CN112824540A

CN112824540A - SNX5 as biological marker for liver cancer prognosis and application thereof

Info

Publication number: CN112824540A
Application number: CN201911151294.7A
Authority: CN
Inventors: 田华; 周青青; 李锦军; 李红; 葛超
Original assignee: Shanghai Cancer Institute
Current assignee: Shanghai Cancer Institute
Priority date: 2019-11-21
Filing date: 2019-11-21
Publication date: 2021-05-21

Abstract

The invention relates to application of SNX5 in diagnosis of liver cancer prognosis and liver cancer treatment. Specifically, the invention provides an SNX5 gene, mRNA, cDNA or protein or a detection reagent thereof, and application of the SNX5 gene, the mRNA, the cDNA or the protein or the detection reagent thereof in diagnosis of liver cancer prognosis and liver cancer treatment, the SNX5 gene or the protein inhibitor thereof can effectively inhibit proliferation, migration and invasion of liver cancer cells, and the SNX5 gene or the protein inhibitor thereof can be optionally combined with chemotherapeutic drugs, and has a remarkable synergistic effect on the treatment of liver cancer. The research of the invention proves that the SNX5 gene and the expression product thereof can be used as a specific marker gene for diagnosing or predicting liver cancer metastasis, and are beneficial to diagnosing and predicting liver cancer metastasis more accurately and earlier.

Description

SNX5 as biological marker for liver cancer prognosis and application thereof

Technical Field

The present invention relates to the field of diagnostics. More specifically, the invention relates to a biological marker for diagnosing liver cancer prognosis and application thereof.

Background

Primary liver cancer is a malignant tumor seriously harming human health, and according to the 2018 global cancer statistical report, the primary liver cancer becomes the sixth most common cancer in the world and is the fourth leading cause of cancer death in the world, and about 841000 new cases and 782000 deaths occur each year. Primary liver cancers include hepatocellular carcinoma and intrahepatic bile duct cancer, among other rare types, with hepatocellular carcinoma being the most common (about 75-85%).

Hepatocellular carcinoma (hereinafter, liver cancer) is one of common malignant tumors in China, has high malignancy degree and is closely related to HBV infection. Because of the difficulty in early diagnosis of liver cancer, most patients are found at a middle or advanced stage, and most patients still have an inevitable death from tumor recurrence and metastasis despite active surgical treatment and other comprehensive treatment. The occurrence and development of liver cancer are extremely complex and multifactorial processes, but the molecular mechanism for the occurrence and development of liver cancer is not clear at present. Therefore, the method further explores and researches the relationship between new gene function change and the occurrence and development of the liver cancer and the malignant characteristics of the liver cancer, and has important significance for disclosing the precise molecular mechanism of the occurrence and development, designing a reasonable treatment scheme, judging prognosis and further improving the treatment level of the liver cancer.

Disclosure of Invention

The invention aims to provide a novel target with diagnosis or prognosis prediction and treatment effects and a novel method for increasing chemotherapeutic drugs.

In a first aspect of the invention, there is provided a use of the SNX5 gene, mRNA, cDNA, or protein or a detection reagent thereof,

(i) as a marker for detecting tissue grading of liver cancer; and/or

(ii) Used as a marker for detecting the risk of liver cancer metastasis; and/or

(iii) Used as a marker for judging the prognosis and survival period of a liver cancer patient; and/or

(iv) A diagnostic reagent or a kit for preparing tissue grading for detecting liver cancer; and/or

(v) The kit is used for preparing a diagnostic reagent or a kit for detecting the risk of liver cancer metastasis; and/or

(vi) Used for preparing a diagnostic reagent or a kit for judging the prognosis and the survival period of a liver cancer patient.

In another preferred embodiment, the diagnostic reagent comprises an antibody, a primer, a probe, a sequencing library, a nucleic acid chip (e.g., a DNA chip), or a protein chip.

In another preferred embodiment, the protein comprises a full-length protein or a protein fragment.

In another preferred embodiment, the SNX5 gene, mRNA, cDNA, or protein is derived from a mammal, preferably from a rodent (e.g., mouse, rat), primate, and human, more preferably from a patient diagnosed with liver cancer.

In another preferred example, the SNX5 gene, mRNA, cDNA, or protein is derived from a liver cancer patient.

In another preferred example, the accession number of the SNX5 Gene is Gene ID: 27131.

In another preferred example, the accession number of the SNX5 mRNA is NM _ 014426.4.

In another preferred example, the accession number of the SNX5 protein is NP _ 055241.1.

In another preferred embodiment, the test is a tissue sample test.

In another preferred embodiment, the detection comprises immunohistochemistry, immunoblotting and fluorescent quantitative PCR detection.

In another preferred embodiment, the metastasis includes intrahepatic metastasis and pulmonary metastasis.

In another preferred embodiment, the detection is the determination of liver cancer tissue, or liver tissue samples in general.

In another preferred embodiment, the general liver tissue includes a tissue adjacent to a cancer.

In another preferred example, the detection reagent comprises an antibody specific for SNX5, a specific binding molecule specific for SNX5, a specific amplification primer, a probe or a chip.

In another preferred embodiment, the SNX5 protein or its specific antibody or specific binding molecule is coupled with or carries a detectable label.

In another preferred embodiment, the detectable label is selected from the group consisting of: a chromophore, a chemiluminescent group, a fluorophore, an isotope, or an enzyme.

In another preferred embodiment, the antibody specific for SNX5 is a monoclonal antibody or a polyclonal antibody.

In another preferred embodiment, the tissue classification of liver cancer comprises stage I, stage II, stage III, and stage IV.

In another preferred embodiment, the transfer risk refers to n years of transfer risk, wherein n is any number (including decimal) from 1 to 5.

In a second aspect of the invention, there is provided a method for (i) detecting a tissue grade of liver cancer; and/or (ii) detecting the risk of metastasis of liver cancer; and/or (iii) a diagnostic kit for determining prognosis and survival of a patient with liver cancer, said kit comprising a container, said container comprising a detection reagent for detecting SNX5 gene, mRNA, cDNA, or protein; and a label or instructions for use of the kit for detecting (i) a tissue grade for detecting liver cancer; and/or (ii) detecting the risk of metastasis of liver cancer; and/or (iii) determining prognosis and survival of a liver cancer patient.

In another preferred embodiment, the detection of the risk of liver cancer metastasis refers to the detection of whether liver cancer has occurred, the metastasis site, and/or the determination of the probability (susceptibility) of liver cancer metastasis.

In another preferred example, the judgment includes a preliminary judgment (prediction).

In another preferred embodiment, the detection reagent for detecting SNX5 gene, mRNA, cDNA or protein comprises:

(a) an antibody specific for an anti-SNX 5 protein; and/or

(b) A specific primer that specifically amplifies mRNA or cDNA of SNX 5.

In another preferred embodiment, the test is a tissue sample test.

In another preferred embodiment, the label or instructions may indicate the following:

(a) when the ratio of the SNX5 expression level E1 in the liver cancer cells or tissues of the detected object to the SNX5 expression level E2 in the common liver cells or tissues is more than or equal to 1.5, the probability that the liver cancer of the detected object has the intrahepatic metastasis risk is higher than that of the common people;

(b) when the ratio of the SNX5 expression level E1 in the liver cancer cells or tissues of the detected object to the SNX5 expression level E2 in the common liver cells or tissues is more than or equal to 1.5, the probability of poor prognosis and shortened survival time of the detected object is higher than that of the common population;

wherein E2 is the expression level of SNX5 of common liver cells or tissues of the general population.

In another preferred embodiment, the general liver cells or tissues include paracancerous liver cells or tissues.

In a third aspect of the invention, there is provided a method of (i) detecting the risk of metastasis of liver cancer; and/or (ii) a method of determining prognosis and survival of a liver cancer patient, the method comprising:

a) providing a test sample from a subject;

b) detecting the expression level of the SNX5 protein in the test sample E1; and

c) comparing the expression level of the SNX5 protein determined in step b) with a control,

wherein an amount of expression of the SNX5 protein in the sample as compared to the control is above a reference value, indicating that the subject has a higher risk of developing metastasis than the general population (control population); and/or the expression level of the SNX5 protein is lower than the reference value, indicating that the probability of the subject developing intrahepatic metastasis is lower than that of the general population (the control group population); and/or

The expression level of the SNX5 protein in the sample is higher than the reference value compared to the control, indicating that the subject has a higher probability of poor prognosis and shortened survival than the general population (control population); and/or the expression level of the SNX5 protein is lower than the reference value, which indicates that the probability of poor prognosis and shortened survival of the subject is lower than that of the general population (the control group population).

In another preferred embodiment, the subject is a human or non-human mammal.

In another preferred embodiment, the test sample is a liver cancer cell or tissue.

In another preferred example, the reference value is a cut-off value (cut-off value).

In another preferred embodiment, the reference value is the relative expression level of SNX5 in the sample.

In another preferred embodiment, the reference value is 1.5.

In another preferred example, the detecting step (ii) comprises detecting the amount of SNX5 mRNA, or the amount of SNX5 cDNA; and/or detecting the amount of SNX5 protein.

In another preferred example, the expression level of SNX5 protein in the sample is detected by fluorescent quantitative PCR or immunohistochemistry.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In a fourth aspect of the invention, there is provided a method of determining a treatment plan, comprising:

a) providing a test sample from a subject;

b) detecting the expression level of SNX5 protein in the test sample; and

c) determining a treatment regimen based on the expression level of SNX5 protein in the sample.

In another preferred embodiment, the subject is a human or non-human mammal.

In another preferred example, when the expression level of the SNX5 protein in the sample is higher than the reference value, the probability that the subject has the risk of liver cancer metastasis is higher than that of the general population (the control population); or the grade of the malignancy degree of the liver cancer suffered by the subject is higher than that of the general population (the control group population); or a subject with a better chance of having a poor prognosis and reduced survival than the general population (control population), the treatment regimen comprising a combination of an SNX5 inhibitor therapy, an SNX5 inhibitor therapy with a chemotherapeutic agent (chemotherapeutic drug), such as sorafenib (sorafenib), erlotinib (Erlotinb).

In another preferred embodiment, the SNX5 inhibitor therapy, the SNX5 inhibitor therapy in combination with a chemotherapeutic agent (e.g., sorafenib, erlotinib) is selected from the group consisting of:

SNX5 inhibitor therapy: an antibody, a small molecule compound, microRNA, siRNA, shRNA, or a combination thereof;

chemotherapeutic agent therapy: a small molecule compound selected from the group consisting of: sorafenib (sorafenib), erlotinib (Erlotinb), regorafenib (regorafenib), lenvatinib (lenvatinib), or combinations thereof.

In another preferred embodiment, when the risk of liver cancer metastasis in the subject is higher than that in the general population (control population); or the grade of the malignancy degree of the liver cancer suffered by the subject is higher than that of the general population (the control group population); or a subject with a greater probability of poor prognosis and reduced survival than the general population (control population), the treatment regimen further comprises a combination of an SNX5 inhibitor therapy, an SNX5 inhibitor therapy with a chemotherapeutic agent (e.g., sorafenib, erlotinib); and other medicines for treating liver cancer.

In another preferred embodiment, the other agent for treating liver cancer is selected from the group consisting of: 5-fluorouracil, doxorubicin, oxaliplatin, or combinations thereof.

In a fifth aspect of the invention, there is provided the use of a SNX5 gene or a protein inhibitor thereof, in the preparation of a composition or formulation for (a) inhibiting the growth or proliferation of liver cancer cells; and/or (b) inhibiting metastasis of hepatoma cells; and/or (c) preventing and/or treating liver cancer; and/or (d) increasing the sensitivity of the hepatoma cells to chemotherapeutic agents.

In another preferred embodiment, the chemotherapeutic agent is selected from the group consisting of: sorafenib (sorafenib), erlotinib (Erlotinb), or a combination thereof.

In another preferred embodiment, the SNX5 gene or protein inhibitor thereof is selected from the group consisting of: antibodies, small molecule compounds, microRNAs, siRNAs, shRNAs, or combinations thereof.

In another preferred embodiment, the inhibitor comprises an inhibitor that inhibits expression of the SNX5 gene or its protein.

In another preferred example, the inhibition of metastasis of hepatoma cells includes intrahepatic metastasis of hepatoma cells and pulmonary metastasis.

In another preferred embodiment, the composition comprises a pharmaceutical composition.

In another preferred embodiment, the composition comprises a therapeutically effective amount of an inhibitor of the SNX5 gene or protein thereof, and a pharmaceutically acceptable carrier.

In another preferred embodiment, the medicament is administered by a mode of administration selected from the group consisting of: oral, intravenous, intramuscular, subcutaneous, sublingual, rectal, nasal spray, oral spray, topical or systemic transdermal administration of the skin.

In another preferred embodiment, the formulation is selected from the group consisting of: tablet, capsule, injection, granule, and spray.

In a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a1) an inhibitor of the SNX5 gene or protein thereof;

(a2) optionally a chemotherapeutic agent; and

(b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition further comprises:

(c) other medicines for preventing and/or treating liver cancer.

In another preferred embodiment, the chemotherapeutic agent is selected from the group consisting of: sorafenib (sorafenib), erlotinib (Erlotinb), regorafenib (regorafenib), lenvatinib (lenvatinib), or combinations thereof.

In another preferred embodiment, the weight ratio of component (a1) to component (a2) is 100:1 to 0.01:1, preferably 10:1 to 0.1:1, more preferably 2:1 to 0.5: 1.

In another preferred embodiment, the content of the component (a1) in the pharmaceutical composition is 1% to 99%, preferably 10% to 90%, more preferably 30% to 70%.

In another preferred embodiment, the content of the component (a2) in the pharmaceutical composition is 1% to 99%, preferably 10% to 90%, more preferably 30% to 70%.

In another preferred embodiment, the content of the component (c) in the pharmaceutical composition is 1% to 99%, preferably 10% to 90%, and more preferably 30% to 70%.

In another preferred embodiment, the component (a1) and optional component (a2) and optional component (c) in the pharmaceutical composition constitute 0.01 to 99.99 wt%, preferably 0.1 to 90 wt%, more preferably 1 to 80 wt% of the total weight of the pharmaceutical composition.

In another preferred embodiment, the dosage form of the pharmaceutical composition comprises an injection dosage form and an oral dosage form.

In another preferred embodiment, the oral dosage form comprises tablets, capsules, films, and granules.

In another preferred embodiment, the dosage form of the pharmaceutical composition comprises a sustained release dosage form and a non-sustained release dosage form.

In a seventh aspect of the invention, there is provided a kit comprising:

(a1) a first container, and an inhibitor of SNX5 gene or a protein thereof, or a drug containing an inhibitor of SNX5 gene or a protein thereof, located in the first container;

(b1) optionally a second container, and a chemotherapeutic agent, or a drug containing a chemotherapeutic agent, located in said second container.

In another preferred embodiment, the kit further comprises:

(c1) a third container and other drugs for preventing and/or treating liver cancer in the third container or other drugs for preventing and/or treating liver cancer.

In another preferred embodiment, the kit further comprises (d1) a fourth container, and a detection reagent for SNX5 located in the fourth container.

In another preferred embodiment, the first container and the second, third and fourth containers are the same or different containers.

In another preferred embodiment, the drug in the first container is a single formulation containing an inhibitor of SNX5 gene or its protein.

In another preferred embodiment, the drug in the second container is a single formulation containing a chemotherapeutic agent.

In another preferred embodiment, the drug in the third container is a single preparation containing other drugs for preventing and/or treating liver cancer.

In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

In another preferred embodiment, the kit further comprises instructions.

In another preferred embodiment, the description recites one or more descriptions selected from the group consisting of:

(a) an inhibitor of the SNX5 gene or its protein is used for (i) inhibiting the growth and proliferation of liver cancer cells; and/or (ii) inhibiting metastasis of hepatoma cells; and/or (iii) preventing and/or treating liver cancer; and/or (iv) a method of increasing the sensitivity of a hepatoma cell to a chemotherapeutic agent;

(b) the inhibitor of the SNX5 gene or its protein is used in combination with a chemotherapeutic agent, and/or optionally other agents for preventing and/or treating liver cancer to (i) inhibit growth, proliferation of liver cancer cells; and/or (ii) inhibiting metastasis of hepatoma cells; and/or (iii) preventing and/or treating liver cancer;

(c) detecting the expression level of SNX5 protein of liver cancer patients, and simultaneously administering an inhibitor of SNX5 gene or protein thereof to (i) inhibit the growth and proliferation of liver cancer cells; and/or (ii) a method of inhibiting metastasis of hepatoma cells; and/or (iii) preventing and/or treating liver cancer; and/or (iv) a method of increasing the sensitivity of a hepatoma cell to a chemotherapeutic agent;

(d) detecting the expression level of SNX5 protein of a liver cancer patient, and combining with an inhibitor of RITI1 gene or protein thereof; and a chemotherapeutic agent, and/or optionally other liver cancer preventing and/or treating agent to (i) inhibit growth, proliferation of liver cancer cells; and/or (ii) a method of inhibiting metastasis of hepatoma cells; and/or (iii) a method for preventing and/or treating liver cancer.

In an eighth aspect of the invention, there is provided a pharmaceutical composition according to the sixth aspect of the invention or a use of a kit according to the seventh aspect of the invention for (a) inhibiting growth, proliferation, or proliferation of a liver cancer cell; and/or (b) inhibiting metastasis of hepatoma cells; and/or (c) preventing and/or treating liver cancer.

In another preferred embodiment, the concentration of the inhibitor of the SNX5 gene or the protein thereof in the pharmaceutical composition is 100-2000ng/ml, preferably 500-1500ng/ml, more preferably 800-1000 ng/ml.

In another preferred embodiment, the concentration of the chemotherapeutic agent in the pharmaceutical composition is 1000-.

In another preferred embodiment, the concentration of the other drugs for preventing and/or treating liver cancer in the pharmaceutical composition is 500-4000ng/ml, preferably 1500-3500ng/ml, more preferably 2000-3000 ng/ml.

In another preferred embodiment, the pharmaceutical composition or kit comprises (a) an inhibitor of the SNX5 gene or a protein thereof; and (b) optionally a chemotherapeutic agent; and (c) optionally other agents for the prevention and/or treatment of liver cancer; and (d) a pharmaceutically acceptable carrier.

In another preferred embodiment, in the pharmaceutical composition or kit, the inhibitor of SNX5 gene or protein thereof; and (b) optionally a chemotherapeutic agent; and (c) optionally other agents for preventing and/or treating liver cancer, in an amount of 0.01-99.99 wt%, preferably 0.1-90 wt%, more preferably 1-80 wt% based on the total weight of the pharmaceutical composition or kit.

In a ninth aspect of the invention, there is provided an in vitro non-therapeutic method of inhibiting growth or proliferation of hepatoma cells, comprising the steps of: culturing the liver cancer cell in the presence of SNX5 gene or its protein inhibitor, thereby inhibiting the growth or proliferation of the liver cancer cell.

In another preferred example, the liver cancer cell highly expresses SNX5 protein.

In another preferred example, the method further comprises adding a chemotherapeutic agent to the culture system of the hepatoma cells; and/or other drugs for preventing and/or treating liver cancer, thereby inhibiting growth or proliferation of liver cancer cells.

In another preferred example, the liver cancer cells are cells cultured in vitro.

In a tenth aspect of the present invention, there is provided a method of screening a candidate compound for preventing and/or treating liver cancer, the method comprising the steps of:

(a) in the test group, adding a test compound to a culture system of cells, and observing the expression amount (E1) and/or activity (A1) of SNX5 in the cells of the test group; in the control group, the test compound was not added to the culture system of the same cells, and the expression amount (E0) and/or activity (a0) of SNX5 in the cells of the control group were observed;

wherein, if the expression level (E1) and/or activity (A1) of SNX5 of the cells in the test group is significantly lower than that of the control group, it indicates that the test compound is a candidate compound for preventing and/or treating liver cancer having an inhibitory effect on the expression level and/or activity of SNX 5.

In another preferred example, the expression level of SNX5 is obtained by fluorescent quantitative PCR or immunohistochemical detection.

In another preferred example, the method further comprises the steps of:

(b) further testing the candidate compound obtained in step (a) for inhibition of growth or proliferation of hepatoma cells; and/or further tested for its effect on the down-regulation of the SNX5 gene.

In another preferred example, step (b) includes the steps of: in the test group, a test compound is added into a culture system of the liver cancer cells, and the number and/or growth condition of the liver cancer cells are observed; in the control group, no test compound is added into the culture system of the liver cancer cells, and the number and/or growth condition of the liver cancer cells are observed; wherein, if the number or growth rate of the liver cancer cells in the test group is less than that of the control group, the test compound is a candidate compound for preventing and/or treating liver cancer, which has an inhibitory effect on the growth or proliferation of the liver cancer cells.

In another preferred embodiment, the method comprises the step (c): administering the candidate compound identified in step (a) to a mammalian model and determining its effect on the mammal.

In another preferred example, the mammal is a mammal having liver cancer.

In another preferred embodiment, the phrase "substantially less than" means E1/E0 ≦ 1/2, preferably ≦ 1/3, more preferably ≦ 1/4.

In another preferred embodiment, the phrase "substantially less than" means A1/A0 ≦ 1/2, preferably ≦ 1/3, more preferably ≦ 1/4.

In another preferred embodiment, the cells comprise hepatoma cells.

In another preferred embodiment, the cells are cultured in vitro.

In an eleventh aspect of the present invention, there is provided a method for preventing and/or treating liver cancer, comprising: administering to a subject in need thereof an inhibitor of the SNX5 gene or a protein thereof; or a pharmaceutical composition according to the sixth aspect of the invention or a kit according to the seventh aspect of the invention.

In another preferred embodiment, the subject comprises a human or non-human mammal having liver cancer.

In another preferred embodiment, the non-human mammal includes rodents and primates, preferably mice, rats, rabbits, monkeys.

In another preferred embodiment, the inhibitor of the SNX5 gene or its protein is administered in a dose of 0.5-5mg/kg body weight, preferably 1-4mg/kg body weight, most preferably 2-3mg/kg body weight.

In another preferred embodiment, the chemotherapeutic agent is administered in a dose of 10-60mg/kg body weight, preferably 20-50mg/kg body weight, most preferably 30-40mg/kg body weight.

In another preferred embodiment, the other agent for preventing and/or treating liver cancer is administered in a dose of 5-70mg/kg body weight, preferably 10-50mg/kg body weight, and most preferably 20-40mg/kg body weight.

In another preferred embodiment, the inhibitor of the SNX5 gene or its protein is administered at a frequency of 1-4 times per week, preferably 2-3 times per week.

In another preferred embodiment, the chemotherapeutic agent is administered at a frequency of 1-5 times per week, preferably 2-3 times per week. In another preferred embodiment, the other drugs for preventing and/or treating liver cancer are administered at a frequency of 1-6 times/day, preferably 3-4 times/week.

In another preferred embodiment, the inhibitor of the SNX5 gene or its protein is administered for 20 to 90 days, preferably 20 to 60 days, and most preferably 30 to 40 days.

In another preferred embodiment, the chemotherapeutic agent is administered for a period of 20 to 90 days, preferably 20 to 60 days, and most preferably 30 to 40 days.

In another preferred embodiment, the other drugs for preventing and/or treating liver cancer are administered for 20 to 90 days, preferably 20 to 60 days, and most preferably 30 to 40 days.

In another preferred embodiment, the inhibitor of the SNX5 gene or protein thereof is administered simultaneously or sequentially with an optional chemotherapeutic agent, and optionally other agents for preventing and/or treating liver cancer.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows that SNX5 is expressed in liver cancer tissues as increased:

(a) analyzing the expression of SNX5 in the TCGA database and GEO database GES36376 and GES56140 liver cancer tissues by heat map, wherein N represents the tissues beside the liver cancer, and T represents the liver cancer tissues;

(b) expression levels of SNX5 mRNA in liver cancer tissues and tissues beside the liver cancer in a TCGA database, wherein N represents the tissues beside the liver cancer, and T represents the tissues beside the liver cancer;

(c) expression level of SNX5 mRNA in GSE36376, wherein N represents a tissue beside liver cancer, and T represents a liver cancer tissue;

(d) the expression level of SNX5 mRNA in GSE 56140;

(e) analyzing the expression level of the SNX5 protein in the liver cancer tissue by a western blot method, wherein the result shows that the expression of SNX5 in the liver cancer tissue is higher than that of the corresponding adjacent tissue, N represents the adjacent tissue of the liver cancer, and T represents the liver cancer tissue;

wherein the beta-actin is beta-actin.

Figure 2 shows that expression of SNX5 in liver cancer tissue affects the prognosis of liver cancer patients:

(a) analyzing the expression of the SNX5 protein in liver cancer tissues by an immunohistochemical method, and selecting 2 representative liver cancer tissues (SNX5 is low in expression and high in expression);

(b) according to the expression level of SNX5 in 143 cases of liver cancer tissues, the liver cancer tissues are correspondingly divided into an SNX5 low expression group and an SNX5 high expression group, the Kaplan-Meier method is adopted to analyze the relation between the expression of SNX5 and the prognosis of a liver cancer patient, and the result shows that the prognosis of the high expression SNX5 patient is poor (p is 0.015);

(c) the TCGA database is analyzed, the expression level of SNX5 is correspondingly divided into a high expression group and a low expression group, the Kaplan-Meier method is adopted to analyze the relation between the expression of SNX5 and the prognosis of the liver cancer patient, and the result shows that the prognosis of the high expression SNX5 patient is poor (p is 0.002).

FIG. 3 shows the results of univariate and multivariate regression analysis performed by the Cox proportional Risk regression model:

(a) carrying out univariate Cox proportional risk regression model analysis to show that the vascular invasion and the expression of SNX5 are risk factors influencing the prognosis of the liver cancer patient;

(b) multivariate Cox proportional hazards regression model analysis showed that vascular invasion and SNX5 expression are risk factors affecting the prognosis of liver cancer patients.

Fig. 4 shows the results of SNX5 promoting growth and proliferation of liver cancer cells in vitro, where vector is empty vector, Mock is blank control, β -actin is β -actin, MHCC-LM3 and Huh7 are liver cancer cell lines, shNC is shRNA interference negative control, shSNX5-1 is shRNA interference SNX5 segment 1, shSNX5-2 is shRNA interference SNX5 segment 2:

(a) SMMC-7721 and Li7 cells overexpress SNX5 through lentivirus infection, and the overexpression level of SNX5 is detected, so that the result shows that the overexpression of SNX5 is successful;

(b) the CCK8 experiment result shows that over-expression of SNX5 promotes the proliferation of SMMC-7721 and Li7 cells;

(c) the results of a clone formation experiment show that over-expressing SNX5 promotes the clone formation capability of SMMC-7721 and Li7 cells;

(d) the expression of SNX5 is interfered by slow virus infected Huh7 and MHCC-LM3 cells, the knock-down level of SNX5 is detected, and the result shows that the expression of SNX5 is obviously reduced;

(e) the CCK8 experiment result shows that the interference of the expression of SNX5 inhibits the proliferation of Huh7 and MHCC-LM3 cells;

(f) the result of a clonogenic experiment shows that the interference of the expression of SNX5 inhibits the clonogenic capacity of Huh7 and MHCC-LM3 cells;

fig. 5 shows the results of SNX5 promoting growth and proliferation of liver cancer cells in vivo, where vector is empty vector, MHCC-LM3 and Huh7 are liver cancer cell lines, shNC is shRNA interference negative control, shSNX5 is shRNA interference SNX5 fragment 1:

(a) MMC-7721 and Li7 cells over expressing SNX5 are injected in situ into a nude mouse liver, after 6-8 weeks, the animal is killed, a tumor forming liver tissue of the mouse is taken and weighed, and the result shows that the over expressing SNX5 promotes the in situ tumor forming capability of the SMMC-7721 and Li7 cells, and the right graph is the statistical result of the liver weight;

(b) taking the Li7 cell to overexpress SNX5 nude mouse liver in-situ tumorigenic tissues, detecting the expression of Ki67 by an immunohistochemical method, and displaying that the overexpression SNX5 promotes the expression of Ki67, wherein the right picture is the statistical result of Ki67 positive rate;

(c) the liver of a naked mouse with MHCC-LM3 cells interfering with SNX5 is injected in situ, the animal is killed after 4 weeks, the tumorigenic liver tissue of the mouse is taken and weighed, the result shows that the expression of the interference SNX5 inhibits the hepatic tumorigenic capacity of the MHCC-LM3 cells in situ, and the right graph is the statistical result of the liver weight.

Fig. 6 shows the results of promoting migration and invasion of liver cancer cells by SNX5, where vector is empty vector, Mock is blank control, shNC is shRNA interference negative control, MHCC-LM3 and Huh7 are liver cancer cell lines, shSNX5-1 is shRNA interference SNX5 segment 1, shSNX5-2 is shRNA interference SNX5 segment 2:

(a) through transwell experiments, SNX5 overexpression promoted SMMC-7721 and Li7 cell migration and invasion, with statistical results on the right;

(b) by transwell experiments, Huh7 and MHCC-LM3 cells interfered with the ability of SNX5 expression to inhibit cell migration and invasion, and the right is the statistical result.

Fig. 7 shows the results of SNX5 promoting the metastasis of hepatoma cells, where vector is empty vector, Mock is blank control, F-actin is F-actin, β -actin is β -actin, DAPI is 4', 6-diamidino-2-phenylindole, merge is merge, MHCC-LM3 and Huh7 are hepatoma cell lines, shNC is shRNA interference negative control, MMP9 is matrix metalloproteinase 9, shSNX5-1 is shRNA interference SNX5 fragment 1, shSNX5-2 is shRNA interference SNX5 fragment 2, snshx 5 is shRNA interference SNX5 fragment 1:

(a) SMMC-7721 and Li7 cells over-expressing SNX5 stain F-actin by phallodin and stain cell nucleus by DAPI, and the result shows that over-expressing SNX5 promotes the formation of invasive pseudopodia of SMMC-7721 and Li7 cells;

(b) through detection of a western blot method, over-expression of SNX5 promotes SMMC-7721 and Li7 cells to promote expression of MMP 9; interfering the expression of SNX5, inhibiting the expression of MMP9 of Huh7 and MHCC-LM3 cells;

(c) the SMMC-7721 cells overexpress SNX5, the liver of a nude mouse is injected in situ, a tumorigenic tissue is taken after 8 weeks, HE staining is carried out to analyze intrahepatic metastasis and pulmonary metastasis, and the result shows that the overexpression SNX5 promotes the intrahepatic metastasis and pulmonary metastasis of the liver cancer cells;

(d) MHCC-LM3 cell interferes with the expression of SNX5, the liver of a nude mouse is injected in situ, a tumorigenic tissue is taken after 4 weeks, HE staining is carried out to analyze the intrahepatic metastasis, and the result shows that the interference SNX5 inhibits the intrahepatic metastasis of the liver cancer cell.

FIG. 8 shows the results of interference with the expression of SNX5 promoting drug sensitivity of Erlotinb (erlotinib) and sorafenib (sorafenib), wherein Huh7 and MHCC-LM3 are hepatoma cell lines:

(a) different doses of Erlotinb are used for treating SNX5 interfered Huh7 and MHCC-LM3 cells, and the clone formation experiment result shows that the drug sensitivity of Erlotinb to liver cancer cells can be obviously improved after SNX5 interference;

(b) different doses of sorafenib are used for treating SNX5 interfered Huh7 and MHCC-LM3 cells, and the clone formation experiment result shows that after SNX5 interference, the drug sensitivity of sorafenib on liver cancer cells can be obviously improved.

Fig. 9 shows the results of interfering with SNX5 expression promoting Erlotinb and sorafenib apoptosis in Huh7 cells:

(a) SNX 5-interfered Huh7 and control cells, sorafenib and Erlotinb treatment, and flow cytometry analysis shows that interference of SNX5 can promote sorafenib and Erlotinb to induce Huh7 cell apoptosis.

Detailed Description

The inventor of the invention has conducted extensive and intensive studies and, for the first time, has surprisingly found that the expression of the gene of SNX5 or the protein thereof in liver cancer tissues is significantly higher than the expression of the gene of SNX5 or the protein thereof in corresponding paracancer tissues and normal tissues, and the applicant has also found that the expression of SNX5 in liver cancer tissues is increased, the overexpression of SNX5 can promote the proliferation, migration and invasion of liver cancer cells, and the expression of gene silencing SNX5 can inhibit the proliferation, migration and invasion of liver cancer cells. And the Kaplan-Meier method analyzes the relation between the expression of SNX5 and the prognosis of the liver cancer patient, which indicates that the prognosis of the high-expression SNX5 patient is poor (p is 0.015), and the SNX5 high-expression patient is closely related to vascular invasion and liver metastasis. Thus, SNX5 can be used as one of the biomarkers for liver cancer prognosis, and SNX5 gene or its protein can be used as (i) to detect the risk of liver cancer developing intrahepatic metastasis and/or lung metastasis; and/or (ii) as a marker for determining prognosis and survival of a patient with liver cancer. In addition, the applicant also unexpectedly discovers that in vivo and in vitro functional experiments show that the SNX5 can promote the proliferation and the metastasis of the liver cancer cells, and inhibitors of the SNX5 gene or the protein thereof (for example, interfering the expression of the SNX 5) can effectively (a) inhibit the metastasis of the liver cancer cells; and/or (b) improving the sensitivity of the hepatoma cells to chemotherapeutic drugs (such as sorafenib and Erlotinb), wherein the inhibitor of the SNX5 gene or the protein thereof can be used together with the chemotherapeutic drugs, and has a remarkable synergistic effect on the treatment of the hepatoma. On this basis, the present inventors have completed the present invention.

Term(s) for

SNX

The snxs (locking nexin) protein family is a family discovered in recent years to regulate the endocytosis process of eukaryotic cells, 33 members exist in mammals, and the protein family is characterized by having a phagocyte oxidase homology (PX domain) structural domain. In addition to the PX domain, the SNXs protein family may have BAR, SH, GAP, FERM, RGS, MIT and some unknown domains that need to be explored further. Because of the different domains contained in the SNXs protein family, the functions of the members within the family are not exactly the same. The SNXs protein mainly depends on a PX structural domain and a BAR structural domain to play biological functions, and mainly comprises transmembrane transport (endocytosis and efflux) of macromolecular substances, protein sorting, degradation, intracellular membrane circulation and the like. At present, researches find that the unlimited proliferation capacity of tumor cells is closely related to the receptor down-regulation defect mediated by endocytosis, and the SNXs protein family members play an important role in the process of mediating the endocytosis of the cells, so that the SNXs protein family is closely related to the occurrence and development of various tumors. SNX1 is used as the first discovered member of SNXs family, and researches show that SNX1 expression is obviously reduced in colon cancer tissues, and the knock-down of expression of SNX1 can obviously increase the phosphorylation level of EGFR to promote the proliferation of colon cancer cells. The gene knockout of SNX10 can promote the occurrence of colon cancer by activating mTOR signaling pathway, and SNX10 can also influence the progression of colon cancer by degrading p21 through molecular Chaperone Mediated Autophagy (CMA). SNX9 promotes metastasis of breast cancer cells by modulating the activity of RhoA and Cdc42 GTPases.

Therefore, all these studies indicate that SNXs protein family members are closely related to tumor proliferation and metastasis, and the main action mechanisms may include interference with receptor endocytosis, deletion of key proteins in endocytosis pathway, escape of ubiquitin-mediated degradation pathway, actin remodeling imbalance during endocytosis, and the like.

Hepatocellular carcinoma

Hepatocellular carcinoma ranks sixth in incidence among all tumors, with nearly 80 million new cases of hepatocellular carcinoma per year. At present, there are many treatment methods for hepatocellular carcinoma, including surgical resection, liver transplantation, tumor ablation, arterial tumor embolization, systemic treatment, etc., but patients with hepatocellular carcinoma in middle and late stages have low benefit from treatment, short survival time and poor prognosis.

There is currently no good treatment for hepatocellular carcinoma.

In the present invention, the classification of hepatocellular carcinoma tissue refers to the classification of TNM, including stage I, stage II, stage III and stage IV.

Intrahepatic metastasis

In the present invention, the intrahepatic metastasis is (1) portal vein tumor thrombus; (2) tumors on liver lobes other than the lobe where the primary tumor is located; (3) a tumor in the same lobe surrounding the primary tumor with multiple satellite nodules and surrounding liver tissue; or a surrounding small isolated tumor that is histologically similar to or less differentiated than the primary tumor.

Metastasis of lung

In the present invention, the lung metastasis refers to the metastasis of primary stem cell cancer to lung tissue via blood or lymph fluid.

Sample (I)

The term "sample" or "specimen" as used herein refers to a material that is specifically associated with a subject from which specific information about the subject can be determined, calculated, or inferred. The sample may be composed in whole or in part of biological material from the subject. The sample may also be a material that has been contacted with the subject in a manner such that the test performed on the sample provides information about the subject. The sample may also be a material that has been contacted with other materials that are not the subject, but that enable the first material to be subsequently tested to determine information about the subject, e.g., the sample may be a probe or scalpel wash. The sample can be a source of biological material other than that contacted with the subject, so long as one of skill in the art is still able to determine information about the subject from the sample.

Expression of

As used herein, the term "expression" includes the production of mRNA from a gene or portion of a gene, and includes the production of protein encoded by an RNA or gene or portion of a gene, as well as the presence of a test substance associated with expression. For example, cDNA, binding of a binding partner (e.g., an antibody) to a gene or other oligonucleotide, protein or protein fragment, and chromogenic moieties of the binding partner are included within the scope of the term "expression". Thus, an increase in the density of half-spots on immunoblots such as western blots is also within the scope of the term "expression" based on biological molecules.

Reference value

As used herein, the term "reference value" refers to a value that is statistically correlated with a particular result when compared to the results of an analysis. In a preferred embodiment, the reference value is determined from a statistical analysis conducted to compare the expression of SNX5 white to studies of known clinical outcome. Some of these studies are shown in the examples section herein. However, studies from the literature and user experience with the methods disclosed herein can also be used to produce or adjust the reference values. The reference value may also be determined by considering conditions and outcomes particularly relevant to the patient's medical history, genetics, age, and other factors.

In the present invention, the reference value refers to a cut-off value (cut-off value), and refers to a relative expression level of SNX5 in a hepatocellular carcinoma cell or tissue, preferably a relative expression level of 0.043 (fluorescent quantitative PCR) or 3.5 (immunohistochemistry).

Sample of non-hepatocellular carcinoma

As used herein, the term "non-hepatocellular carcinoma sample" includes, but is not limited to, a population not suffering from hepatocellular carcinoma, non-hepatocellular carcinoma tissue of a hepatocellular carcinoma patient.

SNX5 proteins and polynucleotides

In the present invention, the terms "protein of the present invention", "SNX 5 protein", "SNX 5 polypeptide" are used interchangeably and all refer to a protein or polypeptide having the amino acid sequence SNX 5. They include SNX5 proteins with or without the initial methionine. In addition, the term also includes full-length SNX5 and fragments thereof. The SNX5 protein referred to in the present invention includes its complete amino acid sequence, its secreted protein, its mutants and functionally active fragments thereof.

SNX5 is a member of the SNXs protein family, located on human chromosome 20p11.23, and encodes a protein with a relative molecular mass of about 50KD, having a PX domain and a BAR domain. The PX domain is a phospholipid binding domain whose main binding target is PI 3P. The protein is phospholipid widely distributed on an endosome membrane, and SNXs is positioned on the surface of the endosome through the combination of a PX structural domain and the phospholipid, so that the various regulation on endosome transportation is exerted. It is presently believed that the function of the BAR structure is related to sensing the tortuosity of the cell membrane, it can bind to the highly curved cell membrane, thereby localizing the whole protein on the organelle, and at the same time, transforming the liposome into a highly curved lipid tube, assisting in transporting the intermediate product, and having the ability to modify the cell membrane. The research finds that SNX5 is positioned in early endosome, SNX5 is recruited to cell membrane after EGF stimulation, and PtdIns (3,4) P2 is also recruited to cell membrane transiently, which indicates that SNX5 not only can influence endocytosis process, but also can regulate phosphatidylinositol signaling pathway.

There are currently fewer reports on SNX5 in tumors. Studies have shown that SNX5 is highly expressed in thyroid papillary carcinomas and that SNX5 knockout mice display a faster proliferative potential and sensitivity to TSH and activate the MAPK and AKT pathways leading to the development of metastatic tumors in the thyroid, indicating that thyroid cells require SNX5 to attenuate TSH-driven tumor signals.

In the present invention, the terms "SNX 5 gene", "SNX 5 polynucleotide" are used interchangeably and all refer to a nucleic acid sequence having the nucleotide sequence SNX 5.

The genome of the human SNX5 Gene has a total length of 27395bp (NCBI GenBank accession number is Gene ID:27131), and the mRNA sequence of the transcription product has a total length of 2341bp (NCBI GenBank accession number is NM-014426.4).

It is understood that nucleotide substitutions in codons are acceptable when encoding the same amino acid. It is also understood that nucleotide changes are also acceptable when conservative amino acid substitutions are made by nucleotide substitutions.

When the amino acid fragment of SNX5 is obtained, a nucleic acid sequence encoding the same can be constructed therefrom, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis. For the PCR amplification method, primers can be designed based on the SNX5 nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and the relevant sequence can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, DNA sequences encoding the proteins of the present invention (or fragments, derivatives thereof) can be obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.

The polynucleotide sequences of the present invention may be used to express or produce recombinant SNX5 polypeptides by conventional recombinant DNA techniques. Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a human SNX5 polypeptide, or with a recombinant expression vector containing the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) isolating and purifying the protein from the culture medium or the cells.

In the present invention, the SNX5 polynucleotide sequence may be inserted into a recombinant expression vector. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing SNX5 encoding DNA sequences and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; an insect cell; animal cells, and the like.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, such as E.coli, competent cells capable of DNA uptake can be harvested after the exponential growth phase and treated by the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Specific antibodies

In the present invention, the terms "antibody of the invention" and "anti-SNX 5-specific antibody" are used interchangeably.

The invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the human SNX5 polypeptide. Herein, "specificity" means that the antibody binds to the human SNX5 gene product or fragment. Preferably, those antibodies that bind to the human SNX5 gene product or fragment, but do not recognize and bind to other unrelated antigenic molecules. Antibodies of the invention include those molecules that bind to and inhibit the human SNX5 protein, as well as those antibodies that do not affect the function of the human SNX5 protein. The invention also includes those antibodies that bind to the human SNX5 gene product in modified or unmodified form.

The present invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab)2 fragments; an antibody heavy chain; an antibody light chain; genetically engineered single chain Fv molecules (Ladner et al, U.S. Pat. No.4,946,778); or chimeric antibodies, such as antibodies that have murine antibody binding specificity but retain portions of the antibody from a human.

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, a purified human SNX5 gene product, or antigenic fragment thereof, can be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing human SNX5 protein or antigenic fragments thereof can be used to immunize animals to produce antibodies. The antibody of the present invention may also be a monoclonal antibody. Such Monoclonal Antibodies can be prepared using hybridoma technology (see Kohler et al, Nature 256; 495, 1975; Kohler et al, Eur.J.Immunol.6: 511,1976; Kohler et al, Eur.J.Immunol.6: 292,1976; Hammerling et al, In Monoclonal Antibodies and T Cell hybrids, Elsevier, N.Y., 1981). The antibody of the invention comprises an antibody capable of blocking the function of the human SNX5 protein and an antibody which does not affect the function of the human SNX5 protein. The antibodies of the invention can be obtained by conventional immunization techniques using fragments or functional regions of the human SNX5 gene product. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to an unmodified form of the human SNX5 gene product can be produced by immunizing an animal with a gene product produced in a prokaryotic cell (e.g., e.coli); antibodies that bind to post-translationally modified forms (e.g., glycosylated or phosphorylated proteins or polypeptides) can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (e.g., a yeast or insect cell).

Antibodies against human SNX5 protein can be used in immunohistochemical techniques to detect human SNX5 protein in a sample, particularly a tissue sample or a serum sample. Because of the presence of the extracellular region of the SNX5 protein, these soluble SNX5 extracellular regions can be targets for serum detection when they shed and enter the blood.

Detection method

The invention also provides a method for detecting liver cancer by utilizing the characteristic that SNX5 exists in liver cancer cells or tissues and is closely related to the risk of liver cancer.

In a preferred embodiment of the invention, the invention provides a high-throughput sequencing-by-generation method for detecting SNX5, Sanger sequencing, quantitative fluorescence PCR (qPCR), in situ immunofluorescence (FISH), immunohistochemistry, and the like.

Detection kit

Based on the correlation between SNX5 and liver cancer, namely that SNX5 exists in liver cancer tissues, SNX5 can be used as a diagnostic marker of liver cancer.

The present invention also provides a method of (i) detecting tissue staging of liver cancer; and/or (ii) detecting the risk of liver cancer developing intrahepatic metastases; and/or (iii) a kit for determining prognosis and survival of a patient with liver cancer, which comprises a detection reagent for detecting SNX5 gene, mRNA, cDNA, or protein; and a label or instructions for use of the kit for detecting (i) a tissue grade for detecting liver cancer; and/or (ii) detecting the risk of liver cancer developing intrahepatic metastases; and/or (iii) determining prognosis and survival of a liver cancer patient.

Wherein the label or instructions recite the following:

Detection method and kit

The present invention relates to diagnostic assays for quantitative and in situ measurement of human SNX5 protein levels or mRNA levels. These assays are well known in the art. Human SNX5 protein levels detected in the assay may be used to diagnose (including aiding diagnosis) tissue grading of liver cancer; and/or risk of metastasis of liver cancer; and/or prognosis and survival of liver cancer patients.

One method for detecting the presence of the SNX5 protein in a sample is to use an antibody specific for the SNX5 protein for detection, which comprises: contacting the sample with an antibody specific for SNX5 protein; observing whether an antibody complex is formed, the formation of an antibody complex indicates the presence of SNX5 protein in the sample.

The SNX5 protein or its polynucleotide can be used for diagnosing and treating SNX5 protein related diseases. A part or all of the polynucleotides of the present invention can be immobilized as probes on a microarray or DNA chip for analysis of differential expression of genes in tissues and gene diagnosis. Antibodies against SNX5 may be immobilized on a protein chip for detecting SNX5 protein in a sample.

The main advantages of the invention include:

(1) the invention discovers for the first time that the expression of the gene or protein of SNX5 in the liver cancer tissue is obviously higher than the expression of the gene or protein of SNX5 in corresponding paracancer tissues and normal tissues, in addition, the expression of SNX5 in the liver cancer tissue is increased, the over-expression of SNX5 can promote the proliferation, migration and invasion of liver cancer cells, and the expression of gene silencing SNX5 can inhibit the proliferation, migration and invasion of the liver cancer cells. And the Kaplan-Meier method analyzes the relation between the expression of SNX5 and the prognosis of the liver cancer patient, which indicates that the prognosis of the high-expression SNX5 patient is poor (p is 0.015), and the SNX5 high-expression patient is closely related to vascular invasion and liver metastasis. Thus, SNX5 can be used as one of the biomarkers for liver cancer prognosis, and SNX5 gene or its protein can be used as (i) to detect the risk of liver cancer developing intrahepatic metastasis and/or lung metastasis; and/or (ii) as a marker for determining prognosis and survival of a patient with liver cancer.

(2) The invention firstly discovers that in vivo and in vitro functional experiments show that SNX5 can promote the proliferation and the metastasis of liver cancer cells, and an inhibitor (for example, the expression of SNX5 is interfered) of SNX5 gene or protein thereof can effectively (a) inhibit the metastasis of the liver cancer cells; and/or (b) increasing the sensitivity of hepatoma cells to chemotherapeutic drugs (e.g., sorafenib and Erlotinb).

(3) The invention discovers for the first time that the inhibitor of the SNX5 gene or the protein thereof can be used together with chemotherapeutic drugs and has obvious synergistic effect on the treatment of liver cancer.

(4) The invention discovers for the first time that the SNX5 is highly expressed in a tissue sample of a liver cancer patient, which indicates that the prognosis of the patient is poor.

(5) The invention discovers for the first time that SNX5 promotes the metastasis potential of liver cancer cells, and SNX5 promotes the intrahepatic metastasis and pulmonary metastasis of the liver cancer cells.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise specified, materials and reagents used in examples of the present invention are commercially available products.

General procedure

1. Cell culture

The cell experiments in the invention are all at 37 ℃ and 5% CO₂And culturing in an incubator with saturated humidity (70-75%). The culture medium is a high-glucose cell culture medium (DMEM) containing streptomycin with the concentration of 100 mu g/mL, penicillin with the concentration of 100IU/mL and 10 percent Fetal Bovine Serum (FBS). When the cells were grown to a confluence of 90%, they were digested with pancreatin digest for passage or treatment.

RNA extraction

1) And taking out the cells with the fusion degree of more than 90% in the cell culture dish, discarding the culture solution in the cell culture dish, washing twice with 1 multiplied by PBS, fully sucking up the residual PBS, adding 2ml of TRIzol solution, fully blowing to ensure that the adherent cells are suspended in the TRIzol solution, sucking 0.5 ml of TRIzol solution by using 1ml of wall head, and putting the solution into a 1.5ml of EP tube. The remaining 1.5ml of TRIzol solution was stored at minus 80 ℃.

2) Standing 0.5 ml of TRIzol solution containing cells at room temperature for 5 minutes, then adding 100ul of chloroform solution, shaking for 15 seconds with force, standing again at room temperature for 2-3 minutes, placing the mixed solution in a 4 ℃ centrifuge, and centrifuging at 12000g at high speed for 15 minutes.

3) After centrifugation at high speed for 15 minutes, the liquid in the EP tube was observed to separate into three layers, a lower protein organic phase, an intermediate DNA solid phase and an upper RNA aqueous phase. Gently pipette the upper aqueous RNA phase into a new EP tube using a pipette tip

4) 0.5 ml of isopropanol was added to the EP tube containing the RNA aqueous phase, and after carefully pipetting the mixture, the mixture was allowed to stand at normal temperature for 10 minutes, and then the mixture was again placed in a 4 ℃ centrifuge and centrifuged at 12000g at high speed for 10 minutes, whereupon white RNA precipitate was observed at the bottom of the EP tube.

5) Discarding the liquid, carefully cleaning the residual liquid by using a gun head, then opening a cover, airing, adding about 20ul DEPC water after a white precipitate becomes transparent, uniformly blowing and dissolving by using the gun head, measuring the concentration by using a NanoDrop machine in a laboratory, then taking 1ug of RNA, carrying out reverse transcription to obtain cDNA, and storing the rest RNA at minus 80 ℃.

3. Reverse transcription

PrimeScript, a kit for reverse transcription, was purchased from TaKaRa, Japan^TMRT reagent Kit (Perfect Real Time). The 20uL RNA reverse transcription system is: 5 XPrimeScript Buffer 4. mu.L, PrimeScript RT Enzyme Mix I1. mu.L, Random 6mers (100. mu.M) 1. mu.L, Oligo dT Primer (50. mu.M) 1. mu.L, total RNA 1. mu.g, and DEPC H was added₂The content of O is filled to 20 mu L. Adding the prepared manifold into a PCR instrument, and performing the following procedures: run at 37 ℃ for 15 minutes; run at 85 ℃ for 5 seconds and finally at 4 ℃.

4. Fluorescent quantitative PCR

The system of real-time quantitative PCR was 20ul in total volume, and then SYBR Premix Ex Taq was added to each component^TMSolutions(2×)10μL，ROX Reference Dye II(50×)0.4μL，Primer R0.8μL，Primer F0.8μL，ddH₂O6. mu.L, and finally 2. mu.L of cDNA was added. And (3) repeatedly arranging three holes in each sample, preparing a header pipe, adding the header pipe into a 96-well plate, and placing the 96-well plate on an ABI 7500 quantitative PCR instrument for operation. The operating temperature program and the number of cycles were: first 95 ℃ for 30 seconds and then 40 cycles of {95 ℃, 5 seconds, 60 ℃, 30 seconds }. After the experiment, the change of the expression level of the expressed mRNA was analyzed as a change in CT value in 7500software using GAPDH as an internal control.

5. Protein extraction

1) Preparing a protein lysate (the lysate with the total volume of 1mL contains 880ul of basic lysate, 100ul of 10 Xphosphatase inhibitor solution and 20 mu L of 50 Xprotease inhibitor solution), taking out a culture dish containing cells from a cell culture box, discarding the culture solution, washing residual serum by precooled 1 XPBS, fully sucking out the 1 XPBS by a gun head, adding a proper amount of prepared protein lysate according to different cell numbers in the culture dish, slightly scraping the cells by a scraper, and placing the cells in a 1.5mL EP tube to be placed on ice for lysis for 40 minutes.

2) After 40 minutes the EP tube containing the protein lysate was placed in a 4 ℃ centrifuge and centrifuged at 12000g for 15 minutes.

3) The centrifuged EP tube was removed and the supernatant was pipetted into a new EP tube, which was then run on ice.

4) Next, the protein concentration was measured and 200ul BCA, 20ul 1 XPBS, 4ul CuSO were added to a new EP tube₄Finally, 5ul of centrifuged protein lysate is added, and the absorbance value is measured at a wavelength of 570nm by using an enzyme-linked immunosorbent assay. After completion, the measured protein concentration was calculated using the standard curve.

5) And finally, performing protein lysate-based separation on the remaining protein lysate supernatant: 5 × loading buffer: lysates were aspirated at a ratio of 7:2:1 with 10 × DTT and the corresponding 5 × loading buffer and DTT were added.

6. Construction of SNX5 overexpression plasmid

1) Extracting liver cancer cell RNA (same steps)

2) Reverse transcription of RNA into cDNA (methods as before)

3) Rpn10 Gene amplification

Downloading a CDS sequence of SNX5 from a NCBI network, introducing a Bam HI/EcoR I enzyme cutting site when designing a clone primer of SNX5, using cDNA extracted in the previous step as a template, amplifying a CDS region of SNX5 by using a PCR instrument, and adding samples according to the following amplification system:

the temperature program in the PCR instrument was:

3) purifying and recovering amplified DNA product fragments and carrying out enzyme digestion and connection:

a 1% agarose gel was prepared: adding 3mg of agar powder into 30ml of 0.5 xTBE buffer solution, putting the agar powder into a microwave oven, heating and boiling the agar powder, cooling the agar powder to about 55-60 ℃, and adding DNA dye ethidium bromide by using a liquid transfer gun. Pouring the mixture into a glue preparation groove in which a comb is inserted after the mixture is fully mixed, cooling and solidifying the mixture in a half hour, carefully pulling out the comb, and putting the glue block into an electrophoresis tank. Following amplification of the DNA fragment: DNA Marker 5:1, adjusting the voltage to 120V, when Marker is assigned to a proper position, observing with an ultraviolet lamp, carefully cutting off the DNA band of the amplified product, placing the DNA band in a 1.5ml EP tube, and recovering the DNA product according to the instruction of DNA purification recovery kit of Tiangen corporation. After recovery, the pWPXL and the PCR amplification product are digested by two high fidelity enzymes MiuI/EcoRI respectively according to the following system.

After the enzyme digestion is finished, recovering the enzyme digestion product of the PCR amplification product according to a DNA purification recovery kit of Tiangen company, and recovering the enzyme digestion product of the pWPXL according to the enzyme digestion product: adding the DNA Marker into the prepared electrophoresis gel block in a ratio of 1:6, cutting the required strip under an ultraviolet lamp after the completion, and recovering the strip by using a DNA purification and recovery kit of Tiangen company according to the instruction. After the concentration was measured, ligation was performed according to a 10. mu.l ligation system: the molar mass ratio of the cleavage products of pWPXL and the PCR amplification product was 1:7, 1. mu. l T4 ligase, 1. mu.l of buffer, and ddH was used for the remainder₂The amount of O was adjusted to 10. mu.l. Connecting in a 22 deg.C water bath for 4 hr, inactivating at 65 deg.C for 10 min, and cooling on ice.

4) Transforming and selecting monoclonal bacteria

Firstly, 50ul of E.coli Ecoli DH5 alpha competent and 10ul of ligation product were added to an autoclaved EP tube, mixed well and placed on ice for half an hour.

② 90 seconds in a water bath kettle at 42 ℃ and 2-3 minutes on ice.

③ Add 200ul of SOCS medium and shake for 1h at 150rpm on a shaker.

And fourthly, uniformly smearing the mixed solution on a bacterial culture plate with ampicillin resistance. The mixture was inverted and placed in a 37 ℃ incubator overnight.

Observing the growth of colony in the culture dish after the next day, picking single colony with sterilized small Tip, placing in 5ml liquid LB solution with ampicillin resistance, placing in 37 deg.C shaking table, and standing overnight at 250 rpm.

5) Extraction of plasmids

500ul of equilibrium liquid BL is added into an adsorption column CP3, then the adsorption column CP3 is placed into a collecting pipe, the collection pipe is centrifuged at 12000rpm for 1 minute, and the adsorption column CP3 is placed into the collecting pipe again after the waste liquid is poured out.

Secondly, putting the bacterial liquid on the overnight shaking table into a centrifugal tube for centrifugation at 12000rpm for 1 minute, and pouring out the supernatant after the centrifugation is finished to leave the bacterial liquid.

③ adding 250ul of P1 solution into the centrifuge tube with the bacteria solution left, and placing the centrifuge tube on a shaker to resuspend the bacteria solution.

Adding 250ul of P2 solution, and turning the mixture up and down gently for 6-8 times until the turbid bacterial liquid becomes clear.

Add 350ul of P3 solution immediately after adding, turn up and down gently to prevent forming local precipitation.

Sixthly, putting the mixture into a centrifugal machine for 1 minute at 12000rpm, sucking the supernatant after the centrifugation is finished, putting the supernatant into CP3, and standing the supernatant for 2 minutes at room temperature.

Seventhly, placing the CP3 into a collecting pipe, centrifuging for 1 minute at 12000rpm in a centrifuge, and discarding the liquid in the collecting pipe after centrifugation.

Adding 600ul of PW solution into CP3, centrifuging at 12000rpm for 1 minute, removing, and discarding the waste liquid in the collecting pipe.

Ninthly, repeatedly operating.

R CP3 was returned to the collection tube and centrifuged at 12000rpm for 2 minutes. After the completion, the cover is opened and the air is dried at room temperature.

Dripping 50ul of the dried product into CP3ddH2O, left to stand at room temperature for 2 minutes, was put into a clean EP tube and centrifuged at 12000rpm for 2 minutes. The concentration was measured and the cells were stored at-20 ℃.

6) Enzyme cleavage identification and sequencing

The plasmid with the correct restriction enzyme band is sent to the EnxWeijie fundic organisms Limited company for sequencing by enzyme digestion identification and agarose gel electrophoresis by using two enzymes MiuI/EcoRI. The sequencing result comparison uses SnapGene software to amplify the plasmid with correct sequence, and glycerol bacteria is stored at-80 ℃ by adding 500ul of 50% glycerol and 500 mul of bacterial liquid with correct sequencing.

7. Protein electrophoresis and Western blot assay

1) Preparing glass plates, preparing 10% polyacrylamide gel separation gel solution according to the formula, pouring the separation gel solution between the two prepared glass plates after fully and uniformly mixing, quickly pouring distilled water on the separation gel solution, flattening the separation gel, reducing the inhibition effect of air on the solidification of the separation gel, and standing at room temperature for about half an hour to be solidified;

2) when the lower layer separation glue is solidified and obvious separation lines appear on the water and glue surfaces, pouring out distilled water, inverting the lower layer separation glue on filter paper for drying, preparing 5% of concentrated glue according to the preparation proportion of the concentrated glue, fully and uniformly mixing, quickly pouring the mixture into a glass clamping plate, immediately inserting a comb with a required aperture, and standing for half an hour at room temperature;

3) after the two layers of glue are solidified, putting the two layers of glue into an electrophoresis tank added with electrophoresis liquid, carefully pulling out a comb, calculating the sample amount according to the concentration of a protein sample and the graduation of different proteins in cells, and adding 1 multiplied loading buffer solution (loading buffer) for supplementing after loading;

4) turning on the power supply of the electrophoresis apparatus, keeping the voltage constant for 80V for half an hour until the protein sample runs to the separation gel, adjusting the voltage to 120V, and keeping the voltage constant for 1 and half an hour;

5) film transfer: preparing a membrane transferring solution (20 times of the membrane transferring solution in a preparation proportion of 1L, 50mL of the membrane transferring solution: 200 mL: 750mL of methanol: distilled water) and placing the membrane transferring solution at four degrees for cooling, placing the gel block and the nitrocellulose membrane after electrophoresis into a membrane transferring clamp, performing constant current of 220mA in a membrane transferring groove placed on ice, and performing wet rotation for 50 minutes;

6) after the membrane is completely transferred, putting the nitrocellulose membrane into 5% freshly prepared skim milk, and slowly shaking the shaker for one and a half hours to seal;

7) after the blocking is finished, the membrane is put into a clean PBST solution (1L 1 multiplied by PBS and 1mL of 0.1 percent Tween 20 is added) to remove residual milk, and the membrane is cleaned;

8) diluting the antibody with blocking solution to an appropriate ratio according to the antibody specification, and incubating the membrane in a refrigerator at 4 ℃ overnight;

9) washing the membrane with clean PBST solution three times in ten minutes each time the next day, diluting horseradish peroxidase-labeled secondary antibody (mouse antibody or rabbit antibody) with 5% skimmed milk solution according to the antibody specification, and incubating for one and a half hours at room temperature after membrane washing;

10) after the secondary antibody is incubated, the membrane is washed by PBST solution for ten minutes each time for three times;

11) luminescence: on a luminometer, a Super Signal chemiluminescent substrate was used, and the appropriate exposure time was adjusted according to the exposure intensity of the antibody, and the photograph was stored and analyzed for bands.

12) After eluting the antibody by using protein eluent, taking beta-actin as a loading internal reference, and diluting the beta-actin antibody by using 5% skimmed milk solution according to the proportion of 1: 10000, PBST solution washing membrane three times, each time for 10 minutes, after washing the membrane, using Super Signal chemiluminescence substrate for exposure.

8. Virus package

1) In the first step, 293T cells with moderate density are inoculated in a 6cm dish, and the growing fusion degree of the 293T cells reaches about 90% on the second day.

2) 500ul of DMEM solution was added to each of the two wells of a 24-well plate, and Lipofectamine was prepared in the following proportions ^TM2000 and plasmid. The mixed plasmid was added to one well, and Lipofectamine was added to the other well ^TM2000, 5min later, the wells contained mixed plasmid and Lipofectamine ^TM2000 in DMEM, and left to stand at room temperature for 20 minutes.

3) The 293T cell culture solution in the 6cm dish was changed to serum-free DMEM solution, and then the plasmid and Lipofectamine were added^TM2000 were carefully added to 293T cells (care was taken not to force, to prevent blowing off of 293T cells).

4) The cells were incubated at 37 ℃ for 6 hours and then replaced with serum-containing DMEM solution.

5) After 48 hours the virus was recovered using a 0.45um filter. And (4) preserving in a refrigerator at the temperature of minus 80 ℃.

9. Lentivirus infection

Firstly, inoculating human hepatocellular carcinoma cells with moderate density into a 6cm dish, and placing the dish in a 37 ℃ incubator for overnight culture, so that the cell fusion degree can reach about 50% on the next day. And step two, taking out the required virus solution from a refrigerator at the temperature of-80 ℃ the next day, and after the virus solution is melted, carrying out cell culture according to the following steps: 1:1, adding polybrene (Sigma-Aldrich) into a culture dish inoculated with human hepatocellular carcinoma cells to enable the final concentration to reach 1 per mill, and changing the liquid after 6-8 hours.

10. Clone formation experiments

1) Taking out the hepatocellular carcinoma cell line growing in the constant-temperature incubator at 37 ℃, after the pancreatin digestion is finished, the counting method is the same as the CCK8 experiment, according to the growth speed of different hepatocellular carcinoma cells, inoculating 1000-3000 cells into each hole of a sterile six-hole plate culture dish, repeating the three holes, and returning to the constant-temperature incubator at 37 DEG C

2) The culture medium is changed every three days, the cell clone size in the six-hole plate is observed after about 2 weeks of culture, and the culture is stopped when the size is proper and the density is moderate.

3) The culture solution was discarded, washed 2 times with 1 × PBS, the residual culture solution was washed off, then 10% neutral formalin solution was added and fixed at room temperature for 1 hour, the formalin solution was poured off, and 2ml Giemsa staining solution was added and stained for one hour. The staining solution was recovered, excess Giemsa staining solution was carefully washed off with distilled water, and dried by inversion at room temperature. After scanning with the scanner, Image J software counted the number of clones formed.

11. Cell migration and invasion assay

Migration experiment:cell digestion and counting, the method is the same as migration experiment, serum-free DMEM is used for resuspending cells at 800rpm and washing twice for 5min, redundant serum is removed as far as possible, serum-free culture medium liquid is used for resuspending cells and counting, and 1 multiplied by 10 is taken⁵Placing each cell into 200 μ L serum-free culture medium, mixing, slowly dripping into the chamber, adding 600 μ L complete culture medium into the lower chamber in advance, observing whether the cells are uniformly spread under the microscope, and culturing in 37 deg.C incubator for 12 h.

Invasion test: the Matrigel matrix glue is thawed at 4 ℃ one day in advance, the Matrigel matrix glue is diluted by a precooled serum-free culture medium according to a ratio of 1:10 (all articles contacting with the melted Matrigel glue are operated in an ice box, a tip head and an EP (EP) tube are precooled at 4 ℃), 20 mu L of the Matrigel matrix glue is uniformly coated on a microporous film at the bottom of a Transwell chamber after being uniformly mixed, the glue on the chamber membrane is solidified in an incubator at 37 ℃ for 30min, the micropores on the membrane are blocked by the glue, and the chamber is placed in a 24-hole culture plate special for a Transwell experiment after being solidified (600 mu L of complete culture medium is added in advance). Cell digestion and counting, the method is the same as migration experiment, serum-free DMEM is used for resuspending cells at 800rpm and washing twice for 5min, redundant serum is removed as far as possible, serum-free culture medium liquid is used for resuspending cells and counting, and 1 multiplied by 10 is taken⁵Placing each cell into 200 μ L serum-free culture medium, mixing, slowly dripping into the chamber, adding 600 μ L complete culture medium into the lower chamber in advance, observing whether the cells are uniformly spread under the microscope, and culturing in 37 deg.C incubator for 24 h.

12. Cell proliferation assay (CCK8 assay)

1) Taking out the cultured hepatocellular carcinoma cell line in a constant temperature incubator at 37 ℃, digesting the hepatocellular carcinoma cell line by pancreatin, sucking 10ul of culture solution and 10ul of trypan blue solution, fully and uniformly mixing the solution, sucking 10ul of culture solution and adding the solution into a counting plate for counting, inoculating the number of cells with different densities in a 96-well plate with the range of 800-3000 cells per hole and three holes in each row for 7 rows in total for seven-day measurement due to different growth speeds of different hepatocellular carcinoma cell lines.

2) The next day, 10. mu.l of CCK8 solution was added to each of the first row of three-well cells, and after 2 hours, the absorbance value was measured by selecting a wavelength of 450nm in a microplate reader.

3) And on the third day, repeating the step two, continuously measuring the absorbance value for seven days, changing the liquid every other day, and drawing a growth curve according to the absorbance value.

13. In situ inoculation experiment of naked mouse liver

Collecting cells with good growth state, digesting with 2.5g/L pancreatin containing EDTA to obtain single cell suspension, counting, and classifying cells according to 2 × 10⁶Dissolving cells in 25 mul of serum-free culture medium, mixing with 25 mul of Matrigel (according to the volume ratio of 1:1), placing on ice for operation, anesthetizing Balb/c nude mice, opening the abdominal cavity, directly injecting cell suspension into the liver, painlessly sacrificing the nude mice after 5-7 weeks, weighing tumor-bearing mice, taking liver and lung tissues, fixing experimental samples in neutral buffered formalin, and performing pathological histological examination after paraffin section and HE staining.

Example 1 SNX5 can be used as a molecular marker for prognosis of liver cancer

By analyzing TCGA and GEO databases, the expression of SNX5 in liver cancer tissues is found to be increased (see figure 1), the expression level of SNX5 protein in the liver cancer tissues is further analyzed by a western blot method by the inventor, the result shows that the expression of SNX5 in the liver cancer tissues is higher than that of corresponding paracarcinoma tissues, the expression of SNX5 protein in the liver cancer tissues is analyzed by an immunohistochemical method by the inventor, and according to the expression level of SNX5 in 143 cases of liver cancer tissues, the SNX5 low expression group and the SNX5 high expression group are correspondingly divided, and the relationship between the expression of SNX5 and the prognosis of a liver cancer patient is analyzed by a Kaplan-Meier method.

The results show (see fig. 2, table 1) that the prognosis of the SNX 5-highly expressed patients is poor (p ═ 0.015), and that the SNX 5-highly expressed patients are closely related to vascular invasion and intrahepatic metastasis. Meanwhile, the TCGA database also shows that the prognosis of the SNX5 high-expression patient is poor. The inventors further analyzed the prognosis risk ratio by univariate and multivariate analysis based on the expression level of SNX5 in 143 liver cancer tissues (see fig. 3), showing that SNX5 is a risk factor affecting the prognosis of liver cancer patients.

Therefore, the expression level of SNX5 may be used as a molecular marker for liver cancer prognosis.

TABLE 1 relationship between SNX5 expression in liver cancer tissue and patient clinical pathology

*p<0.05

Example 2 SNX5 promotion of liver cancer cell proliferation

According to the expression level of SNX5 in liver cancer cells, SNX5 is overexpressed by SMMC-7721 and Li7 cells, and SNX5 is knocked down by Huh7 and MHCC-LM3 cells, and CCK8 and clonogenic experiments show that the liver cancer cell proliferation is promoted by overexpression of SNX5, and the liver cancer cell proliferation is inhibited by knocking down of SNX5, and the results are shown in FIG. 4.

Meanwhile, in vivo animal experiments also show that the over-expression of SNX5 can promote the in vivo proliferation of liver cancer cells, interfere the expression of SNX5 and inhibit the liver in-situ tumorigenicity ability of MHCC-LM3 cells, and the results are shown in figure 5.

Example 3 SNX5 promotion of hepatocyte transfer

Through transwell experiments, over-expression of SNX5 promotes migration and invasion of SMMC-7721 and Li7 cells, whereas knocking down expression of SNX5 inhibits migration and invasion capacity of liver cancer cells, and the result is shown in FIG. 6.

Furthermore, it was found by phallodin staining that overexpression of SNX5 promoted the formation of invasive pseudopodia in SMMC-7721 and Li7 cells, increased expression of MMP9 as an invasive marker, overexpression of SNX5 promoted the expression of MMP9 in liver cancer cells, and reduction of expression of SNX5 suppressed the expression of MMP9 in liver cancer cells. Further, animal experiments show that when SNX5 overexpression cells are injected in situ into the liver, the overexpression of SNX5 can promote liver metastasis and lung metastasis of liver cancer cells, interfere with the expression of SNX5 and inhibit the liver metastasis of the liver cancer cells, and the results are shown in figure 7.

Example 4 interference with SNX5 expression increases drug sensitivity of sorafenib and Erlotinb

The inventor detects the sensitivity of the expression of knocking down SNX5 to chemotherapeutic drugs sorafenib and Erlotinb, and the result (shown in figure 8 and figure 9) shows that the knocking down expression of SNX5 can enhance the drug sensitivity of sorafenib and Erlotinb to liver cancer cells, inhibit the clonogenic capacity of the liver cancer cells and promote the apoptosis of the liver cancer cells.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Use of the SNX5 gene, mRNA, cDNA, or protein or a detection reagent thereof,

(i) as a marker for detecting tissue grading of liver cancer; and/or

(ii) Used as a marker for detecting the risk of liver cancer metastasis; and/or

2. A tissue grade for (i) detecting liver cancer; and/or (ii) detecting the risk of metastasis of liver cancer; and/or (iii) a diagnostic kit for determining prognosis and survival of a patient with liver cancer, wherein the kit comprises a container, and the container contains a detection reagent for detecting SNX5 gene, mRNA, cDNA or protein; and a label or instructions for use of the kit for detecting (i) a tissue grade for detecting liver cancer; and/or (ii) detecting the risk of metastasis of liver cancer; and/or (iii) determining prognosis and survival of a liver cancer patient.

3. A method of (i) detecting the risk of metastasis of liver cancer; and/or (ii) a method of determining prognosis and survival of a liver cancer patient, comprising:

a) providing a test sample from a subject;

4. A method of determining a treatment plan, comprising:

a) providing a test sample from a subject;

b) detecting the expression level of SNX5 protein in the test sample; and

5. Use of the SNX5 gene or a protein inhibitor thereof for the preparation of a composition or formulation for (a) inhibiting the growth or proliferation of a liver cancer cell; and/or (b) inhibiting metastasis of hepatoma cells; and/or (c) preventing and/or treating liver cancer; and/or (d) increasing the sensitivity of the hepatoma cells to chemotherapeutic agents.

6. A pharmaceutical composition, comprising:

(a1) an inhibitor of the SNX5 gene or protein thereof;

(a2) optionally a chemotherapeutic agent; and

(b) a pharmaceutically acceptable carrier.

7. A kit, comprising:

8. Use of the pharmaceutical composition of claim 6 or the kit of claim 7 for (a) inhibiting the growth, proliferation; and/or (b) inhibiting metastasis of hepatoma cells; and/or (c) preventing and/or treating liver cancer.

9. An in vitro non-therapeutic method of inhibiting growth or proliferation of hepatoma cells comprising the steps of: culturing the liver cancer cell in the presence of SNX5 gene or its protein inhibitor, thereby inhibiting the growth or proliferation of the liver cancer cell.

10. A method of screening for a candidate compound for the prevention and/or treatment of liver cancer, said method comprising the steps of: