| ?Background and Objectives?Hepatocellular carcinoma(HCC)is a global health problem with high morbidity and mortality,which is a serious threat to human health.Surgical treatment is usually the first choice for isolated small HCC,with a five-year survival rate of more than 50%.However,the incidence of HCC is hidden,the diagnosis of patients has generally reached the middle and late stage,only some patients have the opportunity to receive liver transplantation(LT),there are still many patients need to receive traditional chemotherapy or new gene therapy,targeted therapy and immunotherapy in recent years.As we all know,traditional chemotherapeutic drugs have low selectivity and strong side effects,and immunotherapy is not effective in the treatment of solid tumors,especially hepatocellular carcinoma.In addition,there is still more than 70% probability of intrahepatic dissemination or extrahepatic metastasis in patients after surgical treatment,which makes the postoperative recurrence of HCC another difficult problem for medical workers.In recent years,with the addition of targeted drugs such as sorafenib and imatinib,the traditional treatment of HCC has been expanded to combination system therapy,and the drug treatment of HCC is being gradually optimized.Based on the fact that tumor is a disease with multiple gene changes and abnormal signal pathway,more biomolecules are expected to become therapeutic targets and need to be studied urgently,so as to improve the therapeutic effect and overall survival of patients with HCC.Suppressors of cytokine signaling 1(SOCS1)gene is a member of the SOCS protein family,all members are very similar in structure,with domains of N-terminal domain,SH2 region and SOCS box,which can participate in the phosphorylation of signal proteins and the ubiquitin pathway of target proteins,respectively.SOCS1 is reported as a tumor suppressor in a variety of tumors,and aberrant methylation exists in HCC,but the specific mechanism of its action in HCC is still limited,especially the mechanism of cell cycle regulation of HCC is worth exploring.?Material and Methods?The purpose of this study is to explain the role and mechanism of SOCS1 in HCC through two parts of experiments.1.To determine the expression level of SCSO1 in human HCC tissues and adjacent normal tissues and liver cancer cell lines,and to analyze the value of SOCS1 in the diagnosis and prognosis of HCC combined with clinicopathological data.(1)The m RNA expression of SOCS1 in normal liver,cirrhotic tissues and HCC tissues was collected by Oncomine international database,and the difference of m RNA expression between normal group and abnormal group was analyzed.(2)The m RNA expression of SOCS1 was detected by real-time fluorescence quantitative polymerase chain reaction(q RT-PCR)in HCC tissues of 159 patients with HCC and matched normal tissues adjacent to HCC.(3)Immunohistochemical staining was used to detect the protein expression of SOCS1 in HCC tissues of 90 patients with HCC and matched normal tissues.(4)Normal liver cell line QSG-7701 and seven hepatoma cell lines(SMMC-7721,HPEG2,HEP3 B,HUH7,HCC-LM3,MHCC-97 H and SK-HEP-1)were selected to verify the consistency of the expression trend of SOCS1 in the cell line and HCC tissues.(5)The relationship between the expression of SOCS1 and the prognosis of patients with HCC was analyzed by Kaplan-Meier survival analysis and log-rank test,and the correlation between the expression of SOCS1 and clinicopathological data was analyzed by chi-square test.2.To explore the role and mechanism of SOCS1 in the cell cycle regulation of HCC,to find the theoretical basis for the progression of HCC proliferation,and to explore new potential targets for the treatment of HCC.(1)Human hepatoma cell lines SMMC-7721,HCC-LM3 and MHCC-97 H with stable overexpression of SOCS1 were constructed by lentivirus transfection,and the overexpression efficiency was verified by Western blot.(2)High-throughput sequencing was used to analyze the differential expression of transcriptome genes to enrich the signal pathways of potential effects of SOCS1,CCK-8,flow cytometry cycle detection and Ed U assay were used to detect the specific effect of SOCS1 on hepatoma cell cycle,and the key regulatory nodes were confirmed by Western blot.(3)Xenografted tumors were established in nude mice with SMMC-7721 cell line stably overexpressing SOCS1 and control cells,to observe the effect of SOCS1 on tumor growth in vivo,and to further verify the expression level of key regulatory proteins by immunohistochemical staining.(4)Combined with the results of sequencing,the regulation pathway of differentially expressed proteins was analyzed.The cytoplasmic and nuclear proteins were separated and extracted to detect the expression level of key proteins in the nucleus,the half-life of protein degradation was detected by adding cycloheximide,the binding relationship in protein interaction was confirmed by co-immunoprecipitation(Co-IP)experiment,the expression of key regulatory molecules was overexpressed,and the reversal of downstream signal pathway was observed.STRING protein interaction database was used to analyze the exact correlation of the proteins.?Results?1.The m RNA and protein expression levels of SOCS1 in liver cancer cell lines and HCC tissues were lower than those in normal liver cell line and adjacent normal tissues(P < 0.0001).2.The analysis of SOCS1 and clinicopathological data showed that the expression level of SOCS1 was correlated with tumor size(P = 0.02),and the patients with high expression of SOCS1 had higher tumor-free survival rate(P = 0.0042)and overall survival rate(P = 0.0265)than the low expression group.3.The results of high-throughput sequencing analyzed the differential genes and enriched the communication pathway,and found that there was a correlation between SOCS1 and cell cycle and ubiquitin pathway,suggesting that there may be a signal pathway in HCC through which SOCS1 regulates the ubiquitin modification of key molecules in cell cycle to inhibit the proliferation of HCC cells.4.After overexpression of SOCS1,the cell expansion rate of SMCC-7721,HCC-LM3 and MHCC-97 H decreased significantly(P < 0.0001),the cell cycle arrest of HCC-LM3 and SMCC-7721 cell lines was in G1 phase(P < 0.05),and the proportion of S phase cells decreased significantly(P < 0.005),but no obvious cell cycle arrest was observed in MHCC-97 H cell line.Ed U assay confirmed that there was a G1-S phase transition block of cell cycle after overexpression of SOCS1 in HCC-LM3 and SMCC-7721 cell lines.5.The phosphorylation level of Retinoblastoma(Rb),the key regulator of G1 phase of cell cycle,was significantly down-regulated after overexpression of SOCS1,which blocked the progression of cell cycle.The protein expression level of another key regulatory molecule Cyclin D1 was increased,while the protein expression levels of P21 and P27 upstream were down-regulated.6.In vivo experiment,the growth of SMCC-7721 cell line overexpressing SOCS1 was significantly inhibited,and the tumor size was significantly smaller than that of the control group(P < 0.001),and the differential expression of key regulatory proteins was confirmed by immunohistochemical staining.7.The transcriptional levels of P21 and P27 did not change significantly after SOCS1 overexpression.Through the time gradient experiment of MG-132 drugs,it was suggested that there was the possibility of ubiquitination modification of P21,and it was confirmed by Co-IP that P21 was regulated by ubiquitination,which further affected the stability of Cyclin D1/CDK4 complex in the nucleus.8.After isolation and extraction of cytoplasmic and nuclear proteins,Western blot showed that the total amount of Cyclin D1 proteins that played a role in the nucleus was down-regulated and contained more inactivated Phospho-Cyclin D1,which could not bind to CDK4.9.After overexpression of SOCS1,the overexpression of P21 could reverse the differential expression of the above-mentioned cell cycle signal pathways,reduce the inactivation of Cyclin D1 in the nucleus and stabilize the Cyclin D1/CDK4 complex.10.Co-IP experiment further confirmed that the amount of Cyclin D1 protein that CDK4 could bind decreased significantly after SOCS1 overexpression.There is a binding relationship between SOCS1 and Ring-box1(RBX1)in ubiquitin ligase E3 complex,which can recruit ubiquitin transferase E2 to promote the degradation of target protein through ubiquitin pathway.?Conclusion?1.SOCS1 is low expressed in liver cancer cells and patients' HCC tissues,and is correlated with tumor size.2.Through high-throughput sequencing analysis and experimental demonstration,it is found that SOCS1 can effectively regulate P21-Cyclin D1/CDK4-Rb cell cycle signal pathway in HCC.3.SOCS1 has a clear inhibitory effect on HCC.It can inhibit the development of HCC through cell cycle arrest.It plays an important role in the cell cycle signal network and may be used as a potential target for the treatment of HCC. |
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