| Research purpose: The incidence of primary hepatocellular carcinoma(HCC)is extremely high,accounting for the fourth place in malignant tumors in China.In recent years,the incidence of HCC has continued to increase in both men and women,and its rising speed has accelerated significantly.Meanwhile,primary liver cancer is currently the third leading cause of cancer-related deaths worldwide.The occurrence and development of hepatocellular carcinoma pose a serious threat to people’s lives and health,especially in China,where it causes a huge economic and social burden.Currently,although antiviral drugs can significantly reduce the risk of HCC caused by viral hepatitis,some patients still progress to HCC.At present,the treatment methods for HCC have the characteristics of multidisciplinary participation and coexistence of multiple treatment methods,including HCC surgical resection,liver transplantation,radiofrequency ablation,transcatheter arterial chemoembolization(TACE),radiation therapy,systemic anti-tumor therapy,and other methods.Despite years of development,the treatment strategies for liver cancer have been significantly improved and developed.However,due to the clinical problems of high recurrence and metastasis rates in some HCC patients after surgical resection,and the limited improvement of survival time by mid to late stage systemic therapy,the overall survival and prognosis of liver cancer are still unsatisfactory.Based on the current status of HCC diagnosis and treatment,it is of great scientific and clinical significance to search for biomarkers for early screening,diagnosis,and prognosis of HCC,and explore new targets for HCC treatment to improve patient survival.Sulfotransferase(SULT)is a functional enzyme that catalyzes the sulfonation process.SULT is involved in a number of important metabolic activities: protein,lipid,carbohydrate or steroid and membrane metabolism in the cytoplasm.It plays an important role in endogenous and exogenous metabolism and is also a key step in the biological activation of some drugs and many original carcinogens.In recent years,studies have confirmed that SULTs are abnormally expressed in a variety of tumors,and the gene polymorphism is closely related to the susceptibility of tumors,which is involved in the occurrence and development of tumors.Therefore,the role of SLUT family members in tumors has attracted more and more attention.Current studies suggest that SULT1B1 also plays a role in the occurrence and development of various tumors,but its specific mechanism is unknown.There was only one report on HCC,which found that SULT1B1 expression was low in HCC tumor samples.However,the expression of SULT1B1 in HCC tumor tissues still needs to be confirmed in large samples,and its role in malignant biological behavior phenotype of HCC remains unclear.Methods: 1.Analyze the expression level of SULT1B1 mRNA in HCC tumor tissue in the HCC data set of TCGA database;the relationship between SULT1B1 mRNA and clinical characteristics and prognosis of HCC patients was analyzed;2.Western Blot was used to detect the expression of SULT1B1 protein in HCC tumor tissues;3.The expression level of SULT1B1 protein in hepatocellular carcinoma was detected by immunohistochemistry;4.The correlation between the expression level of SULT1B1 protein and the clinical phenotype,survival and prognosis of HCC patients was further verified by using the HCC tissue microarray cohort.5.The effects of overexpression of SULT1B1 on HCC cell proliferation,migration,invasion,cloning ability and epithelial-mesenchymal transformation were analyzed by lentivirus transfection;6.The effects of silencing SULT1B1 on the proliferation,apoptosis,migration,invasive clonogenesis and epithelial-mesenchymal transformation of HCC cells were analyzed by using si RNA technology to silence SULT1B1.7.The overexpression cell line of SULT1B1 was constructed by lentivirus transfection technology,and the tumor bearing model of subcutaneous liver cancer in nude mice was established to explore the effect of SULT1B1 on the proliferation of subcutaneous implanted tumor in nude mice;8.The murine hepa1-6 cell line overexpressed with SULT1B1 was constructed by lentivirus transfection technology,and the lung metastasis model of nude mice liver cancer was established to explore the effect of SULT1B1 on HCC metastasis;9.The regulatory effect of SULT1B1 on the desiccation of HCC cells was predicted and verified by cell biology experiments using bioinformatics prediction method;10.Through immunoprecipitation and other cell biological experiments,it was verified that SULT1B1 regulated the expression of DKK1 by binding to the key protein DKK1 of Wnt signaling pathway;11.By silencing DKK1,the complement experiment confirmed that SULT1B1 participated in the occurrence and development of HCC by combining with DKK1.Results: The results showed that the mRNA and protein levels of SULT1B1 in HCC cells and tumor tissues were low;clinical analysis confirmed that the low expression level of SULT1B1 suggested that the clinical prognosis of HCC patients was poor,and SULT1B1 could be used as a potential prognostic marker;In vitro cell experiments confirmed that SULT1B1 significantly inhibited the proliferation of HCC cells and promoted the level of apoptosis of HCC cells.At the same time,SULT1B1 significantly reduced the ability of invasion and migration of HCC cells and the level of EMT;In vivo animal experiments showed that SULT1B1 could inhibit the proliferation and metastasis of HCC tumor;the mechanism exploration experiment found that the overexpression of SULT1B1 might inhibit the stem cell characteristics of HCC;protein immunoprecipitation confirmed that SULT1B1 could bind to DKK1 protein and inhibit its protein degradation;the complement experiment showed that SULT1B1 was involved in the regulation of HCC cell stem and other malignant phenotypes and the occurrence and development of HCC by binding to DKK1 protein and regulating Wnt signaling pathway.Conclusion: In this study,bioinformatics,molecular biology,tumor bearing model of nude mice,lung metastasis model of liver Cancer and other experimental methods were used,combined with TCGA,the expression of SULT1B1 in HCC and its clinical significance were analyzed in HCC expression profile data sets of public databases such as TCGA and GEO and local HCC tumor micromatrix microchips.The effects of SULT1B1 on the proliferation,migration,invasion and clone formation of hepatocellular carcinoma were investigated by in vivo and in vitro functional experiments.Furthermore,biological information analysis and prediction and in vitro experiments were further used to verify that SULT1B1 could combine with DKK1,and functional complement experiments were used to systematically study the role of SULT1B1 combined with DKK1 in regulating Wnt pathway and drying and further influencing HCC progression.We demonstrated that SULT1B1 was low expression in HCC tissues and was associated with poor prognosis in HCC patients.In vitro experiments,SULT1B1 significantly inhibited the proliferation of HCC cells,and significantly reduced the invasion and migration ability and EMT of HCC cells.Moreover,SULT1B1 enhanced apoptosis.The inhibitory effect of SULT1B1 on the proliferation and metastasis of HCC was also confirmed in tumor bearing model,situ liver cancer model and lung metastasis model.Further studies demonstrated the role of SULT1B1 in inhibiting HCC proliferation and metastasis by regulating Wnt pathway and drying by combining with DKK1. |
