Application of PMF1 as target spot in preparation of product for diagnosing, preventing and / or treating hepatocellular carcinoma

Abstract

The invention discloses application of PMF1 as a target spot in preparation of a product for diagnosing, preventing and / or treating hepatocellular carcinoma. According to the invention, the expression conditions of PMF1 in tumor tissues and normal liver tissues in hepatocellular carcinoma specimens are analyzed by using data of an on-line database, and it is found for the first time that the expression of PMF1 in hepatocellular carcinoma is up-regulated and the total survival rate of hepatocellular carcinoma patients with high PMF1 expression is low. Furthermore, experiments prove that the expression of the PMF1 in a hepatocellular carcinoma cell line is obviously higher than that of normal hepatocytes, proliferation, clone formation, migration and invasion of hepatocellular carcinoma cells can be effectively inhibited by reducing the expression level of the PMF1, and it is proved that interference on PMF1 gene expression can become a new way for preventing and / or treating hepatocellular carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of PMF1 as a target in the preparation of products for diagnosing, preventing and / or treating hepatocellular carcinoma. Background Art

[0002] Liver cancer is one of the common malignant tumors and one of the three major causes of cancer-related death. Liver cancer can usually be divided into two categories: secondary liver cancer and primary liver cancer. Secondary liver cancer is a secondary tumor formed by the metastasis of malignant tumors in other parts of the body to the liver through blood, lymph or direct spread. The most common ones are colorectal cancer, gastric cancer, breast cancer, lung cancer, etc.; while primary liver cancer is a malignant tumor originating from the tissue cells of the liver itself, mainly including hepatocellular carcinoma, intrahepatic bile duct carcinoma and mixed liver cancer. Among them, hepatocellular carcinoma accounts for more than 85%-90% of all primary liver cancers and is the most common type of primary liver cancer.

[0003] In the related technologies, local treatments such as surgical resection, radiofrequency ablation, transarterial chemoembolization or liver transplantation can be used when HCC is diagnosed early. However, HCC is usually diagnosed at an advanced stage when the tumor is unresectable, making these treatments ineffective. In addition, like many other cancers, HCC is highly heterogeneous, so it is difficult to achieve good therapeutic effects using a "one-size-fits-all" approach. The choice of liver transplantation, local therapy, targeted therapy or immune checkpoint inhibitors as treatment strategies depends on the disease stage and underlying disease. In addition, patients with similar disease phenotypes may have different molecular causes, resulting in different treatment responses. In recent years, there have been many studies involving the diagnosis and treatment of HCC, which have provided promising research strategies for the exploration of the above fields, but the biomarkers of HCC reported so far are still difficult to meet the needs of clinical diagnosis and treatment.

[0004] Based on this, there is an urgent need to find new and effective biomarkers, which is of great significance for achieving personalized and precise diagnosis and treatment of hepatocellular carcinoma. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes the use of PMF1 as a target in the preparation of products for diagnosing, preventing and / or treating hepatocellular carcinoma. The present invention finds through analysis and research that the expression of PMF1 in hepatocellular carcinoma cell lines is significantly higher than that in normal hepatocytes, and by reducing the expression level of PMF1, the proliferation, cloning, migration and invasion of hepatocellular carcinoma cells can be effectively inhibited.

[0006] The first aspect of the present invention provides use of a PMF1 inhibitor in the preparation of a product for preventing and / or treating hepatocellular carcinoma.

[0007] The application according to the embodiments of the present invention has at least the following beneficial effects: The present invention uses data analysis of an online database to analyze the expression of PMF1 in tumor tissues and normal liver tissues of hepatocellular carcinoma specimens, and for the first time discovers that the expression of PMF1 is up-regulated in hepatocellular carcinoma. The overall survival rate of hepatocellular carcinoma patients with high expression of PMF1 is low, and PMF1 is an independent factor for poor prognosis of hepatocellular carcinoma. Further, the present invention proves through experiments that the expression of PMF1 in hepatocellular carcinoma cell lines is also significantly higher than that of normal hepatocytes, and by reducing the expression level of PMF1, the proliferation, colony formation, migration and invasion of hepatocellular carcinoma cells can be effectively inhibited, indicating that interfering with the expression of the PMF1 gene can become a new way to prevent or treat hepatocellular carcinoma.

[0008] In some embodiments of the present invention, the PMF1 gene has the nucleotide sequence represented by NC_000001.11 in the database GeneBank.

[0009] In some embodiments of the present invention, the PMF1 inhibitor includes a reagent that inhibits the expression of the PMF1 gene and / or reduces the activity of the PMF1 protein.

[0010] In some embodiments of the present invention, the PMF1 inhibitor includes at least one of interfering RNA targeting the PMF1 gene, antisense oligonucleotides targeting the PMF1 gene, CRISPR targeting the PMF1 gene, TALEN targeting the PMF1 gene, and zinc finger nuclease targeting the PMF1 gene.

[0011] In some embodiments of the present invention, the interfering RNA targeting PMF1 includes dsRNA targeting the PMF1 gene, siRNA (small interfering RNA), and shRNA (small hairpin RNA) targeting the PMF1 gene.

[0012] In some embodiments of the present invention, the nucleotide sequence of the shRNA targeting the PMF1 gene is shown in any one of SEQ ID NO.5 to SEQ ID NO.8.

[0013] In some embodiments of the present invention, the reagent for reducing the activity of the PMF1 protein includes a binding molecule that specifically binds to the protein encoded by PMF1 (such as an antibody or ligand capable of inhibiting the activity of the PMF1 protein).

[0014] In some embodiments of the present invention, the uses of the product for preventing and / or treating hepatocellular carcinoma include any one of the following: A) Inhibiting the proliferation of hepatocellular carcinoma cells; B) Inhibiting the cloning of hepatocellular carcinoma cells; C) Inhibiting the migration and / or invasion of hepatocellular carcinoma cells.

[0015] In a second aspect of the present invention, there is provided a pharmaceutical composition for preventing and / or treating hepatocellular carcinoma, wherein the active substance of the pharmaceutical composition comprises a PMF1 inhibitor or a pharmaceutically acceptable salt thereof.

[0016] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0017] In some embodiments of the present invention, the pharmaceutically acceptable excipient comprises at least one of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a solubilizer, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant or a buffer.

[0018] In some embodiments of the present invention, the dosage form of the pharmaceutical composition comprises at least one of a suspension, a granule, a capsule, a powder, a tablet, an emulsion, a solution, a dropping pill, an injection, an oral preparation, a suppository, an enema, an aerosol, a patch or a drop.

[0019] In some embodiments of the present invention, the administration route of the pharmaceutical composition comprises at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, aerosol administration or transdermal administration.

[0020] In a third aspect of the present invention, there is provided the use of the PMF1 gene as a biomarker in the preparation of a candidate compound for screening for preventing and / or treating hepatocellular carcinoma.

[0021] In some embodiments of the present invention, the biomarker comprises, but is not limited to, a hepatocellular carcinoma biomarker, a hepatocellular carcinoma-specific biomarker, a control biomarker, an exogenous biomarker or an endogenous biomarker.

[0022] In some embodiments of the present invention, the steps of screening for a candidate compound for preventing and / or treating hepatocellular carcinoma comprise: In the test group, a test compound is added to the cell culture system, and the expression level and / or activity of PMF1 in the cells of the test group are observed; in the control group, no test compound is added to the cell culture system of the same cells, and the expression level and / or activity of PMF1 in the cells of the control group are observed; Wherein, if the expression level and / or activity of PMF1 in the cells of the test group are lower than those of the control group, it indicates that the test compound is a candidate compound for treating cancer that inhibits the expression and / or activity of PMF1.

[0023] In some embodiments of the present invention, the steps further include: performing further cell experiments and / or animal tests on the obtained candidate compounds to further select and determine substances useful for preventing, alleviating or treating hepatocellular carcinoma from the candidate compounds.

[0024] In some embodiments of the present invention, the system for screening candidate compounds for preventing and / or treating hepatocellular carcinoma is not limited to cell systems, subcellular systems, solution systems, tissue systems, organ systems or animal systems, etc. The system is not limited to the above forms, as long as the system can detect that the test compound can reduce the expression and / or activity of PMF1.

[0025] In some embodiments of the present invention, the candidate compounds include, but are not limited to, interfering molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands), small molecule compounds, etc. designed against the PMF1 gene or the protein encoded thereby or its upstream or downstream genes or proteins.

[0026] In a fourth aspect of the present invention, there is provided the use of a reagent for detecting the expression level of the PMF1 gene in the preparation of hepatocellular carcinoma auxiliary diagnosis products and hepatocellular carcinoma prognosis assessment products The application according to the embodiments of the present invention has at least the following beneficial effects: The present invention has for the first time discovered a new molecular marker of hepatocellular carcinoma - the PMF1 gene. By detecting the expression level of PMF1 in the hepatocellular carcinoma tissue of a subject, it is possible to determine whether the subject has hepatocellular carcinoma and predict the prognosis of the patient, thereby guiding clinicians to provide a prevention plan or a treatment plan for the subject; using a molecular marker to achieve the diagnosis and treatment of a disease is more timely and specific than traditional means.

[0027] In some embodiments of the present invention, the reagent for detecting the expression level of the PMF1 gene includes specific primers for detecting the expression level of the PMF1 gene or an antibody against the PMF1 protein.

[0028] In some embodiments of the present invention, the specific primers for detecting the expression level of the PMF1 gene include primer sequences shown in SEQ ID NO.1 and / or SEQ ID NO.2.

[0029] In some embodiments of the present invention, the hepatocellular carcinoma auxiliary diagnosis products include, but are not limited to, chips, preparations or kits.

[0030] Other features and advantages of the present invention will be described in the subsequent specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further described below with reference to the drawings and embodiments, wherein: Figure 1 This is the expression situation of PMF1 of the present invention in liver cancer tissues and normal liver tissues, and the results of prognosis analysis. Among them, A is the expression situation of PMF1 in the TCGA database, B is the result of prognosis analysis, C is the result of the expression quantity analysis of the GSE76427 data set in the GEO database, and D is the result of the expression quantity analysis of the data set GSE124535 in the GEO database; Figure 2 This is the qPCR detection result of the expression situation of the PMF1 gene of the present invention in hepatocellular carcinoma cells and hepatocytes; Figure 3 This is the statistical result of the expression situation of the PMF1 gene of the present invention in hepatocellular carcinoma cells after gene silencing; Figure 4 This is the detection result of the influence of the PMF1 gene of the present invention on the proliferation ability of different hepatocellular carcinoma cells. Among them, A is the hepatocellular carcinoma cell PLC / PRF / 5, B is the hepatocellular carcinoma cell Huh7, and C is the hepatocellular carcinoma cell Hep3B; Figure 5 This is the influence of the PMF1 gene of the present invention on the colony formation of hepatocellular carcinoma cells. Among them, A is the result diagram of crystal violet staining, and B is the statistical result; Figure 6 This is the influence of the PMF1 gene of the present invention on the migration and invasion abilities of different hepatocellular carcinoma cells. Among them, A is the hepatocellular carcinoma cell PLC / PRF / 5, B is its statistical result; C is the hepatocellular carcinoma cell Huh7, D is its statistical result; E is the hepatocellular carcinoma cell Hep3B, and F is its statistical result; Figure 7 This is the influence of the PMF1 gene of the present invention on the subcutaneous xenograft tumors of hepatocellular carcinoma cells in nude mice. Among them, A is the comparison diagram of tumor volume, B is the statistical result of tumor weight, and C is the statistical result of tumor volume. Detailed implementation manners

[0032] The following will clearly and completely describe the concept of the present invention and the technical effects generated in combination with the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0033] The terms "preferably", "more preferably", etc. in the present invention refer to the embodiments of the present invention that can provide certain beneficial effects in certain situations. However, in the same situation or other situations, other embodiments may also be preferred. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of the present invention.

[0034] When a numerical range is disclosed herein, the above range is considered continuous and includes the minimum and maximum values of the range, as well as every value therebetween. Further, when the range refers to integers, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0035] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the related listed items.

[0036] In the description of the present invention, descriptions with reference to terms such as "one embodiment", "some embodiments", "schematic embodiments", "examples", "specific examples", or "some examples" mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0037]

Term

[0038] The full-length human PMF1 nucleotide sequence or fragments thereof of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis methods. For the PCR amplification method, according to the disclosed relevant nucleotide sequences, a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template to amplify the relevant sequences. When the sequence is relatively long, it is often necessary to perform PCR amplification two or more times, and then splice the fragments amplified each time together in the correct order.

[0039] Those skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the target gene of the present invention. Two sequences are "substantially homologous" (or substantially similar) if, when a nucleic acid or a fragment thereof is optimally aligned with another nucleic acid (or its complementary strand) (with appropriate nucleotide insertions or deletions), nucleotide sequence identity exists in at least about 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95-98% of the nucleotide bases.

[0040] Alternatively, substantial homology or (identity) exists between them when a nucleic acid or a fragment thereof hybridizes with another nucleic acid (or its complementary strand), a strand or its complementary sequence under selective hybridization conditions. Hybridization selectivity exists when hybridization specificity is lost more selectively overall. Typically, selective hybridization occurs when there is at least about 55% identity, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90% identity in a sequence of at least about 14 nucleotides. As described herein, the length of the homologous comparison can be a longer sequence segment, typically at least about 20 nucleotides in some embodiments, more typically at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.

[0041] Inhibitors and pharmaceutical compositions: Based on the discoveries of the present inventors, the present invention provides the use of an inhibitor of PMF1 for preparing a pharmaceutical composition for inhibiting hepatocellular carcinoma. As used herein, the inhibitor of PMF1 includes, but is not limited to, inhibitors, antagonists, blockers, blocking agents, nucleic acid inhibitors, etc.

[0042] The inhibitor of the PMF1 gene or protein refers to any substance that can reduce the activity of the PMF1 protein, reduce the stability of the PMF1 gene or protein, down-regulate the expression of the PMF1 protein, reduce the effective action time of the PMF1 protein, or inhibit the transcription and translation of the PMF1 gene. These substances can all be used in the present invention as substances useful for down-regulating PMF1, and thus can be used for preventing or treating hepatocellular carcinoma. For example, the inhibitor is: a nucleic acid inhibitor, a protein inhibitor, an antibody, a ligand, a protease, a protein-binding molecule, as long as it can down-regulate the expression of the PMF1 protein or its coding gene at the protein or gene level.

[0043] As an alternative of the present invention, the inhibitor of PMF1 is an antibody that specifically binds to PMF1. The antibody can be a monoclonal antibody or a polyclonal antibody. Animals such as rabbits, mice, rats, etc. can be immunized with PMF1 protein to produce polyclonal antibodies; a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant, etc. Similarly, cells expressing PMF1 or its antigenic fragments can be used to immunize animals to produce antibodies. The antibody can also be a monoclonal antibody, and such monoclonal antibodies can be prepared using hybridoma technology. The "specificity" of the antibody refers to the ability of the antibody to bind to the PMF1 gene product or fragment. Preferably, it refers to those antibodies that can bind to the PMF1 gene product or fragment but do not recognize and bind to other non-related antigen molecules. The antibodies of the present invention can be prepared by various techniques known to those skilled in the art. The present invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments such as Fab’ or (Fab)2 fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules; or chimeric antibodies. Antibodies against PMF1 protein can be used in immunohistochemical techniques to detect the content of PMF1 protein in biopsy specimens, and can also be used as specific therapeutic agents for preventing hepatocellular carcinoma. The direct determination of PMF1 protein in blood samples or urine can be used as an auxiliary diagnostic and prognostic observation index for tumors, and can also be used as a basis for early tumor diagnosis. Antibodies can be detected by ELISA, Western Blot analysis, or conjugated with detection groups and detected by methods such as chemiluminescence and isotope tracing.

[0044] As an alternative embodiment of the present invention, the inhibitor of PMF1 is a "Small hairpin RNA (shRNA)", which is a non-coding small RNA molecule capable of forming a hairpin structure. Small hairpin RNA can inhibit gene expression through the RNA interference pathway. As described above, shRNA can be expressed from a double-stranded DNA template. The double-stranded DNA template is inserted into a vector, such as a plasmid or viral vector, and then ligated to a promoter for expression in vitro or in vivo. Under the action of DICER enzyme in eukaryotic cells, shRNA can be cleaved into small interfering RNA molecules, thus entering the RNAi pathway. "shRNA expression vector" refers to some plasmids commonly used in the art to construct shRNA structures. Usually, there is a "spacer sequence" on this plasmid and multiple cloning sites or replacement sequences on both sides of the "spacer sequence", so that people can insert the DNA sequence corresponding to shRNA (or analogs) in a forward and reverse manner into the multiple cloning sites or replace the replacement sequences thereon. The RNA transcribed from this DNA sequence can form a shRNA (Short Hairpin) structure. The "shRNA expression vector" can currently be obtained entirely through commercial purchase, such as some viral vectors.

[0045] When screening for effective shRNA sequences, the present inventors performed a large number of alignment analyses to identify the best effective fragments. The present inventors designed and synthesized a variety of shRNA sequences, and transfected them into hepatocellular carcinoma cell lines using transfection reagents for verification. Two shRNAs with the best interference effects were selected, and they have the sequences shown in SEQ ID NO.5 to SEQ ID NO.8 respectively. Further experiments at the cellular level showed that the inhibition efficiency was very high for cell experiments.

[0046] As a preferred embodiment of the present invention, the inhibitor of PMF1 can also be a PMF1-specific small interfering RNA molecule. As used herein, the "small interfering RNA" refers to a short double-stranded RNA molecule that can target and degrade specific mRNA with a homologous complementary sequence of mRNA, and this process is the RNA interference process. Small interfering RNA can be prepared in the form of double-stranded nucleic acids, which contains a sense strand and an antisense strand, and these two strands form a double-strand only under hybridization conditions. A double-stranded RNA complex can be prepared from the separated sense strand and antisense strand. Thus, for example, the complementary sense strand and antisense strand are chemically synthesized, and then annealed and hybridized to produce a synthetic double-stranded RNA complex.

[0047] The nucleic acid inhibitors of the present invention, such as siRNA, can be chemically synthesized or prepared by transcribing into single-stranded RNA through an expression cassette in a recombinant nucleic acid construct. Nucleic acid inhibitors such as siRNA can be delivered into cells by using appropriate transfection reagents or can also be delivered into cells by using various techniques known in the art.

[0048] Pharmaceutical composition: The present invention also provides a pharmaceutical composition which contains an effective amount of the inhibitor of PMF1 as described above and a pharmaceutically acceptable carrier. The composition can be used for inhibiting hepatocellular carcinoma. Any of the aforementioned inhibitors of PMF1 can be used for preparing the composition.

[0049] 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. The effective amount of the inhibitor may vary with the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by those of ordinary skill in the art according to various factors (such as through clinical trials). The factors include but are not limited to: the pharmacokinetic parameters of the inhibitor of the PMF1 gene, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's body weight, the patient's immune status, the route of administration, etc.

[0050] The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents. This term refers to such pharmaceutical carriers: they are not necessarily the active ingredients themselves and have no excessive toxicity after administration. Suitable carriers are well known to those of ordinary skill in the art. In the composition, the pharmaceutically acceptable carrier may contain a liquid, such as water, saline, buffer solution. In addition, there may also be auxiliary substances in these carriers, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, etc. The carrier may also contain a cell (host cell) transfection reagent.

[0051] The present invention can use various methods well known in the art to administer the inhibitor or its coding gene, or its pharmaceutical composition to mammals. These methods include but are not limited to: subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained-release administration, etc.; preferably, the mode of administration is parenteral administration.

[0052] Preferably, gene therapy can be used. For example, an inhibitor of PMF1 can be directly administered to a subject by methods such as injection; or, an expression unit carrying an inhibitor of PMF1 (such as an expression vector or a virus, or shRNA or siRNA) can be delivered to the target site through a certain route and made to express an active PMF1 inhibitor, and the specific situation depends on the type of the inhibitor, all of which are well known to those skilled in the art.

[0053] The term "host cell" can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS, or 293 cells, etc.

[0054] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, and the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0055] The pharmaceutical composition in the present invention comprises an inhibitor of PMF1, and / or other drugs compatible with the inhibitor, and a pharmaceutically acceptable carrier and / or excipient.

[0056] The pharmaceutical composition of the present invention can also be used in combination with other drugs for treating hepatocellular carcinoma. The other therapeutic compounds can be administered simultaneously with the main active ingredient, or even simultaneously in the same composition.

[0057] The pharmaceutical composition of the present invention can also be administered to other therapeutic compounds in a separate composition or in a dosage form different from the main active ingredient. A partial dose of the main ingredient can be administered simultaneously with the other therapeutic compounds, while other doses can be administered separately. During the treatment process, the dosage of the pharmaceutical composition of the present invention can be adjusted according to the severity of the symptoms, the frequency of recurrence, and the physiological response to the treatment regimen.

[0058] Marker: A "marker" (used alone or in combination with other qualitative terms such as hepatocellular carcinoma marker, hepatocellular carcinoma specific marker, control marker, exogenous marker, endogenous marker) refers to a parameter that can be measured, calculated, or otherwise obtained, associated with any molecule or combination of molecules, and can be used as an indicator of a biological and / or chemical state. In the present invention, a "marker" refers to a parameter associated with one or more biomolecules (i.e., "biomarker") such as nucleic acids (i.e., individual genes, and coding and non-coding DNA and RNA) and proteins (e.g., peptides, polypeptides) produced naturally or synthetically. The "marker" in the present invention also includes a single parameter that can be calculated or otherwise obtained by considering expression data from two or more different markers.

[0059] A hepatocellular carcinoma marker refers to a specific type of marker that can be used as an indicator of hepatocellular carcinoma in a subject (alone or in combination with other markers). In a specific embodiment of the present invention, the hepatocellular carcinoma marker can be used to provide a marker for the clinical evaluation of hepatocellular carcinoma in a subject (alone or in combination with other markers).

[0060] An "endogenous marker" refers to a marker (such as a nucleic acid or polypeptide) derived from the same subject as the sample to be analyzed. More specifically, an "endogenous control marker" refers to a marker that can be used as a control marker (alone or in combination with other control markers) and is derived from the same subject as the sample to be analyzed. In one embodiment, the endogenous control marker may include one or more endogenous genes (i.e., "control genes" or "reference genes") whose expression is relatively stable, such as in hepatocellular carcinoma and non-hepatocellular carcinoma samples, and / or among subjects.

[0061] An "exogenous marker" refers to a marker (such as a nucleic acid or polypeptide) derived from a subject different from the sample to be analyzed. More specifically, an "exogenous control marker" refers to a marker that can be used as a control marker (alone or in combination with other control markers) and is not derived from the same subject as the sample to be analyzed. In the present invention, on the one hand, the exogenous marker or exogenous control marker can be isolated from a different subject or can be produced synthetically and added to the sample to be analyzed. On the other hand, the exogenous control marker can be a molecule added or spiked into the sample to be analyzed as an internal positive or negative control. The exogenous control marker can be used in conjunction with the detection of one or more hepatocellular carcinoma markers to distinguish "true negative" results (e.g., non-hepatocellular carcinoma diagnosis) from "false negative" or "uninformative" results (e.g., due to problems with the amplification reaction).

[0062] "Control marker" or "reference marker" refers to a specific type of marker that is used (alone or in combination with other control markers) to control for possible interfering factors and / or to provide one or more indicators regarding sample quality, effective sample preparation, and / or appropriate reaction setups (e.g., for RT-PCR reactions). In some embodiments, the control marker can be an endogenous control marker, an exogenous control marker, and / or a hepatocellular carcinoma-specific control marker as described herein. The control marker can be co-detected or separately detected with the hepatocellular carcinoma marker of the present invention. The control marker can be one or more endogenous genes, such as housekeeping genes or a combination of hepatocellular carcinoma-specific control markers or genes.

[0063] Drug screening: The present invention provides a method for screening drugs for preventing or treating hepatocellular carcinoma, that is: In the experimental group, a test compound is added to the cell culture system, and the expression level of PMF1 is measured; in the control group, the test compound is not added to the same culture system, and the expression level of PMF1 is measured; wherein, if the expression level of PMF1 in the experimental group is less than that in the control group, it indicates that the candidate compound is an inhibitor of PMF1.

[0064] In the present invention, the method further includes: further testing the candidate compound obtained in the above step for its effect of inhibiting hepatocellular carcinoma. If the test compound has a significant inhibitory effect on hepatocellular carcinoma, it indicates that the compound is a potential substance for preventing or treating hepatocellular carcinoma.

[0065] Detection method: The present invention can measure gene expression using any method known in the art. Those skilled in the art should understand that the means for measuring gene expression is not an important aspect of the present invention. Exemplary methods for quantifying RNA expression in a sample known in the art include, but are not limited to, Southern blotting, Northern blotting, microarray, polymerase chain reaction (PCR), NASBA, TMA, RT-PCR, real-time fluorescence quantitative PCR.

[0066] In the present invention, the terms "upregulated" or "overexpressed" refer to a gene that is expressed (e.g., RNA and / or protein expression) at a higher level in cancer tissues (such as in hepatocellular carcinoma tissues) relative to the level in other corresponding tissues. Genes upregulated in cancer are expressed at a level that is at least 10%, preferably at least 25%, more preferably at least 50%, more preferably at least 100%, more preferably at least 200%, and most preferably at least 300% higher than the expression level in other corresponding tissues (such as normal or non-cancerous liver tissues).

[0067] Chip, kit: The gene chip of the present invention includes: a solid-phase carrier; and oligonucleotide probes orderly immobilized on the solid-phase carrier, and the oligonucleotide probes specifically correspond to part or all of the sequences shown in PMF1.

[0068] Specifically, suitable probes can be designed according to the gene of the present invention, immobilized on a solid-phase carrier to form an "oligonucleotide array". The "oligonucleotide array" refers to an array having addressable positions (i.e., positions characterized by distinguishable, accessible addresses), and each addressable position contains a characteristic oligonucleotide connected thereto. According to needs, the oligonucleotide array can be divided into multiple sub-arrays.

[0069] "Probe" is intended to include a nucleic acid oligomer or aptamer that specifically hybridizes to a target sequence in a nucleic acid or its complement under hybridization-promoting conditions, thereby allowing detection of the target sequence or its amplified nucleic acid. Detection can be direct (i.e., produced by a probe directly hybridizing to the target or amplified sequence) or indirect (i.e., produced by a probe hybridizing to an intermediate molecular structure connecting the probe and the target or amplified sequence). The "target" of a probe generally refers to a sequence in an amplified nucleic acid sequence that specifically hybridizes to at least part of the probe sequence through standard hydrogen bonds or "base pairing". Sequences that are "sufficiently complementary" allow the probe sequence to stably hybridize to the target sequence, even if the two sequences are not completely complementary. The probe can be labeled or unlabeled. The probe can be produced by molecular cloning of a specific DNA sequence or can be synthesized. Those skilled in the art of the present invention can easily determine various primers and probes that can be designed and used in the context of the present invention.

[0070] The solid-phase carrier in the present invention can adopt various common materials in the field of gene chips, such as including but not limited to plastic products, microparticles, membrane carriers, etc. The plastic products can be combined with antibodies or protein antigens through non-covalent or physical adsorption mechanisms. The most commonly used plastic products are small test tubes, beads and microtiter plates made of polystyrene; the microparticles are microspheres or particles polymerized from high-molecular monomers, and their diameters are mostly in micrometers. Due to having functional groups capable of binding to proteins, they are easily chemically coupled with antibodies (antigens) and have a large binding capacity; the membrane carriers include microporous filter membranes such as nitrocellulose membranes, glass fiber membranes and nylon membranes.

[0071] The preparation of the PMF1 chip can adopt the conventional manufacturing methods of biochips known in the art. For example, if the solid-phase carrier is a modified glass slide or silicon wafer, and the 5' end of the probe contains an amino-modified poly dT string, the oligonucleotide probe can be formulated into a solution, and then spotted on the modified glass slide or silicon wafer by a spotter, arranged into a predetermined sequence or array, and then fixed by standing overnight to obtain the gene chip of the present invention.

[0072] The present invention provides a kit, which can be used to detect the expression of PMF1 gene or protein. Preferably, the preparation or kit further contains a marker for labeling RNA samples and a substrate corresponding to the marker. In addition, the kit may further include various reagents required for RNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzymes, control solution, color development solution, washing solution, etc. In addition, the kit also includes an instruction manual and / or chip image analysis software.

[0073] The components of the kit can be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the kit generally include at least one vial, test tube, flask, plastic bottle, syringe or other container, in which one component can be placed, and preferably, can be appropriately aliquoted. When there is more than one component in the kit, the kit will usually also include a second, third or other additional container, in which the additional components are placed separately. However, different combinations of components can be included in one vial. The kit of the present invention will generally also include a container for containing the reactants, sealed for commercial sale. Such a container may include a molded or blow-molded plastic container, in which the required vials can be retained.

[0074] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. For the experimental methods without specific conditions noted in the embodiments, they are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0075] Example 1: Screening of gene markers related to hepatocellular carcinoma The gene expression profiles and clinical relevant information of 371 cases of hepatocellular carcinoma tissues and 50 cases of adjacent normal liver tissues were obtained from the TCGA database as the analysis data set. Based on the above analysis data set, gene expression analysis and Kaplan-Meier survival analysis were performed. The results are shown in Figure 1 A and B as shown below, showing that compared with non-tumor tissues, the expression of PMF1 is up-regulated, and the survival rate of hepatocellular carcinoma patients with high PMF1 expression is lower.

[0076] Expression level analysis was performed in the GSE76427 and GSE124535 data sets in the GEO database. The results are shown in Figure 1 C and D as shown below, showing that the expression of PMF1 in HCC tissues is significantly higher than that in adjacent tissues.

[0077] Example 2: Differential Expression of PMF1 Gene in Hepatocellular Carcinoma Cell Lines and Hepatocyte Cell Lines In this example, the qPCR experimental method was used to detect the differential expression of the PMF1 gene in hepatocellular carcinoma cell lines and hepatocyte cell lines, which specifically includes the following content.

[0078] 1. Cell Culture The human hepatocyte cell line MIHA, as well as the hepatocellular carcinoma cell lines HUH7, HEP3B, PLC, SNU387, SNU182, HEPG2, and HCC-LM3 (all purchased from the Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection) were obtained and inoculated in DMEM medium containing 10% fetal bovine serum and 1% P / S, and cultured in an incubator at 37°C, 5% CO2, and a relative humidity of 90%. The medium was changed every 2 - 3 days, and the cell growth state was observed during this period. The cells grew well and adhered to the wall in a monolayer. Conventional digestion and passage were performed using 0.25% trypsin containing EDTA.

[0079] 2. Detection of the Transcription Level of PMF1 Gene by qPCR (1) Extraction of Total Cellular RNA: The RNA in the above cells was extracted using the ESscience cell RNA extraction kit, and the experimental operation was carried out according to the instructions; The concentration and purity of the extracted RNA were detected using Nanodrop2000. The qualified standard for quality was a concentration ≥200 ng / μL and an OD260 / 280 between 1.8 and 2.2.

[0080] (2) mRNA Reverse Transcription: The Novizan HiScript III reverse transcription kit (product number: R323 - 01) was used for mRNA reverse transcription. The specific steps are as follows: Step S1: Take a 0.2 mL PCR tube and prepare the reaction system on ice. Take 4 μL of 4 × gDNA wiper Mix, 1 μg of RNA, and make up the total volume to 16 μL with nuclease-free water. After thorough mixing, centrifuge briefly for 3 - 5 seconds using a microcentrifuge, and use a PCR instrument to remove genomic DNA.

[0081] Step S2: Set the reaction conditions as: 42°C, 2 min.

[0082] Step S3: After the reaction is completed, directly add 4 μL of 5 × HiScript III qRTSuperMix to the reaction tube, mix well, centrifuge briefly for 3 - 5 seconds using a microcentrifuge, and use a PCR instrument for reverse transcription.

[0083] The reverse transcription reaction conditions were set as follows: heated at 37°C for 15 min, heated at 85°C for 5 s, cooled to 4°C, and stored at -20°C for later use. After reverse transcription, the obtained cDNA was diluted with DEPC water at a ratio of 1:10 for later use.

[0084] (3)qPCR amplification: 1) Primer design: qPCR amplification primers were designed according to the coding sequences of the PMF1 gene and the housekeeping gene GAPDH gene in PrimerBank and synthesized by Tsingke Biotechnology Co., Ltd.

[0085] Primer sequences for the PMF1 gene: The forward primer sequence is 5'- TCGTCTTCGGAATCTGTGCCAC-3' (SFQ ID NO.1); The reverse primer sequence is 5'- TGTGTCATCGCAGGCTGCAACT-3' (SEQ ID NO.2).

[0086] Primer sequences for the GAPDH gene: The forward primer sequence is 5'- GTCTCCTCTGACTTCAACAGCG-3' (SEQ ID NO.3); The reverse primer sequence is 5'- ACCACCCTGTTGCTGTAGCCAA-3' (SEQ ID NO.4).

[0087] 2) qPCR amplification reaction: The reaction system was prepared on ice. 0.2 μL of the forward primer, 0.2 μL of the reverse primer (initial primer concentration was 10 nM), 7 μL of 2 × SYBR Green qPCR Mix, and 3 μL of the diluted cDNA were added to each well.

[0088] The reaction program was set as follows: 95°C, 5 min; (95°C 10 s, 60°C 30 s) × 40 cycles, melting curve.

[0089] After the reaction, the gene expression was calculated using the formula based on the obtained ct value: 2–(ΔΔCt).

[0090] 3. Statistical analysis The above experiments were all completed with 3 replicates. SPSS 17.0 statistical software was used for statistical analysis. The difference between the two was analyzed by t-test, and it was considered statistically significant when P<0.05.

[0091] 4. Results The detection results are as Figure 2As shown, compared with normal hepatocytes, the expression of PMF1 in hepatocellular carcinoma cells is up-regulated, and the difference is statistically significant (P<0.05).

[0092] Example 3: Silencing of PMF1 gene 1. Cell culture Human hepatocellular carcinoma cell lines PLC, HUH7, and HEP3B were obtained and inoculated into DMEM medium containing 10% fetal bovine serum and 1% P / S, and cultured in an incubator at 37°C, 5% CO2, and 90% relative humidity. The medium was changed every 2 - 3 days, and the cell growth status was observed during this period. The cells grew well and adhered to the wall in a monolayer. Conventional digestion and passage were performed using 0.25% trypsin containing EDTA.

[0093] 2. Lentivirus infection (1) Treatment of cells before lentivirus infection: One day before transfection, the above-mentioned human hepatocellular carcinoma cell lines were inoculated into 6-well culture plates at a density of 3×10 5 ~5×10 5 cells / well and cultured in complete medium for 24 h. The cell density at the time of transfection was 30% - 50%.

[0094] (2) Design of shRNA and packaging and synthesis of lentivirus: Based on the PMF1 gene, two shRNAs were screened in the present invention, namely shRNA1-1 and shRNA1-3. Another negative control shRNA sequence (shRNA-NC, hereinafter referred to as shNC) was purchased from Tsingke Biotechnology Co., Ltd., and its sequence information is as follows: shRNA1-1: Sense strand: 5’- CCAGAGATTCACTGACTGCTA-3’ (SEQ ID NO.5); Antisense strand: 5’- TAGCAGTCAGTGAATCTCTGG-3’ (SEQ ID NO.6); shRNA1-3: Sense strand: 5’- GACATCTATCCGGGAGGAAAT-3’ (SEQ ID NO.7); Antisense strand: 5’- ATTTCCTCCCGGATAGATGTC-3’ (SEQ ID NO.8); shRNA-NC: Sense strand: 5’-CCTAAGGTTAAGTCGCCCTC-3’ (SEQ ID NO.9); The antisense strand is 5'-GAGGGCGACTTAACCTTAGG-3' (SEQ ID NO.10); shRNA plasmid vector: pLKO.1-TRC-copGFP-2A-PURO.

[0095] Transfection was carried out using the PEI transfection reagent from Yeasen Biotech Co., Ltd. according to the instructions. The specific steps are as follows: Step S1: Take 20 μg of shRNA plasmid, PSPAX2 plasmid (Addgene#12260), and PMD2.G (Addgene#12259) plasmid in a ratio of 3:2:1 and add them to 500 μL of serum-free medium. Gently mix and incubate at room temperature for 5 min; Step S2: Take 40 μL of PEI and add it to 460 μL of serum-free medium. Gently mix and incubate at room temperature for 5 min; Step S3: Mix the above two mixtures (total volume 1 mL), gently mix, and incubate at room temperature for 25 min to form a complex; Step S4: Add 1 mL of the complex and an appropriate amount of medium to each well of 293T cells cultured in a 6 cm dish. Gently mix, and collect the culture supernatant containing lentivirus at 48 h and 96 h. After filtering through a 0.45 μm pore size PVDF membrane filter, it can be used directly or aliquoted and stored.

[0096] (3)Cell infection: Add the diluted lentivirus suspension with the corresponding titer to the medium, and at the same time add polybrene at a ratio of 1:1000 to enhance the lentivirus infection ability. Place the 6-well plate in the cell culture incubator and culture for 8 h, then change to fresh medium. After 18 h of infection, use 2 μg / mL of puromycin to screen for stable transfected cell lines. Expand, passage, and store the selected stable transfected cell lines. The gene overexpression or knockdown efficiency was detected and confirmed by fluorescence real-time PCR and western blot.

[0097] 3. qPCR detection of the transcriptional level of the PMF1 gene The steps of extracting total RNA, reverse transcription, and qPCR amplification of cells were all carried out according to the method steps of Example 2 above.

[0098] 4. Statistical analysis All experiments were completed with 3 replicates. The SPSS17.0 statistical software was used for statistical analysis. The difference between the two was analyzed by t-test, and it was considered that when P < 0.05, it was statistically significant.

[0099] 5. Results The results are asFigure 3 As shown, compared with the control group, the expression levels of PMF1 in the cells transfected with shRNA1-1 and shRNA1-3 were significantly down-regulated, indicating that the shRNA1-1 and shRNA1-3 screened in the present invention have good efficiency in inhibiting the expression of PMF1.

[0100] Example 4: Effect of PMF1 gene on the proliferation ability of hepatocellular carcinoma cells In this example, the IncuCyte Zoom instrument was used to detect the effect of the PMF1 gene on the proliferation ability of hepatocellular carcinoma cells, which specifically includes the following content.

[0101] 1. Cell proliferation culture First, the above-mentioned cell lines with knocked-down PMF1 (PLC / PRF / 5, Huh7 or Hep3B) and the control group cells (shNC group cells) were routinely digested into single-cell suspensions, and then the cells were counted and diluted into cell suspensions with appropriate concentrations.

[0102] Then, an appropriate amount of cells were fully mixed with the culture medium in proportion, and the cell suspension was added to a 96-well plate at 200 μL per well. The 96-well plate was placed in the IncuCyte Zoom for culture (37 °C, 5% CO2, relative humidity of 90%) for 4 - 6 days. The IncuCyte Zoom's built-in software was used to analyze the cell growth curve.

[0103] 2. Statistical analysis The experiments were all completed with 3 replicates. The SPSS17.0 statistical software was used for statistical analysis. The t-test was used for the difference between the two groups. It was considered that when P < 0.05, it was statistically significant.

[0104] 3. Results The results are as Figure 4 shown. Compared with the control, after transfection with shRNA1-1 and shRNA1-3, the proliferation of the experimental group cells was significantly inhibited, and the difference was statistically significant (P < 0.05), indicating that inhibiting the expression of the PMF1 gene can reduce cell proliferation.

[0105] Example 5: Effect of PMF1 gene on the colony formation of hepatocellular carcinoma cells In this example, the effect of the PMF1 gene on the colony formation of hepatocellular carcinoma cells was detected, which specifically includes the following content.

[0106] 1. Cell culture and staining After digesting the above cells with stable knockdown of PMF1 (PLC / PRF / 5, Huh7 or Hep3B) and control group cells (shNC group cells) into single cell suspensions respectively, count the cells and dilute them into cell suspensions with appropriate concentrations. Then seed the cells in 6-well plates at a density of 500 cells / 2 mL of complete medium per well and culture them in an incubator at 37 °C, 5% CO2 and a relative humidity of 90%, and change the medium every 3 - 4 days.

[0107] After culturing for 10 days until cell clones are visible to the naked eye, discard the medium, add 1 mL of 4% paraformaldehyde to fix for 10 minutes. Then discard the paraformaldehyde and add 1 mL of 0.1% crystal violet staining solution to stain at room temperature for 20 minutes. Finally, pour out the crystal violet and gently wash with PBS until the background is clear. After drying, take pictures and count.

[0108] 2. Statistical analysis All experiments were completed with 3 replicates. The SPSS 17.0 statistical software was used for statistical analysis. The t-test was used for the difference between the two groups, and it was considered that there was statistical significance when P < 0.05.

[0109] 3. Results The results are as Figure 5 shown. Compared with the control, after transfection with shRNA1 - 1 and shRNA1 - 3, the colony formation of cells in the experimental group was significantly inhibited, and the difference was statistically significant (P < 0.05), indicating that PMF1 has the effect of promoting cell colony formation.

[0110] Example 6: Effect of PMF1 gene on the migration and invasion abilities of hepatocellular carcinoma cells This example detected the effect of PMF1 gene on the migration and invasion abilities of hepatocellular carcinoma cells, which specifically includes the following content.

[0111] 1. Cell culture Take out the Transwell chamber in the biosafety cabinet, place it in the supporting 24-well plate, add 200 μL of serum-free medium to each well of the Transwell chamber, and place the Transwell chamber in a 37 °C, 5% CO2 incubator for 30 min.

[0112] After digesting the above cells with stable knockdown of PMF1 (PLC / PRF / 5, Huh7 or Hep3B) and control group cells (shNC group cells) into single cell suspensions respectively, wash the cells twice with PBS, count the cells, and dilute them into cell suspensions with appropriate concentrations.

[0113] 2. Cell migration experiment After adding 680 μL of medium containing 10% FBS into a 24-well plate, place a Transwell chamber into it, avoiding air bubbles. Vertically add 200 μL of cell suspension into each well of the Transwell chamber, and incubate it in an incubator at 37°C and 5% CO2 for 24 - 48 h. Take out the chamber, discard the medium and cells in the upper chamber, carefully wipe off the remaining medium and cells in the upper chamber with a cotton swab dipped in PBS, and then invert it for 1 - 2 min to dry. Then place the chamber in 4% paraformaldehyde and fix it at room temperature for 30 min. After fixation, take out the chamber, invert it for 1 - 2 min to dry. Then place the chamber in 0.1% crystal violet staining solution and stain it at room temperature for 30 min. After staining, wipe off the crystal violet staining solution in the upper chamber with a cotton swab dipped in PBS. The lower chamber can be gently rinsed in PBS. After washing, take out the chamber, invert it for 1 - 2 min to dry.

[0114] Photographing and counting: Take photographs under a 20× objective lens, and use ImageJ software to count the cells that have passed through the Transwell basement membrane.

[0115] 3. Transwell cell invasion assay The specific steps of the Transwell cell invasion assay are as follows: Pretreat the chamber with Matrigel in advance, and the subsequent steps are the same as those of the Transwell cell migration assay. The chamber collection time can be appropriately extended.

[0116] 4. Statistical analysis The experiments were all completed with 3 replicates. The SPSS 17.0 statistical software was used for statistical analysis. The t-test was used for the difference between the two groups. It was considered that when P < 0.05, it was statistically significant.

[0117] 5. Results The results are as Figure 6 shown. Compared with the control group, after transfection with shRNA1 - 1 and shRNA1 - 3 in the experimental group, the migration and invasion abilities of the cells were significantly inhibited, and the difference was statistically significant (P < 0.05), indicating that PMF1 has the effect of promoting cell migration and invasion abilities.

[0118] Example 7: Effect of PMF1 gene on subcutaneous xenograft tumors of hepatocellular carcinoma cells in nude mice In this example, the effect of the PMF1 gene on subcutaneous xenograft tumors of hepatocellular carcinoma cells in nude mice was detected. The specific method is as follows: 1. Weigh 4-week-old male nude mice (balb / c-nu, purchased from Guangdong Zhiyuan Biomedical Technology Co., Ltd.), mark them with ear tags, and randomly divide them into groups with 6 mice in each group.

[0119] 2. Trypsinize and centrifuge Huh7 cells with a growth confluence of 80 - 90%, resuspend the cells in serum-free medium, count the cells, adjust the cell concentration to 500,000 cells / 100 µL, pipette evenly, and place on ice.

[0120] 3. Take the skin in the mid-posterior part of the left axilla of the mouse as the injection site. After alcohol disinfection, aspirate 100 µL of the cell suspension, spread the skin and insert the needle subcutaneously from the injection point, slowly inoculate the cells subcutaneously into the mouse, inject 100 µL of cells into each mouse, quickly withdraw the needle and press the skin puncture site with a cotton swab for 5 seconds to avoid leakage.

[0121] 4. Start observing the tumor formation and the change in tumor size every three days 3 days after inoculation.

[0122] 5. When the tumor volume of the experimental group or the control group grows to 1.5 - 2.0 cm 3 , euthanize the mouse, remove the tumor, weigh it and take a photo.

[0123] The results are as Figure 7 shown, indicating that compared with the control, after transfection with shRNA, the subcutaneous xenograft tumors of hepatocellular carcinoma cells in the experimental group were significantly inhibited, suggesting that PMF1 has the effect of promoting tumor formation in vivo of hepatocellular carcinoma cells.

[0124] In summary, the present invention provides an application of PMF1 as a target in the preparation of products for diagnosing, preventing, and / or treating hepatocellular carcinoma. The present invention analyzes the expression of PMF1 in tumor tissues and normal liver tissues of hepatocellular carcinoma specimens using data analysis of online databases, and for the first time discovers that the expression of PMF1 is up-regulated in hepatocellular carcinoma. The overall survival rate of patients with high expression of PMF1 in hepatocellular carcinoma is low, and PMF1 is an independent factor for poor prognosis of hepatocellular carcinoma. Further, the present invention proves through experiments that the expression of PMF1 in hepatocellular carcinoma cell lines is also significantly higher than that of normal hepatocytes. And it is found that by reducing the expression level of PMF1, the proliferation, colony formation, migration, and invasion of hepatocellular carcinoma cells can be effectively inhibited, suggesting that detecting the expression level of the PMF1 gene can become one of the auxiliary diagnostic indicators for the diagnosis of hepatocellular carcinoma, and interfering with the expression of the PMF1 gene can become a new way to prevent or treat hepatocellular carcinoma.

[0125] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the purpose of the present invention. In addition, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

Claims

1. Use of a PMF1 inhibitor in the preparation of a product for preventing and / or treating hepatocellular carcinoma.

2. The application according to claim 1, characterized in that, The PMF1 inhibitor includes a reagent that inhibits PMF1 gene expression and / or reduces PMF1 protein activity.

3. The application according to claim 1, characterized in that, The PMF1 inhibitor includes at least one of interfering RNA targeting the PMF1 gene, antisense oligonucleotide targeting the PMF1 gene, CRISPR targeting the PMF1 gene, TALEN targeting the PMF1 gene, and zinc finger nuclease targeting the PMF1 gene.

4. The application according to claim 3, wherein The interfering RNA targeting PMF1 includes dsRNA, siRNA, and shRNA targeting the PMF1 gene; Preferably, the nucleotide sequence of the shRNA is shown in any one of SEQ ID NO.5 to SEQ ID NO.

8.

5. The application according to any one of claims 1 to 4, characterized in that, The uses of the product for preventing and / or treating hepatocellular carcinoma include any one of the following: A) Inhibiting the proliferation of hepatocellular carcinoma cells; B) Inhibiting the cloning of hepatocellular carcinoma cells; C) Inhibiting the migration and / or invasion of hepatocellular carcinoma cells.

6. A pharmaceutical composition for preventing and / or treating hepatocellular carcinoma, characterized in that, The active substance of the pharmaceutical composition includes a PMF1 inhibitor or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition according to claim 6, wherein, The pharmaceutical composition further includes pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include at least one of diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, solubilizers, preservatives, pH regulators, osmotic pressure regulators, surfactants, coating materials, antioxidants, or buffers.

8. Use of the PMF1 gene as a marker in the preparation of a candidate compound for screening for preventing and / or treating hepatocellular carcinoma.

9. Use of a reagent for detecting the expression level of the PMF1 gene in the preparation of a product for auxiliary diagnosis of hepatocellular carcinoma and a product for prognosis assessment of hepatocellular carcinoma.

10. The application according to claim 9, characterized in that, The reagent for detecting the expression level of the PMF1 gene includes specific primers for detecting the expression level of the PMF1 gene or an antibody against the PMF1 protein; Preferably, the specific primers for detecting the expression level of the PMF1 gene include primer sequences shown in SEQ ID NO.1 and / or SEQ ID NO.2.

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