CN112143805B

CN112143805B - Application of RIT1 in diagnosis and treatment of hepatocellular carcinoma

Info

Publication number: CN112143805B
Application number: CN201910562848.6A
Authority: CN
Inventors: 李红; 刘腾飞; 宋镇; 陈静; 李锦军; 葛超
Original assignee: Shanghai Cancer Institute
Current assignee: Shanghai Cancer Institute
Priority date: 2019-06-26
Filing date: 2019-06-26
Publication date: 2024-08-06
Anticipated expiration: 2039-06-26
Also published as: CN112143805A

Abstract

The present invention relates to the use of RIT1 in the diagnosis and treatment of hepatocellular carcinoma. Specifically, the invention provides an application of the RIT1 gene, mRNA, cDNA or protein or a detection reagent thereof in diagnosing hepatocellular carcinoma, the RIT1 gene or a protein inhibitor thereof can effectively treat hepatocellular carcinoma, and the RIT1 gene or the protein inhibitor thereof can be optionally combined with a tyrosine kinase inhibitor and/or other drugs for preventing and/or treating hepatocellular carcinoma, and has a remarkable synergistic effect on the treatment of hepatocellular carcinoma.

Description

Application of RIT1 in diagnosis and treatment of hepatocellular carcinoma

Technical Field

The present invention relates to the field of oncology and diagnostics. More particularly, the invention relates to the use of RIT1 in the diagnosis and treatment of hepatocellular carcinoma.

Background

The incidence of hepatocellular carcinoma is sixth among all tumors, and the new incidence of hepatocellular carcinoma is nearly 80 tens of thousands each year. At present, various treatment means for hepatocellular carcinoma are available, including surgical excision, liver transplantation, tumor ablation, arterial tumor embolism operation, systemic treatment and the like, but patients with middle and late hepatocellular carcinoma have lower benefit from treatment, shorter survival time and poor prognosis. First-line targeting drugs for hepatocellular carcinoma are now few and resistant.

Thus, there is an urgent need in the art to develop new targets with diagnostic or prognostic prediction and therapeutic effects and new methods to overcome drug resistance.

Disclosure of Invention

The object of the present invention is to provide new targets with diagnostic or prognostic prediction and therapeutic effects and new methods of overcoming drug resistance.

The first aspect of the present invention provides the use of a RIT1 gene, mRNA, cDNA, or protein or a detection reagent thereof, (i) as a marker for detecting tissue classification of hepatocellular carcinoma; and/or (ii) as a marker for detecting the risk of metastasis of hepatocellular carcinoma; and/or (iii) as a marker for determining prognosis and survival of a hepatocellular carcinoma patient; and/or (iv) for preparing a diagnostic reagent or kit for detecting tissue classification of hepatocellular carcinoma; and/or (v) for preparing a diagnostic reagent or kit for detecting the risk of metastasis of hepatocellular carcinoma; and/or (vi) for the preparation of a diagnostic reagent or kit for use as a diagnostic for determining prognosis and survival of a patient with hepatocellular carcinoma.

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 RIT1 gene, mRNA, cDNA, or protein is derived from a mammal, preferably a rodent (e.g., mouse, rat), primate, or human, and more preferably a patient diagnosed with hepatocellular carcinoma.

In another preferred embodiment, the RIT1 gene, mRNA, cDNA, or protein is derived from a patient with hepatocellular carcinoma.

In another preferred embodiment, the RIT1 gene has accession number 6016.

In another preferred embodiment, the RIT1 mRNA has accession number NM-001256821.2.

In another preferred embodiment, the RIT1 protein has accession number NP-001243750.1.

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

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

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

In another preferred embodiment, the detection is of hepatocellular carcinoma tissue, or a general liver tissue sample.

In another preferred embodiment, the general liver tissue comprises a paracancerous tissue.

In another preferred embodiment, the detection reagent comprises an antibody specific for RIT1, a specific binding molecule for RIT1, a specific amplification primer, a probe or a chip.

In another preferred embodiment, the RIT1 protein or a specific antibody or specific binding molecule thereof is conjugated or provided with a detectable label.

In another preferred embodiment, the detectable label is selected from the group consisting of: chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.

In another preferred embodiment, the specific antibody for RIT1 is a monoclonal or polyclonal antibody.

In another preferred embodiment, the tissue classification of hepatocellular carcinoma includes stage I, stage II, stage III, and stage IV.

In another preferred embodiment, the risk of metastasis refers to a risk of metastasis for n years, where n is any number (including decimal) from 1 to 5.

In a second aspect, the invention provides a method for (i) detecting tissue classification of hepatocellular carcinoma; and/or (ii) detecting a risk of metastasis of hepatocellular carcinoma; and/or (iii) a diagnostic kit for determining prognosis and survival of a patient with hepatocellular carcinoma, said kit comprising a container containing a detection reagent for detecting a RIT1 gene, mRNA, cDNA, or protein; and a label or instruction stating that the kit is for use in detecting (i) tissue classification for detecting hepatocellular carcinoma; and/or (ii) detecting a risk of metastasis of hepatocellular carcinoma; and/or (iii) determining prognosis and survival of a hepatocellular carcinoma patient.

In another preferred embodiment, the detection of the risk of metastasis of hepatocellular carcinoma refers to detection of whether hepatocellular carcinoma has occurred, the site of metastasis of the occurrence, and/or

The size of the possibility (susceptibility) of the occurrence of hepatocellular carcinoma metastasis is determined.

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

In another preferred embodiment, the detection reagent for detecting RIT1 gene, mRNA, cDNA, or protein comprises:

(a) Specific antibodies against the RIT1 protein; and/or

(B) Specific primers for specific amplification of mRNA or cDNA of RIT 1.

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

In another preferred embodiment, the label or description refers to the following:

(a) When the ratio of RIT1 expression quantity E1 in the hepatocellular carcinoma cells or tissues of the detection object to RIT1 expression quantity E2 of general hepatocytes or tissues is more than or equal to 2, prompting that the probability of the risk of hepatic metastasis of hepatocellular carcinoma of the detection object is higher than that of general population;

(b) When the ratio of RIT1 expression quantity E1 in the hepatocellular carcinoma cells or tissues of the detection object to RIT1 expression quantity E2 of general hepatocytes or tissues is more than or equal to 2, prompting that the prognosis of the detection object is poor and the probability of shortening the survival time is higher than that of general population;

Wherein E2 is the RIT1 expression level of general liver cells or tissues of general population.

In another preferred embodiment, the general hepatocytes or tissue comprise paracancestral hepatocytes or tissue.

In a third aspect, the invention provides (i) detecting a risk of metastasis of hepatocellular carcinoma; and/or (ii) a method of determining prognosis and survival of a patient with hepatocellular carcinoma, the method comprising:

a) Providing a test sample from a subject;

b) Detecting the expression quantity E1 of RIT1 protein in a test sample; and

C) Comparing the expression level of the RIT1 protein determined in step b) with a control,

Wherein an expression level of RIT1 protein in said sample that is higher than a reference value compared to said control indicates that the subject has a higher probability of developing a risk of metastasis than the general population (control population); and/or the expression level of the RIT1 protein is lower than the reference value, indicating that the subject has a lower risk of developing intrahepatic metastasis than the general population (control population); and/or

The higher expression level of RIT1 protein in the sample compared to the control compared to the reference value indicates that the subject has a higher probability of poor prognosis and reduced survival than the general population (control population); and/or the expression level of the RIT1 protein is lower than the reference value, indicating that the subject has a lower probability of poor prognosis and shortened survival than the general population (control population).

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

In another preferred embodiment, the test sample is a hepatocellular carcinoma cell or tissue.

In another preferred embodiment, the reference value is a cut-off value.

In another preferred embodiment, the reference value is the relative expression level of RIT1 in the sample.

In another preferred embodiment, the reference value is 3.5.

In another preferred embodiment, the detecting step (ii) comprises detecting the amount of RIT1 mRNA, or the amount of RIT1 cDNA; and/or detecting the amount of the RIT1 protein.

In another preferred embodiment, the expression level of the RIT1 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, the invention provides a method of determining a treatment regimen comprising:

a) Providing a test sample from a subject;

b) Detecting the expression level of the RIT1 protein in the test sample; and

C) Determining a treatment regimen based on the expression level of the RIT1 protein in the sample.

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

In another preferred embodiment, when the expression level of RIT1 protein in the sample is higher than a reference value, it is indicated that the subject has a higher probability of developing a risk of hepatocellular carcinoma metastasis than the general population (control population); or the level of malignancy of hepatocellular carcinoma in the subject is higher than that in the general population (control population); or a subject with a higher chance of poor prognosis and reduced survival than the general population (control population), the treatment regimen comprising a RIT1 inhibitor therapy, a therapy in which a RIT1 inhibitor is combined with a tyrosine kinase inhibitor.

In another preferred embodiment, the RIT1 inhibitor therapy, a therapy in which a RIT1 inhibitor is combined with a tyrosine kinase inhibitor, is selected from the group consisting of:

RIT1 inhibitor therapy: an antibody, a small molecule compound, microRNA, siRNA, shRNA, or a combination thereof;

Tyrosine kinase inhibitor therapy: a small molecule compound selected from the group consisting of: sorafenib, lenvatinib, regorafenib, or a combination thereof.

In another preferred embodiment, when the subject is at a higher risk of developing hepatocellular carcinoma metastasis than the general population (control population); or the level of malignancy of hepatocellular carcinoma in the subject is higher than that in the general population (control population); or when the prognosis and the probability of a reduction in survival of the subject are higher than those of the general population (control population), the treatment regimen further comprises a RIT1 inhibitor therapy, a therapy in which a RIT1 inhibitor is combined with a tyrosine kinase inhibitor; and other medicaments for treating hepatocellular carcinoma.

In another preferred embodiment, the other agent for treating hepatocellular carcinoma is selected from the group consisting of: 5-fluorouracil, doxorubicin, oxaliplatin, or a combination thereof.

In a fifth aspect, the present invention provides the use of a RIT1 gene or a protein inhibitor thereof for the preparation of a composition or formulation for (a) inhibiting the growth or proliferation of hepatocellular carcinoma cells; and/or (b) inhibiting metastasis of hepatocellular carcinoma cells; and/or (c) preventing and/or treating hepatocellular carcinoma; and/or (d) increasing sensitivity of the hepatocellular carcinoma cell to a tyrosine kinase inhibitor.

In another preferred embodiment, the RIT1 gene or its protein inhibitor is selected from the group consisting of: an antibody, a small molecule compound, microRNA, siRNA, shRNA, or a combination thereof.

In another preferred embodiment, the inhibitor comprises an inhibitor that inhibits the expression of the RIT1 gene or a protein thereof.

In another preferred embodiment, the composition or formulation is also used to reduce the level of P-ERK and/or P-AKT.

In another preferred embodiment, the inhibiting metastasis of hepatocellular carcinoma cells comprises intrahepatic metastasis and pulmonary metastasis of hepatocellular carcinoma cells.

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 RIT1 gene or protein thereof, and a pharmaceutically acceptable carrier.

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

In another preferred embodiment, the formulation is selected from the group consisting of: tablets, capsules, injections, granules and sprays.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising:

(a1) Inhibitors of the RIT1 gene or protein thereof;

(a2) Optionally, a tyrosine kinase inhibitor; and

(B) A pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition further comprises:

(c) Other drugs for preventing and/or treating hepatocellular carcinoma.

In another preferred embodiment, the tyrosine kinase inhibitor is selected from the group consisting of: sorafenib, lenvatinib, regorafenib, or a combination thereof.

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

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

In another preferred embodiment, the content of component (a 2) in the pharmaceutical composition is 1% to 99%, preferably 10% to 90%, more preferably 30% to 70%.

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

In another preferred embodiment, the component (a 1) and the optional component (a 2) and the optional component (c) comprise 0.01 to 99.99wt%, preferably 0.1 to 90wt%, more preferably 1 to 80wt% of the total weight of the pharmaceutical composition.

In another preferred embodiment, the dosage form of the pharmaceutical composition includes an injectable dosage form, and an oral dosage form.

In another preferred embodiment, the oral dosage form comprises a tablet, a capsule, a film, and a granule.

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

A seventh aspect of the invention provides a kit comprising:

(a1) A first container, and an inhibitor of the RIT1 gene or its protein or a medicament containing an inhibitor of the RIT1 gene or its protein in the first container;

(b1) An optional second container, and a tyrosine kinase inhibitor, or a medicament containing a tyrosine kinase inhibitor, located in the second container.

In another preferred embodiment, the kit further comprises:

(c1) A third container, and other medicaments for preventing and/or treating hepatocellular carcinoma or medicaments containing other medicaments for preventing and/or treating hepatocellular carcinoma positioned in the third container.

In another preferred embodiment, the kit further comprises (d 1) a fourth container, and a detection reagent for RIT1 in the fourth container.

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

In another preferred embodiment, the first container of medicament is a single formulation comprising an inhibitor of the RIT1 gene or protein thereof.

In another preferred embodiment, the medicament of the second container is a single formulation comprising a tyrosine kinase inhibitor.

In another preferred embodiment, the third container is a single formulation containing other agents for preventing and/or treating hepatocellular carcinoma.

In another preferred embodiment, the pharmaceutical is in the form of an oral dosage form or an injectable dosage form.

In another preferred embodiment, the kit further comprises instructions.

In another preferred embodiment, the instructions recite one or more instructions selected from the group consisting of:

(a) An inhibitor of the RIT1 gene or its protein is used to (i) inhibit the growth of hepatocellular carcinoma cells; and/or (ii) inhibiting metastasis of hepatocellular carcinoma cells; and/or (iii) preventing and/or treating hepatocellular carcinoma; and/or (iv) a method of increasing sensitivity of a hepatocellular carcinoma cell to a tyrosine kinase inhibitor;

(b) The use of an inhibitor of the RIT1 gene or protein thereof in combination with a tyrosine kinase inhibitor, and/or optionally other agents for the prevention and/or treatment of hepatocellular carcinoma to (i) inhibit the growth of hepatocellular carcinoma cells; and/or (ii) inhibiting metastasis of hepatocellular carcinoma cells; and/or (iii) preventing and/or treating hepatocellular carcinoma;

(c) Detecting the expression level of the RIT1 protein in a patient with hepatocellular carcinoma while administering an inhibitor of the RIT1 gene or its protein (i) to inhibit the growth of hepatocellular carcinoma cells; and/or (ii) a method of inhibiting metastasis of hepatocellular carcinoma cells; and/or (iii) preventing and/or treating hepatocellular carcinoma; and/or (iv) a method of increasing sensitivity of a hepatocellular carcinoma cell to a tyrosine kinase inhibitor;

(d) Detecting the expression level of RIT1 protein of a patient suffering from hepatocellular carcinoma, and simultaneously combining an inhibitor of RITI1 gene or a protein thereof; and tyrosine kinase inhibitors, and/or optionally other agents for preventing and/or treating hepatocellular carcinoma to (i) inhibit the growth of hepatocellular carcinoma cells; and/or (ii) a method of inhibiting metastasis of hepatocellular carcinoma cells; and/or (iii) a method of preventing and/or treating hepatocellular carcinoma.

The eighth aspect of the present invention provides the use of a pharmaceutical composition according to the sixth aspect of the present invention or a kit according to the seventh aspect of the present invention for (a) inhibiting the growth of hepatocellular carcinoma cells; and/or (b) inhibiting metastasis of hepatocellular carcinoma cells; and/or (c) preventing and/or treating hepatocellular carcinoma.

In another preferred embodiment, the concentration of the inhibitor of the RIT1 gene or its protein in the pharmaceutical composition is between 100 and 2000ng/ml, preferably between 500 and 1500ng/ml, more preferably between 800 and 1000ng/ml.

In another preferred embodiment, the concentration of the tyrosine kinase inhibitor in the pharmaceutical composition is in the range of 1000 to 5000ng/ml, preferably 2000 to 4000ng/ml, more preferably 3000 to 3500ng/ml.

In another preferred embodiment, the concentration of the other agents for preventing and/or treating hepatocellular carcinoma is between 500 and 4000ng/ml, preferably between 1500 and 3500ng/ml, more preferably between 2000 and 3000ng/ml.

In another preferred embodiment, the pharmaceutical composition or kit comprises (a) an inhibitor of the RIT1 gene or protein thereof; and (b) optionally, a tyrosine kinase inhibitor; and (c) optionally, other agents for preventing and/or treating hepatocellular carcinoma; and (d) a pharmaceutically acceptable carrier.

In another preferred embodiment, in the pharmaceutical composition or kit, the inhibitor of the RIT1 gene or protein thereof; and (b) optionally, a tyrosine kinase inhibitor; and (c) optionally, other agents for preventing and/or treating hepatocellular carcinoma, in an amount of 0.01 to 99.99wt%, preferably 0.1 to 90wt%, more preferably 1 to 80wt%, based on the total weight of the pharmaceutical composition or kit.

In a ninth aspect, the present invention provides a method for preventing and/or treating hepatocellular carcinoma, comprising:

administering to a subject in need thereof an inhibitor of the RIT1 gene or 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 hepatocellular carcinoma.

In another preferred embodiment, the non-human mammal comprises a rodent and primate, preferably a mouse, rat, rabbit, monkey.

In another preferred embodiment, the inhibitor of the RITI gene or a protein thereof is administered at 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 tyrosine kinase inhibitor is administered at 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 hepatocellular carcinoma is administered at 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 RIT1 gene or protein thereof is administered at a frequency of 1-4 times per week, preferably 2-3 times per week.

In another preferred embodiment, the tyrosine kinase inhibitor is administered at a frequency of 1-5 times per week, preferably 2-3 times per week. In another preferred embodiment, the other agent for preventing and/or treating hepatocellular carcinoma is administered at a frequency of 1 to 6 times per day, preferably 3 to 4 times per week.

In another preferred embodiment, the inhibitor of the RIT1 gene or protein thereof is administered for a period of 20-90 days, preferably 20-60 days, most preferably 30-40 days.

In another preferred embodiment, the tyrosine kinase inhibitor is administered for a period of 20 to 90 days, preferably 20 to 60 days, most preferably 30 to 40 days.

In another preferred embodiment, the additional agent for preventing and/or treating hepatocellular carcinoma is administered for a period of 20-90 days, preferably 20-60 days, and most preferably 30-40 days.

In another preferred embodiment, the inhibitor of the RIT1 gene or protein thereof is administered simultaneously or sequentially with an optional tyrosine kinase inhibitor, and optionally with other agents for the prevention and/or treatment of hepatocellular carcinoma.

In a tenth aspect, the present invention provides a method of non-therapeutically inhibiting the growth or proliferation of hepatocellular carcinoma cells in vitro, comprising the steps of: culturing the hepatocellular carcinoma cells in the presence of the RIT1 gene or a protein inhibitor thereof, thereby inhibiting the growth or proliferation of the hepatocellular carcinoma cells.

In another preferred embodiment, the hepatocellular carcinoma cell highly expresses a RIT1 protein.

In another preferred embodiment, the method further comprises adding a tyrosine kinase inhibitor to the culture system of hepatocellular carcinoma cells; and/or other agents that prevent and/or treat hepatocellular carcinoma, thereby inhibiting the growth or proliferation of hepatocellular carcinoma cells.

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

In an eleventh aspect, the present invention provides a method for screening candidate compounds for the prevention and/or treatment of hepatocellular carcinoma, the method comprising the steps of:

(a) In a test group, adding a test compound to a culture system of cells, and observing the expression level (E1) and/or activity (A1) of RIT1 in the cells of the test group; in the control group, no test compound was added to the culture system of the same cells, and the expression amount (E0) and/or activity (A0) of RIT1 in the cells of the control group was observed;

Wherein, if the expression amount (E1) and/or activity (A1) of RIT1 of cells in the test group is significantly lower than that of the control group, it is indicated that the test compound is a candidate compound for preventing and/or treating hepatocellular carcinoma having an inhibitory effect on the expression and/or activity of RIT 1.

In another preferred embodiment, the RIT1 expression level is obtained by fluorescent quantitative PCR or immunohistochemical detection.

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

(b) Further testing the candidate compound obtained in step (a) for its inhibitory effect on the growth or proliferation of hepatocellular carcinoma cells; and/or further testing for its effect on down-regulation of the RIT1 gene.

In another preferred embodiment, step (b) includes the steps of: in the test group, a test compound is added into a culture system of the hepatocellular carcinoma cells, and the quantity and/or growth condition of the hepatocellular carcinoma cells are observed; in the control group, no test compound is added in a culture system of the hepatocellular carcinoma cells, and the quantity and/or growth condition of the hepatocellular carcinoma cells are observed; wherein, if the number or growth rate of hepatocellular carcinoma cells in the test group is smaller than that in the control group, it is indicated that the test compound is a candidate compound for preventing and/or treating hepatocellular carcinoma having an inhibitory effect on the growth or proliferation of hepatocellular carcinoma cells.

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

In another preferred embodiment, the mammal is a mammal having hepatocellular carcinoma.

In another preferred embodiment, the term "substantially lower" means E1/E0.ltoreq.1/2, preferably.ltoreq.1/3, more preferably.ltoreq.1/4.

In another preferred embodiment, the term "significantly lower" means A1/A0.ltoreq.1/2, preferably.ltoreq.1/3, more preferably.ltoreq.1/4.

In another preferred embodiment, the cells comprise hepatocellular carcinoma cells.

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

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the expression and clinical significance of RIT1 in hepatocellular carcinoma tissue, wherein 50 in the a TCGA database is for RIT1 expression in hepatocellular carcinoma tissue and paracancestral carcinoma tissue. b expression of RIT1 in hepatocellular carcinoma tissue and paracancerous liver tissue by the present laboratory 90. The TCGA database analyzes the expression of RIT1 in hepatocellular carcinoma tissues of different pathological grades. d analyzing the relationship between RIT1 expression and the overall survival of the hepatocellular carcinoma patient by using a Kaplan-Meier method. e representative image of immunohistochemical staining of RIT1 in hepatocellular carcinoma tissue (left: 40X; right: 400X). * P <0.001.* P <0.0001.

FIG. 2 shows mutations in RIT1 and tissue in a hepatocellular carcinoma cell line, wherein a Western Blot detects RIT1 expression in hepatocellular carcinoma tissue and in paired paracancerous liver tissue. b Western Blot detects RIT1 expression in hepatocellular carcinoma cell lines. Mutation of c RIT1 in hepatocellular carcinoma tissue and leukocytes. dqpcr detects the expression of RIT1 in hepatocellular carcinoma cell lines.

FIG. 3 shows RIT1 overexpression and interference efficiency detection in hepatocellular carcinoma cell lines. Wherein, aqPCR and Western Blot detect expression of RTI1 in over-expressed RIT1 cell lines. bqPCR and Western Blot detection interfere with RTI1 expression in RIT1 cell lines.

FIG. 4 shows that RIT1 promotes the growth of hepatocellular carcinoma cells. Among them, the aCCK 8 method clearly overexpresses the effect of RIT1 on the growth of hepatocellular carcinoma cells. b clone formation experiments the effect of RIT1 on the growth of hepatocellular carcinoma cells was examined. The c CCK8 assay detects the effect of interfering RIT1 on the growth of hepatocellular carcinoma cells. The effect of interfering RIT1 on hepatocellular carcinoma growth was examined in a d-clone formation assay. * P <0.05; * P <0.001.* P <0.0001.

FIG. 5 shows that RIT1 promotes soft agarose clone formation. Among them, a over-expression RIT1 ^E11、RIT1^Q11 and RIT1 Isoform 2 affect the soft agarose clonality of NIH 3T3 and Li-7 cells. Effect of b-knockdown RIT1 on the soft agarose clonogenic capacity of mcc-97L and HCC-LY10 cells. c in situ inoculation of SMMC-7721-pWPXL, SMMC-7721-RIT1 ^Q11, li-7-pWPXL and Li-7-RIT1 ^Q11 cells into tumors (left) and statistical results of tumor-bearing liver weights (right). d Western blot detects RIT1 expression in neoplastic tissue. * P <0.0001.

Figure 6 shows that RIT1 promotes migration and invasion of hepatocellular carcinoma cells in vitro. Wherein, the migration and invasion capacity of a stable over-expression RIT1 to promote the hepatocellular carcinoma cells SMMC-7721, li-7 and Huh7 represent graphs and statistical result graphs. b the ability of stable interference RIT1 to inhibit migration and invasion of hepatocellular carcinoma cell lines HCC-LY10 and MHCC-97L represents a graph and statistical analysis results. c in situ liver tumor and lung metastasis of nude mice inoculated with SMMC-7721-pWPXL and SMMC-7721-RIT1 ^Q11 cells. d Western blot detection of levels of AKT, p-AKT, ERK and p-ERK after overexpression of RIT1 ^E11、RIT1^Q11 and RIT1 Isoform 2 and interference with RIT 1.* P <0.05; * P <0.001.* P <0.0001.

FIG. 7 shows that sorafenib treatment upregulates RIT1 expression in MHCC-97L and HCC-LY10 cells. Wherein the expression of RIT1 in Sorafenib treated MHCC-97L and HCC-LY10 is detected by aqPCR. b Western Blot examined the expression of RIT1 in Sorafenib-treated MHCC-97L and HCC-LY 10.

FIG. 8 shows that interfering RIT1 increases the sensitivity of MHCC-97L and HCC-LY10 to sorafenib. Wherein a detects IC50 of sorafenib after MHCC-97L and HCC-LY10 stably interfere with RIT 1. b CCK8 detects the effect of combined sorafenib treatment and stable interference RIT1 on proliferation of MHCC-97L and HCC-LY 10. c RIT1 interference and corresponding control cells were inoculated subcutaneously into nude mice, and when tumors grew to around 50mm ³, sorafenib (20 mg/kg/d) and corresponding solvents were intraperitoneally injected 5 days per week, and after 20 days the sacrificial mice were weighed and analyzed for tumor taking. d is a representative graph. Tumor volumes were calculated and plotted analytically for 2 tumor diameters per week during e-sorafenib and solvent treatment. * Refer to P <0.05.

Detailed Description

The present inventors have conducted extensive and intensive studies, and for the first time, have unexpectedly found that the expression of the RIT1 gene or its protein in hepatocellular carcinoma cells or tissues is significantly higher than the expression of the RIT1 gene or its protein in normal tissues, and that the expression of RIT1 increases as the pathological grading of hepatocellular carcinoma tissues progresses, and that Kaplan-Meier survival analysis suggests that the survival of hepatocellular carcinoma patients with high RIT1 expression is significantly shorter than that of hepatocellular carcinoma patients with low RIT1 expression, and therefore, the RIT1 gene or its protein can be used as (i) a tissue grade for detecting hepatocellular carcinoma; and/or (ii) detecting a risk of liver metastasis of hepatocellular carcinoma; and/or (iii) as a marker for determining prognosis and survival of a hepatocellular carcinoma patient. Furthermore, the applicant has unexpectedly found that inhibitors of the RIT1 gene or its protein are effective in (a) inhibiting the growth or proliferation of hepatocellular carcinoma cells; and/or (b) inhibiting metastasis of hepatocellular carcinoma cells; and/or (c) preventing and/or treating hepatocellular carcinoma; and/or (d) increase the sensitivity of hepatocellular carcinoma cells to tyrosine kinase inhibitors, and inhibitors of the RIT1 gene or its protein may be used in combination with tyrosine kinase inhibitors, and/or optionally other agents for the prevention and/or treatment of hepatocellular carcinoma, and have a significant synergistic effect on the treatment of hepatocellular carcinoma. On this basis, the present inventors have completed the present invention.

Hepatocellular carcinoma

The incidence of hepatocellular carcinoma is sixth among all tumors, and the new incidence of hepatocellular carcinoma is nearly 80 tens of thousands each year. At present, various treatment means for hepatocellular carcinoma are available, including surgical excision, liver transplantation, tumor ablation, arterial tumor embolism operation, systemic treatment and the like, but patients with middle and late hepatocellular carcinoma have lower benefit from treatment, shorter 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 TNM stage, including stage I, stage II, stage III, and stage IV.

Intrahepatic metastasis

In the present invention, the intrahepatic transfer refers to (1) portal tumor plugs; (2) Tumors on other lobes than the lobe where the primary tumor is located; (3) Tumors surrounding the primary tumor in the same lobe, which have multiple satellite nodules and liver tissue surrounding them; or surrounding micro-isolated tumors that are histologically similar to the primary tumor or differentiate to a lesser extent.

Pulmonary metastasis

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

Sample of

The term "sample" or "specimen" as used herein refers to a material that is specifically associated with a subject from which particular 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 that allows the test performed on the sample to provide information about the subject. The sample may also be a material that has been contacted with another material that is not the subject, but that enables the first material to be subsequently tested to determine information about the subject, e.g., the sample may be a cleaning solution for a probe or scalpel. The sample may be a source of biological material other than that contacting the subject, so long as one skilled 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 gene portion, and includes the production of a protein encoded by RNA or gene portion, and also includes the presence of a detection substance associated with expression. For example, cDNA, binding of a binding ligand (e.g., an antibody) to a gene or other oligonucleotide, protein or protein fragment, and chromogenic portions of the binding ligand are included within the term "expressed". Thus, an increase in half-pel density in immunoblots, such as western blots, is also within the term "expression" based on biological molecules.

Reference value

As used herein, the term "reference value" refers to a value that is statistically relevant to a particular result when compared to the result of an analysis. In a preferred embodiment, the reference value is determined based on statistical analysis of the expression of RIT1 white against studies of known clinical outcomes. Some of these studies are shown in the examples section herein. But the studies from the literature and the user experience of the methods disclosed herein can also be used to produce or adjust the reference value. Reference values may also be determined by considering conditions and results that are 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, which refers to the relative expression level of RIT1 in a hepatocellular carcinoma cell or tissue, preferably 0.043 (fluorescent quantitative PCR) or 3.5 (immunohistochemistry).

Samples of non-hepatocellular carcinoma

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

RIT1 proteins and polynucleotides

In the present invention, the terms "protein of the invention", "RIT1 protein", "RIT1 polypeptide" are used interchangeably and refer to a protein or polypeptide having the amino acid sequence RIT 1. They include RIT1 proteins with or without an initiating methionine. In addition, the term also includes full length RIT1 and fragments thereof. The RIT1 protein comprises the complete amino acid sequence, secreted protein, mutant and functional active fragment.

RIT1 (Ras like without CAAX 1) maps to chromosome 1q22, the gene comprises 6 exons, 4 introns, and the mRNA comprises three subtypes Isoform, isoform 2 and Isoform 3.Isoform 2 is composed of 236 amino acids, has a space structure similar to that of a small G protein, comprises a SWITCH I domain and a SWITCH II domain, and can play a role in signal transduction by adjusting the conformation of the protein to enable the protein to be in an activated state and combined with a downstream effector molecule when the protein is in a GTP binding state; whereas signal transduction ceases when GTP hydrolysis is converted to GDP. RIT1 itself has GPT enzyme activity, can hydrolyze GTP into GDP, and can be combined with new GTP to be activated again after the protein coded by RIT1 releases GDP. Gtpase Activating Proteins (GAPs) inactivate most gtpases, whereas guanylate activators (GEFs) promote reactivation of gtpases by accelerating the conversion of GDP to GTP.

The full length of the human RIT1 protein is 236 amino acids (accession NP-001243750.1). The murine RIT1 protein is 219 amino acids in full length (accession NP-033095.1).

In the present invention, the terms "RIT1 gene", "RIT1 polynucleotide" are used interchangeably and refer to a nucleic acid sequence having a RIT1 nucleotide sequence.

The genome of the human RIT1 gene is 13541bp (NCBI GenBank accession No. NM-001256821.2), and the mRNA sequence of the transcription product is 3324bp (NCBI GenBank accession No. NM-001256821.2).

The genome of the murine RIT1 gene is 14207bp (NCBI GenBank accession No. NM-009069.4) in length and the mRNA sequence of the transcript is 2509bp (NCBI GenBank accession No. NM-009069.4) in length.

Human and murine RIT1 have a DNA level of 85.89% similarity and a protein sequence similarity of 94.98%.

It is understood that substitution of nucleotides in the codon is acceptable when encoding the same amino acid. It is further understood that nucleotide substitutions are also acceptable when conservative amino acid substitutions are made by the nucleotide substitutions.

In the case of obtaining an amino acid fragment of RIT1, a nucleic acid sequence encoding it 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, recombinant methods or artificial synthesis. For the PCR amplification method, primers can be designed based on the RIT1 nucleotide sequence disclosed in the present invention, particularly the open reading frame sequence, and amplified to obtain the relevant sequence using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. 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.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, it is entirely possible to obtain the DNA sequences encoding the proteins of the invention (or fragments, derivatives thereof) by chemical synthesis. The DNA sequence may then be introduced into a variety of existing DNA molecules (e.g., vectors) and cells known in the art.

The polynucleotide sequences of the invention may be used to express or produce recombinant RIT1 polypeptides by conventional recombinant DNA techniques. Generally, there are the following steps:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a human RIT1 polypeptide of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) Host cells cultured in a suitable medium;

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

In the present invention, the RIT1 polynucleotide sequence may be inserted into a recombinant expression vector. In general, any plasmid or vector can be used as long as it replicates 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 translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the RIT1 encoding DNA sequence and appropriate transcriptional/translational 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 an appropriate 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.

In addition, 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 as described above, as well as 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; insect cells; animal cells, and the like.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl ₂ method using procedures well known in the art. Another approach is to use MgCl ₂. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformant obtained 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 culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such 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 (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

Specific antibodies

In the present invention, the terms "antibody of the present invention" and "specific antibody against RIT 1" are used interchangeably.

The invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for a human RIT1 polypeptide. Here, "specific" means that the antibody is capable of binding to a human RIT1 gene product or fragment. Preferably, those antibodies that bind to human RIT1 gene products or fragments but do not recognize and bind to other non-related antigenic molecules. Antibodies of the invention include those molecules that bind to and inhibit the human RIT1 protein, as well as those that do not affect the function of the human RIT1 protein. The invention also includes antibodies that bind to the modified or unmodified form of the human RIT1 gene product.

The invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments such as the Fab' or (Fab) ₂ fragments; 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 having murine antibody binding specificity but retaining antibody portions derived from humans.

Antibodies of the invention may be prepared by various techniques known to those skilled in the art. For example, purified human RIT1 gene products, or antigenic fragments thereof, may be administered to animals to induce polyclonal antibody production. Similarly, cells expressing the human RIT1 protein or an antigenic fragment thereof can be used to immunize animals to produce antibodies. The antibodies of the invention may also be monoclonal antibodies. 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 Hybridomas, elsevier, N.Y., 1981). The antibodies of the present invention include antibodies that block the function of human RIT1 protein and antibodies that do not affect the function of human RIT1 protein. The various antibodies of the invention can be obtained by conventional immunization techniques using fragments or functional regions of the human RIT1 gene product. These fragments or functional regions may be prepared by recombinant methods or synthesized by a polypeptide synthesizer. Antibodies that bind to unmodified forms of the human RIT1 gene product may be produced by immunizing an animal with the gene product produced in a prokaryotic cell (e.g., e.coli); antibodies (e.g., glycosylated or phosphorylated proteins or polypeptides) that bind to post-translational modifications 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 RIT1 protein are useful in immunohistochemical techniques to detect the presence of human RIT1 protein in a sample, particularly a tissue sample or serum sample. Since the RIT1 protein has an extracellular region, these soluble RIT1 extracellular regions can be targets for serum detection when the extracellular region is detached and enters the blood.

Detection method

The invention also provides a method for detecting the hepatocellular carcinoma by utilizing the characteristic that RIT1 exists in hepatocellular carcinoma cells or tissues and is closely related to the risk of the hepatocellular carcinoma.

In a preferred embodiment of the invention, the invention provides a high throughput second generation sequencing method for detecting RIT1, and Sanger sequencing, fluorescent quantitative PCR (qPCR), in situ immunofluorescence (FISH), immunohistochemistry and the like.

Detection kit

RIT1 is a diagnostic marker for hepatocellular carcinoma because of its correlation with hepatocellular carcinoma, i.e., the presence of RIT1 in hepatocellular carcinoma tissue.

The invention also provides (i) tissue classification for detecting hepatocellular carcinoma; and/or (ii) detecting a risk of liver metastasis of hepatocellular carcinoma; and/or (iii) a kit for determining prognosis and survival of a patient with hepatocellular carcinoma comprising a detection reagent for detecting RIT1 gene, mRNA, cDNA, or protein; and a label or instruction stating that the kit is for use in detecting (i) tissue classification for detecting hepatocellular carcinoma; and/or (ii) detecting a risk of liver metastasis of hepatocellular carcinoma; and/or (iii) determining prognosis and survival of a hepatocellular carcinoma patient.

Wherein the label or the instruction notes the following:

Detection method and kit

The present invention relates to diagnostic assays for the quantitative and positional detection of human RIT1 protein levels or mRNA levels. Such tests are well known in the art. The levels of human RIT1 protein detected in the assay can be used for diagnosis (including assisted diagnosis) of tissue classification of hepatocellular carcinoma; and/or a risk of metastasis of hepatocellular carcinoma; and/or prognosis and survival of hepatocellular carcinoma patients.

One method of detecting the presence or absence of RIT1 protein in a sample is by using an antibody specific for RIT1 protein, which comprises: contacting the sample with an antibody specific for a RIT1 protein; observing whether an antibody complex is formed, the formation of which indicates the presence of RIT1 protein in the sample.

The RIT1 protein or its polynucleotide can be used for diagnosing and treating RIT1 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 diagnosis of genes. Antibodies against RIT1 may be immobilized on a protein chip for detection of RIT1 protein in a sample.

The main advantages of the invention include:

(1) The invention discovers for the first time that the expression of RIT1 gene or protein thereof in hepatocellular carcinoma cells or tissues is obviously higher than that of RIT1 gene or protein thereof in normal tissues, the expression of RIT1 can be increased along with pathological grading progress of the hepatocellular carcinoma tissues, and Kaplan-Meier survival analysis indicates that the survival of hepatocellular carcinoma patients with high RIT1 expression is obviously shorter than that of hepatocellular carcinoma patients with low RIT1 expression, so that RIT1 can be used as (i) the tissue grading for detecting hepatocellular carcinoma; and/or (ii) detecting a risk of liver metastasis of hepatocellular carcinoma; and/or (iii) as a marker for determining prognosis and survival of a hepatocellular carcinoma patient.

(2) The invention is found for the first time. Inhibitors of the RIT1 gene or its protein are effective in (a) inhibiting the growth or proliferation of hepatocellular carcinoma cells; and/or (b) inhibiting metastasis of hepatocellular carcinoma cells; and/or (c) preventing and/or treating hepatocellular carcinoma; and/or (d) increasing sensitivity of the hepatocellular carcinoma cell to a tyrosine kinase inhibitor.

(3) The present invention has found for the first time that inhibitors of the RIT1 gene or its protein can be used in combination with tyrosine kinase inhibitors, and/or optionally other drugs for preventing and/or treating hepatocellular carcinoma, and have a remarkable synergistic effect on the treatment of hepatocellular carcinoma.

(4) The invention discovers for the first time that the high expression of RIT1 in a tissue sample of a hepatocellular carcinoma patient indicates that the patient is in middle and late stages and has poor prognosis.

(5) The invention discovers for the first time that RIT1 promotes the growth of hepatocellular carcinoma cells in vivo and in vitro.

(6) The invention discovers for the first time that RIT1 promotes the metastatic potential of hepatocellular carcinoma cells.

(7) The invention discovers for the first time that the tyrosine kinase inhibitor (such as sorafenib) can up-regulate RIT1 expression when treating hepatocellular carcinoma cells (hepatocellular carcinoma cells)

(8) The invention discovers for the first time that the interference RIT1 can improve the sensitivity of hepatocellular carcinoma cells to sorafenib.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

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

General method

1. Cell culture

The human hepatocellular carcinoma cell lines and normal hepatocellular carcinoma cell lines used in the study were placed in an incubator at 37℃under 5% CO ₂ and saturated with humidity, and the cell culture broth contained 10% fetal bovine serum, 100 IU/mL penicillin and 100. Mu.g/mL streptomycin. Nunc polystyrene plates and multi-well plates were purchased from Thermo Scientific.

RNA extraction

1) When the cell density in a cell culture dish with the diameter of 6cm is 90%, the upper layer culture medium is discarded, 1mL TRIzol is added for repeated blowing, then 1mL to 1.5mL centrifuge tubes are sucked, and the culture medium is kept stand for 5min.

2) 0.2ML of chloroform was added thereto, the mixture was vigorously shaken for 15 seconds, and the mixture was allowed to stand at room temperature for 2 to 3 minutes. Centrifuging in a centrifuge at 4deg.C under 12000g for 15min. The liquid is divided into three layers: the lower layer is protein organic phase, the middle layer is DNA solid phase, and the upper layer is RNA aqueous phase.

3) The supernatant was aspirated and transferred to a new 1.5mL centrifuge tube, 0.5mL isopropanol was added, gently blown and mixed, allowed to stand at room temperature for 10min, centrifuged in a centrifuge at 12000g at 4℃for 10min, and RNA was precipitated to the bottom of the EP tube.

4) The supernatant was aspirated, 0.5mL of 75% ethanol was added, and RNA pellet was gently swirled. The RNA was deposited onto the bottom of the EP tube by centrifugation in a centrifuge at 7500g at 4℃for 5 min.

5) The supernatant was removed by pipetting and the RNA pellet was dried at room temperature. 20. Mu.L to 50. Mu. L Diethy Pyrocarbonate (DEPC) of water was added and the mixture was gently blown to dissolve (stored at-80 ℃). RNA concentration was then measured using a Nano Drop 2000.

3. Reverse transcription

The 20. Mu.L system of reverse transcription kit PRIME SCRIPT ^TM RT REAGENT KIT (PERFECT REAL TIME) is as follows:

the reaction conditions are as follows: 37 ℃ for 15min;

85℃，5s；

4℃，……

4. Fluorescent quantitative PCR

The reverse transcribed cDNA was diluted 1:50 with ddH ₂ O and the 20. Mu.L system for the fluorescent quantitative PCR assay was as follows:

Each sample was run in parallel in a 96-well plate with 3 wells and PCR reactions were performed on an ABI 7500 fluorescent quantitative PCR instrument. The PCR reaction conditions were 95℃for 30s, then 95℃for 5s and 60℃for 30s for 40 cycles. GAPDH (GLYCERALDEHYDE-3-phosphate dehydrogenase) was used as a reference.

5. Protein extraction

1) When the cells were grown to 90% density, the culture medium in the dish was discarded, washed twice with pre-chilled 1 XPBS, and after the residual liquid was aspirated, an appropriate amount of protein lysate (1 mL of protein lysate including 880. Mu. L T-PER tissue protein extraction reagent, 20. Mu.L of protease inhibitor, and 100. Mu.L of phosphatase inhibitor) was added and the cells were scraped with a spatula.

2) An appropriate amount of cell lysate was pipetted into a 1.5mL centrifuge tube and lysed in an ice bath for 40min.

3) High speed centrifugation at 12000g for 15min at 4℃carefully aspirate the supernatant to a new 1.5mL centrifuge tube.

4) Protein quantification: mu.L of sample or 5. Mu.L of PBS (blank), 200. Mu.L of BCA and 20. Mu.L of 1 XPBS mix (BCA: cuSO ₄ = 200:4) were pipetted and the metal bath was incubated at 37℃for 30min. And measuring absorbance at 570nm wavelength by using a multifunctional enzyme-labeled instrument, and calculating the protein concentration according to a standard concentration curve.

5) Adding 5×loading buffer and 10×DTT into the rest supernatant, gently blowing, standing in boiling water at 100deg.C for 10min, immediately cooling in ice bath for 10min, and storing at-20deg.C (long-term storage is at-80deg.C).

6. Construction of pWPXL-RIT1 overexpression plasmid

1) And (2) PCR: the CDS region of RIT1 ^E11/RIT1^Q11/RIT 1 Isoform was amplified by PCR according to the cloning primers for the RIT1 ^E11/RIT1^Q11/RIT 1 Isoform gene described above (introduction of MluI/NdeI cleavage site).

2) Agarose gel electrophoresis: preparing 1% agarose gel and 0.5 XTBE electrophoresis buffer; heating with a microwave oven to melt agarose, cooling to room temperature, adding ethidium bromide, shaking gently, and pouring; pulling out the comb, putting the gel into TBE buffer solution, and carrying out electrophoresis under 140-160V voltage after sample loading; the gel strips of the PCR products at the target sites were excised under UV light, mashed and placed in a 1.5mL centrifuge tube.

3) Purification recovery, cleavage and ligation: according to the DNA purification recovery kit instruction of Tiangen biochemical technology Co., ltd, performing a purified PCR amplification product test; double-enzyme digestion is carried out on the pWPXL and the DNA purification recovery product by MluI/NdeI according to an enzyme digestion system, and after enzyme digestion is completed for 12 hours, the DNA purification recovery kit is used according to the same method to carry out enzyme digestion product purification recovery; the digested pWPXL was then ligated to the target DNA in a metal bath at 22℃for 3h, and left to cool at room temperature.

4) Conversion: mixing 10 mu L of the connection product and 50 mu L of DH5 alpha competent bacteria uniformly, ice-bathing for 30min, putting the tube into a water bath kettle at 42 ℃, after 90s of shaking-free (strict control time is noted), rapidly putting the tube on ice to cool for 2-3 min, adding a proper amount of SOC culture medium into the mixed solution after heat shock, shaking the tube for 40min at 37 ℃, uniformly smearing the obtained mixed solution on an Ampicillin (AMP) -resistant LB plate, inverting the plate, and culturing the tube in an incubator at 37 ℃ for overnight (12-16 h).

5) Selecting monoclonal: individual colonies grown in LB plates were picked in 5mL of ampicillin-containing LB solution and shaken at 37℃for 12-16 h at 250 rpm.

6) Plasmid extraction: plasmids were extracted according to the instructions of plasmid minipump kit from Tiangen Biochemical Co., ltd, and the concentration of the extracted plasmids was measured using Nano Drop. And finally, carrying out double digestion for 3 hours by using MluI/NdeI, identifying the digested products by using agarose gel electrophoresis, and sending plasmids with correct enzyme digestion identification to Shanghai Sanny biotechnology Co., ltd for sequencing, and retaining corresponding strains for later use after the sequencing is correct.

7. Protein electrophoresis and Western blot assay

1) And (3) glue preparation: preparing 12% separating glue according to the above formula, mixing, and rapidly adding the glue into the glass plate to a height of about 1.5cm from the upper edge of the thin glass plate. Immediately adding a proper amount of distilled water, and standing at room temperature for 30min; after the separation gel is solidified, sucking residual distilled water by using clean filter paper, preparing 5% concentrated gel according to the above, mixing uniformly, quickly adding into a glass plate, inserting a comb with 10 teeth or 15 teeth, taking care to prevent bubble generation, and after 20-30 min, finishing the upper layer gelation and solidification for later use.

2) Loading: the extracted protein samples were taken and protein markers and protein samples (typically 30. Mu.g loading was made up to an equal volume per well with a1 Xloading buffer) were added to the wells using a pipette and a loading gun head.

3) Electrophoresis: constant pressure 80V 0.5h and constant pressure 120V1h (corresponding adjustment according to the target molecular weight), the blue line moves to the bottom end of the glass plate, the end of electrophoresis is prompted, and the power supply is turned off.

4) Transferring: the transfer buffer solution is prepared in advance according to the formula and stored at 4 ℃; after the film transfer device is assembled, the transfer tank is filled with transfer liquid, ice cubes are put into the transfer tank, the transfer tank is placed into a foam box filled with ice, a power supply is turned on, and film transfer is carried out at constant current of 220mA for 65 min. ( When the film rotating clamp is assembled, bubbles cannot be necessarily formed among contact surfaces of the filter paper, the gel and the film; PVDF membrane is soaked in methanol for 1min before use )

5) Antibody hybridization: sealing the transferred membrane in 5% skimmed milk, and incubating for 1h at room temperature; discarding skimmed milk, diluting the primary antibody to a certain proportion with a sealing solution, and incubating at 4 ℃ overnight (12 h); the membranes were washed 3 times with PBST (PBS pH7.4, 0.1% Tween 20) for 10min each; diluting horseradish peroxidase-labeled secondary antibody to a certain concentration (attention is paid to distinguishing species) by using 5% skimmed milk, and incubating for 0.5-1 h at room temperature; the membranes were washed 3 times, 10min each, using PBST (PBS pH7.4, 0.1% Tween 20).

6) Chemiluminescent detection: super Signal chemiluminescent substrates were dropped onto the membrane, developed using a chemiluminescent fluorescence imager and the bands analyzed. All Western blot experiments were referenced to beta-actin.

8. Construction of GV248-shRIT1 plasmid

Plasmids containing shRIT.sup.1 fragment were synthesized by Shanghai Ji Kai gene. The sequence of the targeted fragment is shown in Table 5:

TABLE 5 interference of RIT1 sequences

Table 5 Sequences used to silence RIT1

9. Virus package

1) HEK 293T is inoculated into a 10cm cell culture dish, cultured overnight and transfected when the cells grow to about 80% of fusion degree;

2) Preparing jetPRIME liposome and plasmid transfection solution:

The mixture of plasmid and jet PRIME was added to 0.5mL serum-free medium, mixed well and left at room temperature for 10min, the transfection mixture was slowly added to a 10cm 293T cell culture dish, and the cell culture medium was added to a final volume of 10mL and incubated in a 37 ℃ incubator for 48h.

3) Then observing green fluorescence intensity and area of the cells by using a fluorescence microscope, photographing and judging transfection efficiency, collecting culture solution supernatant, filtering (0.45 μm) by using a microporous filter, sub-packaging and storing at-80 ℃ for later use.

10. Lentiviral infection

Human hepatocellular carcinoma cells were inoculated into 46 cm cell culture plates on day 1, and after the cells had adhered to the wall and grown to about 40% -50% confluence, the cell culture medium and virus liquid were mixed according to a ratio of 1:3, adding the mixture into a culture dish, simultaneously adding 1%polybrene (Sigma-Aldrich) with a final volume of 5mL, culturing for 6-8 hours, changing the liquid, adding fresh cell culture medium, culturing in a constant temperature box at 37 ℃ for 48 hours, and observing the green fluorescence intensity and percentage of cells in a control group and an experimental group under a fluorescence microscope to preliminarily judge the infection efficiency.

11. Cloning formation experiments

1) The hepatocellular carcinoma cells are counted after being digested by pancreatin, then about 1000-3000 cells are inoculated in each hole in a 6-hole cell culture plate, three holes are repeated in parallel for each sample, the culture solution is added to 2mL in each hole, and the cells are uniformly dispersed by cross shaking.

2) The 6-well plate is placed in a constant temperature incubator at 37 ℃ for culture, when the single clone grows to about 50 cells at maximum and is visible to naked eyes, the culture is stopped, and the culture solution is discarded.

3) Wash 2 times with 1×pbs, fix with 10% neutral formalin for 30min, giemsa stain for 30min, rinse with tap water, air dry. The scanner scans and generates images, counts the number of clones and makes statistical analysis.

12. Cell migration and invasion experiments

1) Coating a substrate film

Pre-cooling the serum-free medium, and then performing a Matrigel: serum-free medium (V/V) =1: 9 ratio dilution, coating the diluted Matrigel on the upper chamber surface of the microporous membrane at the bottom of the Transwell chamber, and air-drying at room temperature (migration experiment does not need to carry out the step).

2) Hepatocellular carcinoma cell count and resuspension

Cells were routinely digested, after termination of digestion, cell pellet was collected by centrifugation, washed 3 times with serum-free medium, resuspended and counted in serum-free medium, cell suspension containing 1X 10 ⁵ cells was added to the upper chamber of the Transwell chamber, supplemented to 200. Mu.L with serum-free medium, 600. Mu.L of medium containing fetal bovine serum was added to the lower chamber of the 24-well plate, and incubated in an incubator at 37℃for 24-48 h.

3) Fixation and statistics

According to the characteristics of different cells, taking out the transwell chamber after a certain time, discarding the culture solution of the upper chamber of the chamber, washing twice with 1 XPBS, fixing with 10% neutral formalin solution for 30min, and staining with crystal violet for 15min. And lightly wiping the upper cells in the inner chamber of the Transwell chamber by using a cotton swab, airing, and taking down and fixing the microporous film at the bottom of the chamber on a glass slide by using a blade. Dried at room temperature and then observed under a microscope. 6 fields were randomly selected for taking photographs and counted for statistical analysis.

13. Cell proliferation assay (CCK 8 assay)

1) Hepatocellular carcinoma cells in the cell dish were digested with pancreatin, counted, seeded at about 800-3000 cells per well in a 96-well cell culture plate, shaken well with a shaker, and repeated in triplicate for each sample.

2) After the inoculated 96-well cells are placed in a 37 ℃ incubator for culturing for 24 hours, fresh cell culture medium is added by changing liquid (the action is required to be gentle when changing liquid, and the cell loss caused by improper operation is avoided), 10 mu L of CCK8 solution is added to each well, the incubation is carried out for 2 hours in the 37 ℃ constant temperature incubator, and then the value of absorbance A450nm is measured by using an enzyme-labeled instrument.

3) The CCK8 assay was run for 7 days, and the values were recorded and analyzed.

14. Hepatocellular carcinoma tissue microarray

236 Cases of liver cell cancer tissues and corresponding paracancestral liver tissue specimens used in the research institute are all from the research institute for prevention and treatment of liver cancer in the open. After fixation with 4% neutral buffered formalin and paraffin embedding for 72h, donor blocks were prepared, and then selected sites in the donor blocks were sampled and embedded into the well-perforated microarray blocks. Each tissue core on the microarray wax block has a diameter of about 1.5mm, is well-arranged, and has substantially no displacement spots. Immunohistochemical staining intensities (3: tan; 2: tan; 1: pale yellow; 0: colorless) were considered comprehensively and the immunohistochemical scores were calculated as 0,1,2,3 and 4 by the percentage of positive cells (4: >75%;3: 50-75%; 2: 25-50%; 1: 10-25%; 0: < 10%). All sample and patient data collection procedures were performed with informed consent of the patient and were approved by the ethical review board of the research institute for prevention and treatment of liver cancer at the open-east people hospital.

15. In-situ inoculation experiment of nude mouse liver

SMMC-7721-pWPXL and SMMC-7721-RIT1 ^Q11 cells in a 10cm dish with good growth state are digested with pancreatin, after counting, 1X 10 ⁶ cells are resuspended in 20. Mu.L serum-free DMEM and 40. Mu.L of mixed solution is prepared with Matrigel 1:1, and the mixed solution is evenly mixed on ice (without bubbles); 20 nude mice of 6-8 weeks of age were randomly divided into experimental and control groups of 10 cells each, and cells were inoculated into left liver lobes of the nude mice. Mice are bred in SPF-class animal houses after inoculation, the mice are sacrificed after about 42 days without pain, liver and lung tissues of the mice are collected, pathological histology experiments are carried out, and the liver weight, intrahepatic tumor metastasis and lung metastasis conditions of nude mice in experimental groups and control groups are counted respectively. In situ experiments of Li-7-pWPXL and Li-7-RIT1 ^Q11 in nude mice were performed as described above.

Balb/c nude mice used in this study were supplied by the laboratory animal platform of this study, and all animal experiments were performed in SPF (Specific pathogen Free) animal laboratories. All animal experiments were reviewed and approved by the animal ethics committee of the tumor institute, shanghai.

16. Sorafenib treatment of hepatocellular carcinoma cells

When the cell fusion degree reaches 60%, adding a proper amount of sorafenib into each cell of the hepatocellular carcinoma, respectively processing for 24 hours and 48 hours at the final concentrations of 0, 3, 6 and 9 mu M, and respectively collecting protein and RNA for detection;

17. Sorafenib IC50 in hepatocellular carcinoma cells (Half maximal inhibitory concentration)

HCC-LY10 and mhc-97L cells stably interfering with RIT1 were seeded in 96-well plates, 3000 cells per well, and appropriate amount of sorafenib was added to the wells 48h after seeding to a maximum concentration of 40 μm, and a double dilution was performed, followed by CCK8 measurement for 48h, and an IC50 curve was drawn, noting that 3 wells were required for each concentration in parallel.

Example 1 high expression of RIT1 in tissue samples from patients with hepatocellular carcinoma suggests that the patient is at middle-late stage and has poor prognosis

Analysis of 50 differential expression of RIT1mRNA levels in hepatocellular carcinoma tissue and its paired paracancerous liver tissue using the TCGA database revealed that RIT1mRNA was significantly more expressed in hepatocellular carcinoma tissue than in paired paracancerous liver tissue (FIG. 1 a). The qPCR detection results of 90 pairs of liver cell cancer tissues and liver tissues beside the paired cancers collected by the invention are consistent with the TCGA database, and the mRNA expression level of RIT1 in the liver cell cancer tissues is obviously increased (figure 1 b). Further analysis of the TCGA database found that the expression of RIT1 increased with progression of pathological grading of hepatocellular carcinoma tissue (fig. 1 c). Analysis of Kaplan-Meier survival suggests that RIT1 high expressing hepatocellular carcinoma patients have significantly shorter survival than RIT1 low expressing hepatocellular carcinoma patients (FIG. 1 d)

To further analyze the clinical significance of RIT1 expression in hepatocellular carcinoma, we examined RIT1 expression in 236 hepatocellular carcinoma tissues using immunohistochemical methods, scoring RIT1 expression as 0,1,2,3 and 4 based on immunohistochemical staining intensity and positive cell percentage, with scores of 0,1 and 2 as the RIT1 low expression group (103, 43.64%) and scores of 3 and 4 as the RIT1 high expression group (133, 56.36%) (FIG. 1 e). Analysis of the correlation of RIT1 expression in hepatocellular carcinoma tissue with patient clinical pathology, including age, sex, serum Alpha Fetoprotein (AFP), hepatitis B Virus (HBV) infection status, tumor size, histological grade, whether concomitant intrahepatic transfer and whether concomitant cirrhosis, revealed that RIT1 expression levels were significantly positively correlated with patient tissue grade, whether concomitant intrahepatic transfer (Table 1). In conclusion, the expression of RIT1 in the liver cell cancer tissue is obviously increased, and is positively correlated with the tissue classification of liver cell cancer patients, whether liver transfer occurs or not, and the high expression of RIT1 indicates that the liver cell cancer patients have poor prognosis and short survival time.

TABLE 1 relationship between RIT1 expression levels and clinical pathological characteristics of hepatocellular carcinoma

The P-value represents the probability of χ ² test from the RIT1 expression level between the variable subgroups. AFP, alpha fetoprotein. * Represents P <0.05.

EXAMPLE 2 RIT1 promotes the growth of hepatocellular carcinoma cells in vivo and in vitro

RIT1 has 3 shear variants: isoform 1, isoform2 and Isoform 3, wherein Isoform2 is 18 amino acids more than Ras at the N-terminus, isoform 1 is 17 amino acids more than Isoform2 at the N-terminus comprising an exon, isoform 3 initiates the translation initiation site downstream of the G1 domain, and therefore lacks a critical nucleotide binding site. The existing studies were based mainly on the variant at Isoform2, whereas RIT1 Isoform 1 and Isoform2 were expressed in hepatocellular carcinoma cell lines and tissues according to our Western Blot results (FIGS. 2, a and b). The TCGA database was used to analyze the mutation of RIT1 in hepatocellular carcinoma tissue (http:// www.cbioportal.org), and no mutation was found in Isoform and no previously reported E81Q mutation was found. Since TCGA database only provided RIT1 Isoform data, we detected Isoform mutations in 21 hepatocellular carcinoma tissues collected in this laboratory, the sequence was normalized to the sequence of RIT1 isosporm 1 in white blood cells of 4 healthy persons, and as a result, it was found that there were mutations in Q11E in 5 hepatocellular carcinoma tissues relative to the sequence of RIT1 isosporm 1 in healthy persons (fig. 2 c). The expression level of RIT1 in hepatocellular carcinoma cell lines was examined using qPCR and Western Blot, and the results showed that RIT1 was expressed higher in hepatocellular carcinoma cell lines MHCC-97H, MHCC-LM3, MHCC-97L, moderately expressed in hepatocellular carcinoma cell lines HCC-LY10 and HepG2, and lower in hepatocellular carcinoma cell lines Li-7, MHCC-7721, huh7 (FIGS. 2, b and d).

To study the function of RIT1 in hepatocellular carcinoma cells, stable overexpressing cell lines were established in the RIT1 overexpressing RIT1 ^E11,RIT1^Q11 and RIT1 isoport 2 in the RIT1 overexpressing hepatocellular carcinoma cell lines SMMC-7721, li-7 and Huh 7; the expression of RIT1 is interfered by a liver cell cancer cell line MHCC-97L with high RIT1 expression and a liver cell cancer cell HCC-LY10 with moderate expression, and a RIT1 stable interference cell line is established. Overexpression and interference efficiency of RIT1 was detected using qPCR and Western Blot (FIGS. 3, a and b).

The results of the clone formation experiment and the CCK8 experiment show that the over-expression of RIT1 ^E11、RIT1^Q11 and RIT1Isoform can obviously promote the proliferation of the hepatocellular carcinoma cell line in vitro, and no obvious difference exists between the three (figures 4, a and b). Interfering with endogenous RIT1 expression significantly inhibited the growth of hepatocellular carcinoma cells in vitro (fig. 4, c and d). Previously reported in literature that RIT1is capable of transforming NIH3T3 cells, we performed soft agarose clone formation experiments on NIH3T3 and Li-7 cells that overexpress RIT1 ^E11、RIT1^Q11 and RIT1Isoform 2, and found that RIT1 ^E11、RIT1^Q11 and RIT1Isoform both promote the formation of soft agarose clones of NIH3T3 and Li-7 cells; in contrast, the knock-down of RIT1 in MHCC-97L and HCC-LY10 inhibited the soft agar clonality (FIG. 5, a-b). To further clarify the function of RIT1 in vivo nodulation in nude mice with hepatocellular carcinoma cells, we vaccinated in situ with hepatocellular carcinoma cell lines overexpressing RIT1 ^Q11 and the corresponding controls, sacrificed mice painlessly after about 6 weeks, tumor-bearing liver tissues were removed and weighed, and the results showed that over-expression of RIT1 ^Q11 significantly promoted proliferation of SMMC-7721 and Li-7 cells compared to the corresponding control group (fig. 5, c-d).

Example 3 RIT1 promotes metastatic potential of hepatocellular carcinoma cells

As previously mentioned, the expression level of RIT1 is closely related to whether or not the patient has developed intrahepatic metastasis, and it has also been reported that over-expression of RIT1 can promote cell migration and invasion. In vitro and in vivo experiments examined the effect of RIT1 on the metastatic potential of hepatocellular carcinoma. The results indicate that overexpression of both RIT1 ^E11、RIT1^Q11 and RIT1 Isoform 2 significantly promoted migration and invasiveness of the hepatocellular carcinoma cell lines SMMC-7721, li-7, and Huh7 (FIG. 6 a). Interfering with RIT1 expression significantly inhibited migration and invasion capacity of hepatocellular carcinoma cell lines HCC-LY10 and mhc c-97L (fig. 6 b). Liver was inoculated in situ with a hepatocellular carcinoma cell line overexpressing RIT1 ^Q11 and the corresponding control, mice were sacrificed painlessly after about 6 weeks and liver and lung tissues were harvested, serial sections and HE staining were performed, and the cases of lung metastasis in nude mice of experimental and control groups were observed (fig. 6 c). The results showed that in SMMC-7721 cells, lung metastasis occurred in1 nude mice (10%) in the control group and 6 nude mice (60%) in the over-expressed RIT1 isosporm 1 (RIT 1 ^Q11) group. In conclusion, RIT1 can significantly promote in vitro migration invasion of hepatocellular carcinoma cells and lung metastasis in nude mice.

Like other Ras family members, RIT1 transduces cellular signals to specific effector molecules, activating different signaling pathways, including MAPK and PI3K/AKT pathways, and Western Blot detection of over-expressed RIT1 and interfering with the expression of RIT1 cell lines p-ERK and p-AKT (FIG. 6 d), indicating that RIT1 is capable of increasing p-ERK and p-AKT levels.

Example 4 Sorafenib treatment of hepatocellular carcinoma cells capable of up-regulating RIT1 expression

Sorafenib is a multiple kinase inhibitor, a first-line drug in the treatment of hepatocellular carcinoma, which can extend the survival of patients. After treatment of MHCC-97L and HCC-LY10 cells with different concentrations of 0, 3,6, 9. Mu.M for 24h and 48h, qPCR and western blot examined RIT1 expression, which indicated that after sorafenib treatment, RIT1 mRNA and protein levels were significantly increased and time and concentration dependent (FIGS. 7,a and b). RIT1, like other Ras family members, can transduce membrane signals to specific effector molecules, activating different signaling pathways, including the MAPK signaling pathway and the PI3K/AKT signaling pathway. RIT1 is able to up-regulate the levels of p-ERK and p-AKT as previously described. Whether RIT1 affects sorafenib drug sensitivity by modulating MAPK signaling pathways and PI3K/AKT signaling pathways remains to be studied further. In conclusion, RIT1 mRNA and protein levels were significantly elevated after Sorafenib treatment of hepatocellular carcinoma cells.

Example 5 interference of RIT1 increases sensitivity of hepatocellular carcinoma cells to sorafenib

To determine if RIT1 is associated with sorafenib resistance in hepatocellular carcinoma cells, the IC50 of sorafenib was tested in MHCC-97L and HCC-LY10 cells (FIG. 8 a). As can be seen from the results, both MHCC-97L and HCC-LY10 have an IC50 of more than 10. Mu.M, indicating that both cells are not sensitive to sorafenib by themselves. However, when RIT1 was knocked down, the IC50 was significantly reduced. CCK8 experiments showed that sorafenib (3. Mu.M) in combination with interfering RIT1 inhibited proliferation capacity of MHCC-97L and HCC-LY10 cells more significantly than sorafenib alone (3. Mu.M) or interfering RIT1 alone (FIG. 8 b). RIT1 interference and corresponding control hepatocellular carcinoma cells were inoculated subcutaneously into nude mice, and when tumors grew to about 50mm ³ days per week, sorafenib (20 mg/kg/d) and corresponding solvents were intraperitoneally injected 5 days per week, tumor volumes were calculated 2 times per week, and after 20 days the mice were sacrificed for tumor weighing and analysis. The results show that interfering RIT1 can significantly improve the tumor growth inhibiting effect of sorafenib (FIG. 8,c-e). In conclusion, interfering with the expression of RIT1 may increase the sensitivity of hepatocellular carcinoma cells to sorafenib.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

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Claims

1. Use of a reagent for the detection of a RIT1 gene, mRNA, cDNA, or protein for the preparation of a diagnostic reagent or kit for the detection of the risk of hepatocellular carcinoma metastasis, including intrahepatic and pulmonary metastasis.

2. Use of an inhibitor of the RIT1 gene for the preparation of a composition or formulation for (b) inhibiting metastasis of hepatocellular carcinoma cells; and/or (d) increasing sensitivity of the hepatocellular carcinoma cell to a tyrosine kinase inhibitor;

and the metastasis includes intrahepatic metastasis and pulmonary metastasis, the inhibitor is an interfering RIT1 sequence selected from SEQ ID nos. 2 and 3.

3. The use according to claim 2, wherein the composition or formulation is further for reducing the level of P-ERK and/or P-AKT.

4. A pharmaceutical composition comprising:

(a1) An inhibitor of the RIT1 gene, said inhibitor being an inhibitor of the RIT1 sequence selected from SEQ ID NO.2 and 3;

(a2) Tyrosine kinase inhibitors; and

(B) A pharmaceutically acceptable carrier.

5. A kit, comprising:

(a1) A first container, and an inhibitor of the RIT1 gene or a medicament containing an inhibitor of the RIT1 gene located in said first container, said inhibitor being a polypeptide that interferes with the RIT1 sequence selected from the group consisting of SEQ ID nos. 2 and 3;

(b1) A second container, and a tyrosine kinase inhibitor, or a medicament containing a tyrosine kinase inhibitor, located in the second container.

6. The kit of claim 2, further comprising (d 1) a fourth container, and a detection reagent for RIT1 located in the fourth container.

7. Use of the pharmaceutical composition of claim 3 or the kit of claim 5 for the preparation of a medicament for inhibiting metastasis of hepatocellular carcinoma cells;

and the metastasis includes intrahepatic metastasis and pulmonary metastasis.