A biomarker related to liver cancer and its application

Abstract

The present invention relates to the field of molecular biology, and in particular to a biomarker related to liver cancer and its application. The present invention first discovered that the expression of CYSTM1 gene is upregulated in liver cancer, and the total survival of liver cancer patients with high expression of CYSTM1 gene is significantly reduced, suggesting that detecting the expression level of CYSTM1 gene can become one of the auxiliary diagnostic indicators for liver cancer diagnosis, and high expression of CYSTM1 gene has high accuracy as a liver cancer diagnostic factor. Moreover, the present invention also discovered that inhibiting the expression of CYSTM1 gene can effectively inhibit the proliferation and cloning of liver cancer, and overexpressing CYSTM1 gene can significantly enhance the proliferation and cloning of liver cancer. Therefore, interfering with the expression of CYSTM1 gene can become a new way to prevent or treat liver cancer, providing a new idea for the development of related drugs and clinical treatment in the later stage.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a biomarker associated with liver cancer and application thereof. Background Art

[0002] The incidence of liver cancer is on the rise, and it is also one of the most common malignant tumors in my country. Liver cancer is usually divided into two categories: primary liver cancer and secondary liver cancer. Primary liver cancer mainly includes hepatocellular carcinoma, intrahepatic bile duct carcinoma and mixed liver cancer. Hepatocellular carcinoma accounts for more than 85%-90%, and the incidence and mortality rate rank fourth and second among cancers, respectively, which seriously threatens human health. In the early stage of liver cancer, local treatments such as surgical resection, radiofrequency ablation, transarterial chemoembolization or liver transplantation can be used. However, more than 80% of liver cancer patients are in the middle and late stages when they are diagnosed, and the best time for surgical treatment is lost, and the five-year survival rate is only 15%. Although the systemic treatment of advanced liver cancer with multi-target tyrosine kinase inhibitors such as soratinib, lenvatinib, regorafenib and ramucirumab has improved the survival rate to a certain extent, its total survival period is only about 10 months. Therefore, finding new and effective biomarkers is of great significance for the realization of individualized and precise diagnosis and treatment of hepatocellular carcinoma.

[0003] Although biomarkers of liver cancer, such as angiopoietin 2, vascular endothelial growth factor, Golgi protein 73, insulin growth factor 1 and hepatocyte growth factor receptor, were proposed in early studies, their application in the diagnosis and treatment of liver cancer still needs further research and discussion.

[0004] CYSTM1 protein, also known as C5orf32, is a leucine-rich transmembrane protein. The CYSTM1 gene is a relatively unknown gene. Current studies have found that it mainly plays a certain role in stress response and has a certain relationship with the occurrence of Huntington's disease. However, the function of the CYSTM1 gene in tumors has not been reported yet. Summary of the invention

[0005] The purpose of the present invention is to provide a biomarker related to liver cancer and its application to solve the problems existing in the above-mentioned prior art. The CYSTM1 gene of the present invention is helpful for effective diagnosis of early-stage liver cancer patients.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a biomarker associated with liver cancer, wherein the biomarker is the CYSTM1 gene.

[0008] The present invention provides the use of a reagent for detecting the expression amount of the above-mentioned biomarker in the preparation of a product for early diagnosis of liver cancer.

[0009] Preferably, the product comprises a chip or a kit.

[0010] The present invention provides a kit for early diagnosis of liver cancer, which comprises a reagent for detecting the expression amount of the above-mentioned biomarker.

[0011] The present invention provides the use of a reagent for detecting the expression amount of the above-mentioned biomarker in preparing a product for predicting the prognosis of liver cancer.

[0012] Preferably, the expression level of the biomarker is negatively correlated with the prognosis of liver cancer patients.

[0013] Preferably, the product comprises a chip or a kit.

[0014] The present invention provides a kit for predicting the prognosis of liver cancer, which comprises a reagent for detecting the expression amount of the above-mentioned biomarker.

[0015] The present invention provides the use of the reagent for inhibiting the expression of the above-mentioned biomarker in the preparation of a drug for preventing and / or treating liver cancer.

[0016] The present invention provides a drug for preventing and / or treating liver cancer, comprising an agent for inhibiting the expression of the above-mentioned biomarkers.

[0017] The present invention discloses the following technical effects:

[0018] The present invention uses data from an online database to analyze the expression of the CYSTM1 gene in tumor tissues and normal tissues in liver cancer specimens, and analyzes its correlation with the patient's clinical tissue subtype, individual tumor grade, and TP53 mutation status, and explores its relationship with the occurrence and development of liver cancer, thereby finding better ways and methods for early detection and targeted treatment of liver cancer. Through analysis, the present invention first discovered that the CYSTM1 gene is upregulated in liver cancer, and the overall survival of liver cancer patients with high expression of the CYSTM1 gene is significantly reduced, suggesting that detecting the expression level of the CYSTM1 gene can become one of the auxiliary diagnostic indicators for liver cancer diagnosis, and that high expression of the CYSTM1 gene as a liver cancer diagnostic factor has a high accuracy (AUC = 0.935, sensitivity 0.848, specificity 0.94). The results of the specific embodiments of the present invention show that in human liver cancer specimens, the expression of the CYSTM1 gene in cancer cell tissue is significantly higher than that in normal tissue, and the expression of the CYSTM1 gene in liver cancer cell lines is also significantly higher than that in normal liver cells, and high expression of CYSTM1 is positively correlated with poor prognosis of patients, further proving that the CYSTM1 gene can be used as a biomarker for early diagnosis of liver cancer. At the same time, the specific embodiments of the present invention also verify the conclusion that inhibiting the expression of the CYSTM1 gene can effectively inhibit the proliferation and cloning of liver cancer, and overexpressing the CYSTM1 gene can significantly enhance the proliferation and cloning of liver cancer. It can be seen that interfering with the expression of the CYSTM1 gene can become a new way to prevent or treat liver cancer. At the same time, inhibiting the expression of the CYSTM1 gene can effectively inhibit the proliferation and cloning of liver cancer, and overexpressing the CYSTM1 gene can significantly enhance the proliferation and cloning of liver cancer. This discovery provides a sufficient theoretical basis for targeted treatment of liver cancer and provides new ideas for the development of related drugs and clinical treatment in the later stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 Analyze the pan-cancer expression of CYSTM1 gene for the TIMER database;

[0021] Figure 2 and Figure 3 GEPIA and UALCAN were used to analyze the expression of CYSTM1 gene in liver cancer;

[0022] Figure 4 Use R language to analyze the expression of CYSTM1 gene in paired specimens of TCGA database;

[0023] Figure 5 and Figure 6 The expression of CYSTM1 gene in the datasets GSE87630 and GSE84598, respectively;

[0024] Figure 7 To analyze the signal differences of CYSTM1 gene in liver cancer tissues and normal tissues for HPA;

[0025] Figure 8-Figure 13 The correlation between CYSTM1 gene and age, gender, individual tumor stage, tumor grade, histological subtype and TP53 mutation status in liver cancer is shown in order;

[0026] Fig.14 and Fig.15 The relationship between CYSTM1 gene expression and overall survival was analyzed using KM plotter and HPA database respectively;

[0027] Figure 16-Figure 21 The prognoses of the CYSTM1 gene high and low groups in patients with Stage I & Stage II, Stage III & Stage IV, G1 & G2, G3 & G4, AFP ≤ 400, and AFP > 400 were compared in order;

[0028] Fig. 22 The receiver operating curve is the curve;

[0029] Fig.23Figure 2 shows the effect of abnormal expression of CYSTM1 gene on cell proliferation, where A and E show the expression of CYSTM1 gene in liver cancer tissues, the 1st to 10th bands in A show the first pair of para-cancer tissues, the first pair of liver cancer tissues, the second pair of para-cancer tissues, the second pair of liver cancer tissues, the third pair of para-cancer tissues, the third pair of liver cancer tissues, the fourth pair of para-cancer tissues, the fourth pair of liver cancer tissues, the fifth pair of para-cancer tissues and the fifth pair of liver cancer tissues, E shows the expression of CYSTM1 gene in 5 pairs of para-cancer tissues and 5 pairs of liver cancer tissues, B and F show the expression of CYSTM1 gene in cells, the 1st to 4th bands in B show normal liver cells Lo2, liver cancer cells Huh-7, liver cancer cells LM3 and liver cancer cells HepG2, C and G show the effect of 3 siRNA transfected cells on CYSTM1 protein, the 1st to 4th bands in C show NC siRNA (Negative control siRNA), CYSTM1i-1 (siCYSTM1-1), CYSTM1i-2 (siCYSTM1-2) and CYSTM1i-3 (siCYSTM1-1), D and H are the effects of lentivirus-infected cells on CYSTM1 protein expression, the two bands in D are Vector (lentivirus-negative control infected cells) and CYSTM1OV (lentivirus-infected cells), I and J are the effects of CYSTM1 gene interference and overexpression on cell proliferation detected by CCK8, K and L are the effects of CYSTM1 gene interference and overexpression on cell clone formation, respectively. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Example 1

[0036] The present invention uses data from an online database to analyze the expression of the CYSTM1 gene in tumor tissues and normal tissues in liver cancer specimens, and analyzes its correlation with the patient's clinical tissue subtype, individual tumor grade, and TP53 mutation status, to explore its relationship with the occurrence and development of liver cancer, thereby finding better ways and methods for early detection and targeted treatment of hepatocellular carcinoma. The results are as follows Figure 1-Figure 22 shown.

[0037] Depend on Figure 1 It can be seen that TIMER analysis showed that the CYSTM1 gene was highly expressed in patients with liver cancer; Figure 2 and Figure 3 It can be seen that GEPIA and UALCAN analysis showed that the CYSTM1 gene was highly expressed in liver cancer patients; Figure 4-Figure 6 It can be seen that the analysis of the database TCGA, data set GSE87630 and data set GSE84598 showed that the CYSTM1 gene was significantly overexpressed in liver cancer tissues; Figure 7 It can be seen that the signal difference of CYSTM1 gene in liver cancer tissue and normal tissue analyzed by HPA showed that the signal of CYSTM1 gene in liver cancer tissue was significantly enhanced.

[0038] Depend on Figure 8-Figure 13 It can be seen that the CYSTM1 gene has a certain correlation with the patient's age, gender, individual tumor grade, tumor stage, histological subtype and TP53 mutation status.

[0039] Depend on Fig.14 and Fig.15 It can be seen that patients with high expression of CYSTM1 gene showed poor prognosis; Figure 16-Figure 21 It can be seen that in patients with Stage I&Stage II, G3&G4 and AFP≤400, the overall survival was significantly shortened in patients with high expression of CYSTM1 gene.

[0040] Example 2 Detection of the expression level of CYSTM1 gene protein in liver cancer tissues and cells

[0041] 1. Experimental materials: Liver cancer tissue: The 5 pairs (10 samples) of human early liver cancer tissues included in the study were derived from patients diagnosed with early liver cancer at Qilu Hospital of Shandong University (Qingdao) from January 1, 2020 to December 31, 2023. All human tissue specimens involved in this experiment were applied for by the research team and approved by the Ethics Committee of Qilu Hospital of Shandong University (Qingdao), and are limited to laboratory research.

[0042] Liver cancer cells: Normal liver cells LO2 were cultured in RPMI1640 medium containing 10% fetal bovine serum; liver cancer cells LM3 and Huh-7 were cultured in DMEM medium containing 10% fetal bovine serum; HepG2 cells were cultured in MEM medium containing 10% fetal bovine serum; all cells were cultured in a 37°C, 5% CO2 incubator; all the above cells were sourced from Shanghai Zhongqiao Xinzhou Biological Co., Ltd.

[0043] 2. Experimental method: western blot, steps are:

[0044] (1) Preparation of protein samples

[0045] For tissue: Take 70 mg of tissue and add 1 mL of pre-cooled lysis buffer, homogenize with a tissue homogenizer on ice, lyse on ice for 30 minutes, mix several times during the process, and then centrifuge with a pre-cooled high-speed low-temperature centrifuge at 12000 rpm and 4°C for 15 minutes, take the supernatant, quantify the protein by BCA method, take an appropriate amount of protein and add 5× loading buffer at a ratio of 1:5, and cook the sample in a 100°C metal bath for 10 minutes. Store at -80°C for later use.

[0046] For cells: For cells in a 6-well plate that have grown well, discard the culture medium, rinse with 1×PBS, discard the PBS, then add 100μL of cell lysis buffer to each well, shake at 4℃ for 20min, centrifuge at 12000rpm and 4℃ for 15min in a pre-cooled high-speed low-temperature centrifuge, take the supernatant, quantify the protein by BCA method, take an appropriate amount of protein and add 5× loading buffer at a ratio of 1:5, boil the sample in a 100℃ metal bath for 10min. Store at -80℃ for later use.

[0047] (2) Prepare 10% WB gel in advance, add protein samples and markers to the sample wells according to the experimental requirements, perform electrophoresis at a constant voltage of 80V, adjust the voltage to 120V after the marker strips are clearly separated, and perform electrophoresis for about 1 hour; after the electrophoresis is completed, separate the gel plates and soak the gel blocks in transfer buffer, follow the transfer clamp, place in an ice bath, transfer the membrane at a constant current of 200mA for 120min, then wash the membrane with TBST for 5min, and then use 5% BS prepared with TBST. After A was blocked for 1 hour, the corresponding I antibody (CYSTM1 antibody manufacturer: Abnova, catalog number: H00084418-M02; GAPDH antibody manufacturer: Hua'an Biotechnology, catalog number: SA30-01) was added and incubated overnight at 4°C on a shaker; the I antibody was recovered on the next day, and the membrane was washed 3 times with TBST, each time for 10 minutes, and then the corresponding II antibody (manufacturer: Hua'an Biotechnology, catalog number: HA1006) was added. After incubation at room temperature for 1 hour, the II antibody was recovered and the membrane was washed 3 times.

[0048] (3) Prepare the color developing solution by mixing solution A and solution B of the enhanced ECL chemiluminescence detection kit in a volume ratio of 1:1 and keep it away from light for later use; after absorbing the TBST on the membrane, add the color developing solution and store it for color development.

[0049] (4) Results: Fig.23 As recorded in A, B, E and F, the CYSTM1 gene was significantly overexpressed in liver cancer tissues compared with adjacent adjacent tissues; and the CYSTM1 gene was overexpressed in liver cancer cells Huh-7, LM3 and HepG2 compared with normal liver cells.

[0050] Example 3

[0051] In order to evaluate the clinical diagnostic value of the expression level of the CYSTM1 gene, the receiver operating curve was drawn. Fig. 22 As shown. Fig. 22 As recorded in A, the receiver operating curve analysis found that AUC = 0.935, the cut-off value was 5.8959, the sensitivity was 0.848, and the specificity was 0.94, which shows that the prognosis of liver cancer patients can be predicted with high accuracy based on the expression level of the CYSTM1 gene.

[0052] Example 4 Effects of CYSTM1 gene silencing and CYSTM1 gene overexpression on liver cancer cell proliferation

[0053] (I) Effect of CYSTM1 gene silencing on the proliferation of hepatocellular carcinoma cells

[0054] 1. Experimental materials: liver cancer cell Huh-7, siRNA, RNATransmate transfection reagent (Shanghai Bioengineering), CCK-8 detection kit (ABclonal);

[0055] The siRNAs are NC siRNA, siCYSTM1-1, siCYSTM1-2 and siCYSTM1-3, wherein the sequence of the sense strand of NC siRNA is: 5'-uucuccgaacgugucacgutt-3' (SEQ ID NO.1), and the nucleotide sequence of the antisense strand is: 5'-acgugacacguucggagaatt-3' (SEQ ID NO.2); the sequence of the sense strand of siCYSTM1-1 is: 5'-caccuuauccaccacaaccaatt-3' (SEQ ID NO.3), and the sequence of the antisense strand is: 5'-uugguugugguggauaaggugtt-3' (SEQ ID NO.4); the nucleotide sequence of the sense strand of siCYSTM1-2 is: 5'-ccuaccaaggauacccacagutt-3' (SEQ ID NO.5), and the sequence of the antisense strand is: 5'-acuguggguauccuugguaggtt-3' (SEQ ID NO.6); the sequence of the sense strand of siCYSTM1-3 is: 5'-uguaugugguagaagaccaaatt-3' (SEQ ID NO.7), and the sequence of the antisense strand is 5'-uuuggucuucuaccacauacatt-3' (SEQ ID NO.8).

[0056] 2. Experimental methods:

[0057] (1) siRNA transient transfection

[0058] ① Take well-growing Huh-7 cells and evenly spread them on a 6-well plate. After 12 hours, when the cell confluence grows to about 60-80%, perform siRNA transfection;

[0059] ② Dilution of siRNA and RNAtransmate transfection reagent: Take EP tubes and add 200 μL serum-free DMEM medium to each tube, add 1 μL siRNA to one tube and 6 μL RNAtransmate to another tube, mix well with a pipette, and place at room temperature for 5 minutes;

[0060] ③ Transfection: Mix the diluted siRNA and RNAtransmate, and then incubate at room temperature for 20 minutes; first add 1.6mL of DMEM culture medium to each well, then add 0.4mL of the mixed siRNA and RNAtransmate mixture, and continue to culture in the incubator.

[0061] (2) CCK-8 cell proliferation assay

[0062] Digest the adherent cells into a uniform cell suspension, take 10 μL of the uniform cell suspension and add it to the cell counting plate to measure its concentration, and dilute it into a cell suspension with a concentration of 3000 / mL. After taking a 96-well plate, add 3000 cells to each well, quantify 100 μL, set 5 replicates for each treatment, shake well and place in a constant temperature incubator. After culturing for 0, 1, 2, 3 and 4 days, discard the original culture medium, add 100 μL of fresh DMEM culture medium containing 10 μL CCK8 reagent to each well, and after constant temperature incubation for 2 hours, set the wavelength of the microplate reader to 450nm, measure the absorbance of each well, reread 3 times, and record and organize the data.

[0063] (3) Clone formation

[0064] Digest the adherent cells into a uniform cell suspension, take 10 μL of the uniform cell suspension and add it to the cell counting plate to measure its concentration, and dilute the cell suspension according to a certain ratio. After taking a 6-well plate, add 1000 cells to each well, quantify 2 mL, set up 3 replicates for each treatment group, mix well and place in a constant temperature incubator, replace the culture medium every 5 days, and culture for 15 days. After the cell colonies are visible to the naked eye in the six-well plate, discard the original culture medium, wash twice with PBS, add an appropriate amount of 4% paraformaldehyde to each well to fix the cells, place for 30 minutes, discard the fixative, add 800 μL of 0.1% crystal violet to each well, after 10 minutes at room temperature, recover the dye, wash and dry, and take pictures for statistical data.

[0065] (II) Effect of CYSTM1 gene overexpression on liver cancer growth

[0066] 1. Experimental materials: LM3 liver cancer cells; lentivirus overexpressing the CYSTM1 gene (LV-CYSTM1), wherein the overexpression vector is Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin, and the lentivirus is synthesized by GeneCare (Shanghai, China); the accession number of the nucleotide sequence of the CYSTM1 gene is Gene ID: 84418.

[0067] 2. Experimental methods:

[0068] (1) Screening of the MOI of lentiviral infection

[0069] LM3 cells with good growth status were plated in 96-well plates, with 100 μL of cell suspension per well and 3×10 4 Cells / well, after the cell density reaches 20-30%, lentiviral infection is performed. The virus is diluted to a titer of 1×10 8 TU / mL, 5×10 7 TU / mL and 1×10 7TU / mL, discard the original culture medium, add DMEM complete culture medium and 25× infection enhancement solution HiTransGA, mix well and continue to culture, and replace the culture medium after 16 hours. The culture medium can be replaced according to the growth of cells in the middle to maintain cell activity. About 72 hours after infection, the infection efficiency is observed under a microscope to reach about 80%, and the infection conditions and MOI corresponding to the group with good cell growth can be used as the experimental basis for subsequent infection. The infection conditions screened are: MOI=10.

[0070] Then, the infection conditions obtained by the above screening were used to infect LM3 cells in good growth state to obtain LM3 cells infected with lentivirus.

[0071] (2) Puromycin screening of stable cell lines

[0072] LM3 cells were evenly plated in a 12-well plate, with 2×10 cells per well. 5 Cells were infected with lentivirus when the cell confluence was about 30-50%. After 60 hours of infection, the infection rate was about 80% under fluorescence microscopy. The cells were treated with different concentrations of puromycin, with a concentration gradient of 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0 μL / mL; after 48 hours of puromycin treatment, the lowest concentration with a cell death rate of more than 90% was selected for subsequent experiments. The final concentration was determined to be 6 μL / mL puromycin as the lowest concentration.

[0073] Then, according to the growth rate of LM3 cells infected with lentivirus, puromycin with a concentration of 6 μL / mL was used for regular drug screening, and finally LM3 cells stably overexpressing the CYSTM1 gene were obtained. After the cells were fully grown, they were subcultured according to an appropriate ratio.

[0074] (3) CCK-8 cell proliferation assay

[0075] Digest the adherent cells into a uniform cell suspension (LM3 cells infected with lentivirus), take 10 μL of the uniform cell suspension and add it to the cell counting plate to measure its concentration, and dilute it into a cell suspension with a concentration of 3000 / mL. After taking a 96-well plate, add 3000 cells to each well, quantify 100 μL, set 5 replicates for each treatment, shake well and place in a constant temperature incubator. After culturing for 0, 1, 2, 3 and 4 days, discard the original culture medium, add 100 μL of fresh DMEM culture medium containing 10 μL CCK8 reagent to each well, and after constant temperature culture for 2 hours, set the wavelength of the microplate reader to 450nm, measure the absorbance of each well, read it again 3 times, and use the negative control lentivirus infected cell group as a control (Vector), record and organize the data.

[0076] (4) Clone formation

[0077] Digest the adherent cells into a uniform cell suspension, take 10 μL of the uniform cell suspension and add it to the cell counting plate to measure its concentration, and dilute the cell suspension according to a certain ratio. After taking a 6-well plate, add 1000 cells to each well, quantify 2 mL, set up 3 replicates for each treatment group, mix well and place in a constant temperature incubator, replace the culture medium every 5 days, and culture for 15 days. After the cell colonies are visible to the naked eye in the six-well plate, discard the original culture medium, wash twice with PBS, add an appropriate amount of 4% paraformaldehyde to each well to fix the cells, place for 30 minutes, discard the fixative, add 800 μL of 0.1% crystal violet to each well, after 10 minutes at room temperature, recover the dye, wash and dry, and take pictures for statistical data.

[0078] (III) Results

[0079] The results showed that Huh-7 cells were transfected with three siRNAs respectively, and it was found that siRNA1 and siRNA2 of CYSTM1 gene significantly inhibited the expression of CYSTM1 protein ( Fig.23 C and G in the figure); LM3 cells were infected with Vector and LV-CYSTM1, respectively. Compared with the control group Vector, LV-CYSTM1 could significantly enhance the CYSTM1 protein level ( Fig.23 D and H in the figure); Huh-7 cells were transfected with NCsiRNA, siRNA1 and siRNA2 respectively, and CCK8 and clone formation analysis showed that cell proliferation and clone formation were significantly inhibited after CYSTM1 gene was silenced ( Fig.23 I and K in ); CYSTM1 gene overexpression can significantly enhance cell proliferation and clone formation ( Fig.23 J and L in ).

[0080] In summary, the CYSTM1 gene is highly expressed in patients with liver cancer, and patients with high expression of the CYSTM1 gene have a poor prognosis, which indicates that the CYSTM1 gene has certain diagnostic and prognostic value in liver cancer.

[0081] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of an agent for inhibiting CYSTM1 gene expression in the preparation of a drug for treating liver cancer, characterized in that: The reagent for inhibiting CYSTM1 gene expression is a reagent containing siCYSTM1-1 or siCYSTM1-2; the nucleotide sequence of the sense chain of the siCYSTM1-1 is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense chain is shown in SEQ ID NO.4; the nucleotide sequence of the sense chain of the siCYSTM1-2 is shown in SEQ ID NO.5, and the nucleotide sequence of the antisense chain is shown in SEQ ID NO.

6.

2. A drug for treating liver cancer, characterized in that: It includes a reagent for inhibiting the expression of the CYSTM1 gene, wherein the reagent for inhibiting the expression of the CYSTM1 gene is a reagent containing siCYSTM1-1 or siCYSTM1-2; the nucleotide sequence of the sense chain of the siCYSTM1-1 is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense chain is shown in SEQ ID NO.4; the nucleotide sequence of the sense chain of the siCYSTM1-2 is shown in SEQ ID NO.5, and the nucleotide sequence of the antisense chain is shown in SEQ ID NO.6.

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