic reticulum is one of the most abundant organelles in the cell. In neurons, dendrites contain both rough and smooth endoplasmic reticulum, while axons contain only smooth endoplasmic reticulum. The smooth endoplasmic reticulum lacks ribosomes and does not participate in protein translation, but it is sensitive to Ca. 2+ Homeostasis, lipid synthesis and transport, and signal transduction are crucial. Endoplasmic reticulum transmembrane protein (VAMP associated protein, VAP) is a key molecule that promotes tight membrane connections between the endoplasmic reticulum and other intracellular membranes and is widely expressed in different cell types. VAP contains a C-terminal transmembrane domain inserted into the endoplasmic reticulum membrane, an N-terminal domain in the cytoplasm, and a sperm protein (MSP) domain. The MSP domain has a VAP protein-specific FFAT motif binding site, which is also the structural basis for its binding to SCRN1. SCRN1 can interact with VAP to maintain interneuronal Ca 2+Exchange and lipid transfer (Lindhout FW et al. VAP-SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function [J]. EMBO J, 2019, 38 (20): e101345.). In addition, SCRN1 has been identified as a novel amyloid plaque-associated neuronal protein that accumulates in large quantities in neurofibrils and axons of neurodegenerative diseases such as Alzheimer's disease and Down syndrome. Therefore, SCRN1 also has the potential to serve as a new biomarker and therapeutic target for Alzheimer's disease.

[0007] In addition, previous studies have suggested potential links between SCRN1 and tumor progression. For example, SCRN1 is a susceptibility gene for colorectal cancer, gastric cancer, prostate cancer, leukemia, and other cancers. In colon cancer, SCRN1 may promote tumor cell growth and migration by enhancing the secretion of matrix metalloproteinases 2 / 9 (Lin S et al., Secernin-1 contributes to colon cancer progression through enhancing matrix metalloproteinase-2 / 9 exocytosis [J]. Dis Markers, 2015, 2015: 230703). These studies suggest that SCRN1 may be involved in various tumor biological regulation. However, no studies have yet reported a direct involvement of SCRN1 in regulating liver cancer progression or sorafenib resistance. There is an urgent need to identify molecules that can be used effectively in the treatment of hepatocellular carcinoma, particularly inhibitors of acquired drug resistance, and to apply them to these applications. Summary of the Invention

[0008] The present invention aims to provide the use of SCRN1 inhibitors in tumor treatment, particularly in combination with sorafenib for tumor treatment. A second objective of the present invention is to provide a therapeutic agent for hepatocellular carcinoma, particularly an acquired resistance inhibitor molecule for use in combination with a hepatocellular carcinoma therapeutic agent, which can be used to enhance sorafenib-induced ferroptosis in tumor cells, inhibit acquired resistance, and improve the efficacy and responsiveness of sorafenib treatment.

[0009] Through long-term and in-depth research, the inventors discovered that the SCRN1 molecule is highly expressed in tumor tissue cells of patients with hepatocellular carcinoma, and its expression level is correlated with tumor prognosis. Tumors with high SCRN1 expression have a milder degree of cell ferroptosis after sorafenib treatment, a lower response to treatment, and a shortened survival period. Reducing the level or activity of SCRN1 in tumor tissue or cells can increase the sensitivity of liver cancer cells to sorafenib and effectively improve the treatment effect. This application reveals for the first time the role of SCRN1 inhibitors in reducing acquired drug resistance in tumors, thereby providing the use of SCRN1 inhibitors as acquired drug resistance inhibitors, which can be used in the treatment of tumors in subjects, especially in combination with tumor treatment.

[0010] The first aspect of the present invention provides the use of a SCRN1 inhibitor in the preparation of a drug for treating hepatocellular carcinoma.

[0011] The second aspect of the present invention provides the use of SCRN1 in the preparation of acquired drug resistance inhibitors for the treatment of hepatocellular carcinoma.

[0012] The drugs of the present invention are targeted at patients selected from the following groups: patients with acquired drug resistance; patients whose tumors are ineffective, ineffective, or expected to be ineffective with sorafenib treatment; patients who are currently receiving, will receive, or have received sorafenib treatment for their tumors; or patients with two or more of the foregoing conditions. Therefore, the SCRN1 inhibitor is preferably used in combination with a therapeutic agent, such as sorafenib, to improve patients who have a poor response to sorafenib treatment or who need to improve their response to treatment.

[0013] Preferably, SCRN1 is selected from any one of the following substances: SCRN1 gene, SCRN1 mRNA or cDNA, SCRN1 protein, or active or characteristic fragments of any of the foregoing.

[0014] Furthermore, the SCRN1 is a molecule comprising the following sequence:

[0015] (a) SCRN1 molecule having the sequence shown in Gene ID: 9805;

[0016] (b) a molecule that hybridizes under stringent conditions to the sequence defined in (a);

[0017] (c) an SCRN1 molecule having a sequence homology of 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or more, or any value or range of values ​​therebetween) with the sequence in (a) or (b), and the encoded polypeptide or protein having acquired drug resistance inhibitory activity, such as a SCRN1 molecule obtained by codon optimization;

[0018] (d) A polypeptide encoded by any of the above-mentioned SCRN1 molecules, or a derivative protein of the polypeptide by substitution, deletion or addition of one or more amino acids.

[0019] As used herein, the term "stringent conditions" refers to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between two sequences is at least 50%, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, and more preferably 95% or more. For example, the sequence may be the complement of the sequence defined in (a).

[0020] Preferably, the SCRN1 inhibitor is selected from any of the following substances: substances that reduce SCRN1 expression levels, reduce SCRN1 activity, or promote SCRN1 metabolism. For example, the SCRN1 inhibitor includes any one of SCRN1 sgRNA and Crisper-CAS9 mRNA, small interfering RNA molecules, short hairpin RNA, antisense nucleotides, or nanoparticles, viral vectors, PEG-modified proteins, protein microspheres, liposomes, or extracellular vesicles carrying any of the above substances.

[0021] The full-length SCRN1 sgRNA and CRISPR-Cas9 mRNA sequences described herein, or fragments thereof, can typically be obtained using PCR amplification, recombination, or synthetic methods. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein to amplify the relevant sequences. For longer sequences, two or more PCR amplifications are often required, followed by splicing the fragments generated in the correct order.

[0022] It should be understood that the SCRN1 molecules herein are preferably obtained from humans, and other SCRN1 molecules obtained from other animals that are highly homologous to human SCRN1 (e.g., having a sequence identity of greater than 70%, greater than 75%, greater than 80%, more preferably greater than 85%, such as 85%, 90%, 95%, 98%, or even 99% or greater) are also within the scope of equivalence preferably contemplated herein. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0023] The SCRN1 sgRNA and other inhibitors described herein can promote the sensitivity and efficacy of anti-tumor therapy and can be further used in anti-tumor therapy by increasing sorafenib-induced ferroptosis in tumor cells and inhibiting acquired drug resistance. In addition, by increasing the sensitivity of anti-tumor drugs (such as sorafenib treatment), the combined use of the SCRN1 sgRNA and other inhibitors described herein in tumor treatment can reduce the dosage or frequency of anti-tumor drugs, thereby reducing toxic side effects and improving patient compliance.

[0024] In terms of source, the inhibitor of the present invention is selected from: natural purified substances, modified natural purified substances, semi-synthetic substances, and chemically synthesized substances; further, the inhibitor is derived from mammals, such as humans, non-human primates (such as orangutans, apes), rodents (such as rats, mice, guinea pigs), pets (such as cats, dogs), and livestock (such as horses, cattle, sheep, pigs, rabbits).

[0025] In a second aspect of the present invention, a SCRN1 inhibitor recombinant vector is provided, comprising an expression vector and SCRN1 sgRNA and Crisper-CAS9 mRNA, SCRN1 siRNA, SCRN1 shRNA or STRIP antisense nucleotide inserted into the expression vector.

[0026] In a third aspect, the present invention provides use of the above-mentioned SCRN1 inhibitor recombinant vector in the preparation of a drug for treating hepatocellular carcinoma.

[0027] In a fourth aspect, the present invention provides a pharmaceutical composition for treating hepatocellular carcinoma, comprising an active ingredient and a medically acceptable excipient, carrier, or diluent, wherein the active ingredient is the above-mentioned SCRN1 inhibitor or SCRN1 inhibitor recombinant vector.

[0028] Preferably, the pharmaceutical composition is an acquired drug resistance inhibitor, used in combination with other active anti-tumor ingredients. Tumor treatments include, but are not limited to, surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, with chemotherapy being preferred. In some embodiments, targeted therapy includes, but is not limited to, sorafenib, lenvatinib, and regorafenib.

[0029] That is, the present invention provides an acquired drug resistance inhibition product comprising:

[0030] (A) Inhibitors of SCRN1;

[0031] (B) a pharmaceutically or immunologically acceptable carrier or excipient;

[0032] (C) optionally, one or more other anti-tumor active ingredients.

[0033] The term "pharmaceutically / immunologically acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. As used herein, the term "effective amount" refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0034] The term "pharmaceutically acceptable carrier" refers to a vehicle for administering a therapeutic agent, including various excipients and diluents. The term refers to pharmaceutical carriers that are not themselves essential active ingredients and that are not unduly toxic upon administration. Suitable carriers are well known to those of ordinary skill in the art. A comprehensive discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0035] The pharmaceutically acceptable carrier in the composition may contain a liquid such as water, saline, glycerol, and ethanol. In addition, these carriers may also contain auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffering substances, etc. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably, about 6-8.

[0036] The active substance in the composition of this invention accounts for 0.001 to 99.9 wt% of the total weight of the composition; preferably 1 to 95 wt% of the total weight of the composition, more preferably 5 to 90 wt%, and even more preferably 10 to 80 wt%, with the remainder being pharmaceutically acceptable carriers and other additives.

[0037] The composition of the present invention may be in solid form (such as granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid form (such as oral solution) or other suitable forms. The administration routes can be: (1) direct naked DNA / RNA injection; (2) connecting SCRN1 molecule-related sgRNA and CRISPR-Cas9 mRNA to transferrin / poly-L-lysine complexes to enhance their biological effects; (3) forming complexes between SCRN1 sgRNA and CRISPR-Cas9 mRNA and positively charged lipids to overcome the difficulty of crossing the cell membrane caused by the negative charge of the phosphate backbone; (4) encapsulating SCRN1 sgRNA and CRISPR-Cas9 mRNA in liposomes and mediating their entry into cells, which is conducive to the smooth entry of large molecules and protects them from hydrolysis by various extracellular enzymes; (5) combining SCRN1 sgRNA and CRISPR-Cas9 mRNA with cholesterol to increase their cytoplasmic retention time by 10 times; (6) transporting SCRN1 sgRNA and CRISPR-Cas9 mRNA using immunoliposomes to enable their specific delivery to target tissues and target cells; (7) In vitro transfection of mRNA into target cells (such as fibroblasts) can also effectively load relevant drugs into target cells; (8) electroporation, that is, using electric current to introduce SCRN1 sgRNA and CRISPR-Cas9 mRNA into target cells.

[0038] As used in the present invention, the term "unit dosage form" refers to the preparation of the composition of the present invention into a dosage form required for single administration for the convenience of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained-release preparations.

[0039] In some embodiments of the present invention, the composition is in unit dosage form or multiple dosage form. "Unit dosage form" refers to a dosage form in which the product is prepared into a single dosage form for ease of administration, including but not limited to various solid doses (such as tablets), liquids, capsules, and sustained-release formulations. In some embodiments herein, the composition herein is administered at an appropriate frequency, for example, daily, every other day, weekly, every other week or two weeks, monthly, every other month or two months, for example, 1 to 6 doses are administered.

[0040] It should be understood that the effective dose of the active substance used may vary with the severity of the subject to be administered or treated. The specific situation is determined according to the individual circumstances of the subject (e.g., subject weight, age, physical condition, desired effect), which is within the scope of the judgment of a skilled physician.

[0041] In some embodiments, the present invention further provides an anti-tumor method comprising administering a therapeutically effective amount of SCRN1 sgRNA and / or its inhibitor to a subject in need thereof. Preferably, other anti-tumor drugs, particularly sorafenib, are administered before, during, or after administration of the SCRN1 sgRNA and / or its inhibitor of the present application.

[0042] The administration methods of the present invention are diverse, including oral administration, injection (e.g., direct naked DNA or protein injection, liposome-encapsulated DNA, RNA, or protein injection), gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene-encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal administration.

[0043] In a fifth aspect, the present invention provides an anti-tumor method comprising administering a therapeutically effective amount of SCRN1 sgRNA and / or its inhibitor to a subject in need thereof. Preferably, other anti-tumor drugs, particularly sorafenib, are administered before, during, or after administration of the SCRN1 sgRNA and / or its inhibitor of the present application.

[0044] The beneficial protection and effects of the present invention are as follows:

[0045] The applicant obtained a large number of tumor tissue specimens from hepatocellular carcinoma patients from the cooperating hospital, which provided a strong guarantee for the research of the present invention.

[0046] In terms of technology, the detection of SCRN1 is essentially a quantitative PCR test, which has the characteristics of simple operation, sensitive detection, good specificity and high repeatability. It is now increasingly used in clinical testing technology.

[0047] The experimental results showed that SCRN1 expression was elevated in tumor tissues, which did not affect the normal proliferation of liver cancer cells, but reduced the sensitivity of cells to sorafenib and promoted the occurrence of acquired drug resistance. - / - Tumor cells are more sensitive to sorafenib treatment, and patients with high SCRN1 expression are less likely to benefit from sorafenib treatment. Therefore, the present invention reveals a new use of the SCRN1 molecule in the classification of hepatocellular carcinoma treatment and the selection of treatment plans, providing new ideas and approaches for the research, development and utilization of SCRN1. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : qRT-PCR detection of SCRN1 expression in HCC tissues and adjacent adjacent tissues; P values ​​were calculated using Ttest.

[0049] Figure 2: SCRN1 knockout liver cancer cell lines.

[0050] Figure 3 : Comparison of tumor growth curves after SCRN1 knockout liver cancer cells and control cells were inoculated into mice and treated with sorafenib.

[0051] Figure 4 : Lipid peroxidation in SCRN1-knockout liver cancer cells and control cells. Lipid peroxidation in tumor tissues of mice inoculated with SCRN1-knockout liver cancer cells and control cells and treated with sorafenib.

[0052] Figure 5 : Kaplan-Meier survival curve comparison of overall survival time in patients with HCC with high and low SCRN1 expression;

[0053] Figure 6 : Comparison of Kaplan-Meier survival curves of patients with liver cancer with high and low SCRN1 expression after sorafenib treatment, that is, comparison of the effectiveness of sorafenib treatment in patients with high and low SCRN1 expression. DETAILED DESCRIPTION

[0054] This article provides the use of SCRN1 inhibitors in tumor treatment, particularly in combination with tumor immunotherapy. The drugs, pharmaceutical compositions, or products of the present invention can be used to inhibit resistance to sorafenib treatment in hepatocellular carcinoma, inhibit acquired tumor resistance, and improve the efficacy and responsiveness of immunotherapy.

[0055] All numerical ranges provided herein are intended to expressly include all values ​​falling between the endpoints of the ranges and ranges therebetween. Features described herein or in the embodiments may be combined. All features disclosed herein may be used in any combination, and any feature disclosed herein may be replaced by any alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are intended only to be general examples of equivalent or similar features.

[0056] As used herein, “containing,” “having,” or “including” encompasses “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of”; “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0057] The numerical ranges herein include their endpoints and each specific numerical point and sub-range within the numerical range. For example, 1 to 3 includes endpoints 1 and 3, the specific integer numerical point 2 and non-integer numerical point therein (for example, but not limited to: 1.2, 1.5, 1.8, 2.1, 2.3, 2.4, 2.8, etc.), and sub-ranges thereof (for example, but not limited to: 1 to 2, 2 to 3, 1 to 1.2, 1.5 to 1.8, etc.).

[0058] All reagents and raw materials used in the present invention are commercially available or can be prepared according to literature methods. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed according to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are calculated by volume.

[0059] All reagents and raw materials used in the present invention are commercially available or can be prepared according to literature methods. Experimental methods in the following examples, where specific conditions are not specified, generally follow conventional conditions such as those described in Molecular Cloning: A Laboratory Manual, 4th edition, by Michael R. Green et al., Cold Spring Harbor Laboratory Press, New York, 2017, or according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to this application. The preferred embodiments and materials described herein are for illustrative purposes only.

[0061] Example 1: Preparation of SCRN1 detection kit

[0062] A SCRN1 detection kit was prepared according to the following composition. The kit is suitable for detecting the expression of SCRN1 in biological samples by real-time quantitative reverse transcription PCR (qRT-PCR):

[0063] (a) A container containing reverse transcriptase and RNase inhibitor (Takara, Catalog No. RR037);

[0064] (b) a container filled with reverse transcription 5 buffer (75 mM KCl, 500 mM Tris-Cl, pH 8.3, 25°C, 3 mM MgCl2, 10 mM DTT);

[0065] (c) container containing reverse transcription primer (10 μM) of target gene;

[0066] SCRN1 reverse transcription primers:

[0067] Oligo(dT): 5'-TTTTTTTTTTTTTTTTTT-3' (SEQ ID NO.7);

[0068] (d) Container containing target gene PCR upstream primer and downstream primer (10 μM each):

[0069] Quantitative PCR primers:

[0070] 5'-CCATCGTCCACCGCAAAT-3' (upstream, SEQ ID NO. 1); and

[0071] 5′-GCTGTCACCTTCACCGTTCC-3′ (downstream, SEQ ID NO. 2);

[0072] (e) Container containing internal reference reverse transcription primer and PCR upstream primer and downstream primer (10 μM):

[0073] Reverse transcription primers for internal reference β-actin:

[0074] Oligo(dT): 5'-TTTTTTTTTTTTTTTTTT-3' (SEQ ID NO.7);

[0075] Quantitative PCR primers:

[0076] 5'-CCATCGTCCACCGCAAAT-3' (upstream, SEQ ID NO. 3); and

[0077] 5′-GCTGTCACCTTCACCGTTCC-3′ (downstream, SEQ ID NO. 4);

[0078] (f) A container containing Taq DNA polymerase, dNTPs, fluorescent dye TBGreen, and 2X PCR buffer (Takara, catalog number RR430);

[0079] (g) Instructions for use.

[0080] Example 2: Expression of SCRN1 in hepatocellular carcinoma

[0081] The real-time quantitative reverse transcription PCR (qRT-PCR) method was used to analyze the expression of SCRN1 mRNA levels in tumor tissues and adjacent cancer tissues of 120 patients with hepatocellular carcinoma who received sorafenib treatment from Shanghai Oriental Hepatobiliary Surgery Hospital from 2016 to 2020 using the detection kit in Example 1.

[0082] Total RNA was extracted from the tissue using TRIzol (Invitrogen). qRT-PCR was performed using the test kit of Example 1 on a LightCycler 1.5 (Roche) real-time quantitative PCR instrument.

[0083] The relative quantification of SCRN1 was calculated using the 2-ΔΔCt method (β-actin was used as an internal reference) (Livak, KJ. ​​et al., Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method. Methods. 2001; 25: 402-408).

[0084] qRT-PCR analysis results showed that SCRN1 expression was increased in tumor tissues compared with adjacent non-tumor tissues ( Figure 1 ).

[0085] Example 3: Relationship between SCRN1 expression and sorafenib sensitivity.

[0086] According to the literature (Genome engineering using the CRISPR-Cas9 system. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Nat Protoc. 2013 Nov; 8(11): 2281-308.), CRISPR-cas9 gene editing technology was used to knock out SCRN1 expression in hepatocellular carcinoma cells HepG2 to obtain SCRN1 - / - Cells and control cells without SCRN1 knockout. The specific method is as follows:

[0087] The upstream and downstream sequences of SCRN1 gRNA were mixed and inserted into PX458 (purchased from Addgene, Plasmid #48138) as the SCRN1 gRNA vector. HepG2 cells were transfected using JetPEI transfection reagent (purchased from PolyPlus, catalog number 101000053) with a final transfection concentration of 1 μg / ml.

[0088] SCRN1 gRNA upstream primer:

[0089] 5'-caccCACCGTTTCAATCGACCAAGTTCCA-3'(SEQ ID NO.5)

[0090] SCRN1 gRNA downstream primers:

[0091] 5'-aaacAAACTGGAACTTGGTCGATTGAAAC-3'(SEQ ID NO.6)

[0092] After transfection, single cells were sorted into well plates using a flow cytometer (Sony SH800 Cell Sorter). Monoclonal cells were collected 3 weeks later, and proteins were extracted for Western Blot analysis.

[0093] Western Blot was performed according to the literature (Tao Y et al., UFL1 promotes antiviral immune response by maintaining STING stability independent of UFMylation. Cell Death Differ (2022). https: / / doi.org / 10.1038 / s41418-022-01041-9), and SCRN1 antibody (purchased from LSBio, catalog number #LS-C338451) was diluted 1:1000. SCRN1 successfully knocked out cell clones were selected as SCRN1 - / - cells, and cells without SCRN1 knockout served as SCRN1 control cells ( Figure 2 ).

[0094] Stimulation of SCRN1 using different concentrations of sorafenib - / - Cells and control cells were tested using CCK8 (purchased from Selleck, catalog number B34302) to detect the cell survival rate and calculate the half cell lethal dose (IC50). - / - The IC50 of cells was significantly lower than that of control cells, indicating that SCRN1 - / - Cells are more sensitive to sorafenib ( Figure 3 ).

[0095] Example 4: Relationship between SCRN1 expression and the efficacy of sorafenib in treating liver cancer

[0096] Nude mice are mutant mice with congenital thymus defects and are good tool mice for inoculating human cells. - / -HepG2 cells and control HepG2 cells were inoculated subcutaneously into mice, and tumor volumes were measured regularly to compare the growth rates of the two tumors. Tumor volumes were calculated according to the literature (Qi, R. et al., Notch1 signaling inhibits growth of human hepatocellular carcinoma through induction of cell cycle arrest and apoptosis. Cancer Res. 2003; 63: 8323-8329).

[0097] The results showed that there was no difference in the size of the two tumors in nude mice ( Figure 4 ), indicating that SCRN1 - / - There was no significant difference in proliferation and apoptosis between the HepG2 cells and the control cells. 3 Sorafenib was administered orally (30 mg / kg) every 48 hours and the tumor size was monitored. - / - The growth rate of tumor cells is significantly slower than that of control cells, indicating that SCRN1 - / - Tumor cells are more sensitive to sorafenib treatment.

[0098] The above results suggest that SCRN1 does not affect the normal proliferation of liver cancer cells, but can reduce the sensitivity of cells to sorafenib and promote the occurrence of acquired drug resistance.

[0099] Example 5: Relationship between SCRN1 expression and the severity of ferroptosis in liver cancer cells

[0100] Tumor tissues of the subcutaneous tumor-bearing nude mice in Example 3 were collected on day 21 after treatment, and tissue lipid peroxides were detected and histochemically stained.

[0101] Tissue peroxide assay (purchased from Nanjing Jiancheng Biological Research Institute, catalog number A106-1-2): Mouse tumor tissue was accurately weighed and added to normal saline at a weight (g): volume (ml) ratio of 1:9. The mixture was homogenized in an ice-water bath and centrifuged at 2500 rpm for 10 minutes. The supernatant was then assayed. 200 μl of supernatant was added to 650 μl of reaction solution, mixed, and incubated at 45°C for 60 minutes. The supernatant was removed and centrifuged at 4000 rpm for 10 minutes. 200 μl of supernatant was added to a 96-well plate and the OD value was measured at 586 nm.

[0102] Histochemical staining: Obtain mouse tumor tissue, embed and slice it before starting the experiment. Insert the slide into a dedicated slide rack for dewaxing and hydration, bake in a 65°C oven for 1.5-3 hours; dewax in xylene for 30 minutes, 30 minutes, and 20 minutes (optional); wash with gradient alcohol: 100%, 100%, 95%, 85%, 75%, distilled water, and distilled water for 5 minutes each. Then, perform antigen retrieval, block endogenous catalase with 3% H2O2 in PBS, and perform antigen retrieval at room temperature for 20 minutes in the dark. Add 700-800 ml of antigen retrieval solution to the pressure cooker and time for 2.5 minutes. Then, put the slide rack back into the box, pour in the hot antigen retrieval solution, cool naturally to room temperature, and wash twice with PBS on a shaker. After removing the slides, circle the tissue location with a histochemical pen and block each sample with 100-200 μl of goat serum. Allow to stand at room temperature for 30-60 minutes. Dilute the primary antibody to the working concentration (1:50, 1:100, 1:200, 1:500) in antibody diluent and incubate overnight in a humidified chamber. Wash three times with PBS on a shaker, then incubate with the secondary antibody for 40 minutes at 37°C. Wash three times with PBS. DAB is added dropwise for color development. Once the sections appear light brown, the DAB is aspirated and the slides are returned to the slide rack. Rinse the slide rack under running tap water and stain with hematoxylin for 40 seconds. Rinse the slide rack several times in hydrochloric acid-alcohol to remove nonspecific hematoxylin. Return the slides to tap water; the slides should now appear light purple with a hint of brown. After completion, rinse with a gentle stream of tap water for approximately five minutes and dehydrate with a reverse gradient of alcohol: 75%, 80%, 95% for 2 minutes each, and 100% for 5 minutes each twice. Finally, seal the slides with a resin adhesive containing xylene.

[0103] The results showed that SCRN1 - / - The levels of total lipid peroxide (LPO) and histochemical MDA (purchased from Abcam, catalog number ab283311) in tumor tissues were significantly higher than those in control cells ( Figure 5 ), suggesting that SCRN1 deficiency promotes lipid peroxidation in HCC cells during sorafenib treatment.

[0104] The above results suggest that SCRN1 - / - Tumor cells were more sensitive to rafenib treatment, and the degree of intracellular lipid peroxidation was higher than that of control cells.

[0105] Example 6: Relationship between SCRN1 expression and clinical HCC prognosis

[0106] 120 patients with liver cancer receiving sorafenib were treated with real-time quantitative PCR to detect the expression of SCRN1 in tumor tissue according to the kit method in Example 1. The patients were divided into two groups, high and low, with 60 patients in each group, based on the median SCRN1 expression. The correlation between SCRN1 expression and patient survival time was analyzed, and the P value was calculated using the log-rank test. The results are as follows: Figure 6 The results showed that high expression of SCRN1 was significantly associated with lower overall survival time of patients.

[0107] Cox regression analysis was performed on the risk factors affecting HCC prognosis, and the hazard ratio (95% confidence interval) and P value were calculated using univariate and multivariate Cox regression analysis in SPSS 17.0. Table 1 shows the results of univariate and multivariate Cox regression analysis of risk factors affecting the prognosis of HCC patients.

[0108] Table 1 Univariate and multivariate Cox regression analysis of risk factors affecting the prognosis of HCC patients

[0109]

[0110]

[0111] Univariate and multivariate analyses revealed that high expression of SCRN1 was a significant independent risk factor for predicting lower survival time in HCC patients.

[0112] The above results show that the expression of SCRN1 is significantly increased in HCC patients, and its high expression is significantly correlated with the patients' poor prognosis. This result suggests that there is a close correlation between SCRN1 and the occurrence and development of HCC tumors, and thus it can be used as a marker for tumor development judgment, treatment plan selection and / or prognosis assessment.

[0113] These results demonstrate that SCRN1 expression is significantly elevated in patients with liver cancer, and that elevated SCRN1 expression can inhibit sorafenib-induced ferroptosis in tumor cells, leading to acquired drug resistance. Clinically, elevated SCRN1 expression is significantly associated with a poorer prognosis, and patients with high SCRN1 expression are less likely to benefit from sorafenib treatment. These results suggest a close correlation between SCRN1 and tumor development and progression, and that SCRN1 can reduce the responsiveness and efficacy of sorafenib treatment by inhibiting sorafenib-induced ferroptosis.

[0114] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Use of a SCRN1 inhibitor in the preparation of an acquired drug resistance inhibitor for hepatocellular carcinoma, characterized in that: The hepatocellular carcinoma acquired drug resistance inhibitor is a preparation that increases the sensitivity of liver cancer cells to sorafenib. The SCRN1 inhibitor is a SCRN1 gRNA vector, which is prepared by mixing the upstream and downstream sequences of SCRN1 gRNA and inserting them into PX458. The upstream and downstream sequences of the SCRN1 gRNA are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

2. The use according to claim 1, characterized in that The SCRN1 inhibitor is used in combination with sorafenib.

3. An inhibitor of acquired drug resistance in hepatocellular carcinoma, characterized in that comprising an active ingredient and a pharmaceutically or immunologically acceptable excipient, Among them, the HCC acquired resistance inhibitor is a preparation that increases the sensitivity of liver cancer cells to sorafenib. The active component is the SCRN1 inhibitor according to any one of claims 1 to 2.

4. The hepatocellular carcinoma acquired drug resistance inhibitor according to claim 3, characterized in that Combined with sorafenib, a drug for the treatment of hepatocellular carcinoma.

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