Regulation Of Proliferation And Metastasis Of Hepatocellular Carcinoma By GCNT3

Objective:Hepatocellular carcinoma(HCC)is one of the leading causes of cancer-related deaths worldwide,and its high incidence and recurrence rates remain an intractable challenge,hence the urgent need for in-depth studies on the complex mechanisms behind its development and recurrence.The aim of this study is to investigate the biological function and clinical significance of mucin-type N-acetylglucosaminyltransferase 3(GCNT3)in HCC and to analyse the specific mechanisms by which this gene affects the development of hepatocellular carcinoma,in the hope of providing new targets for the treatment of hepatocellular carcinoma and the diagnosis of early metastasis.MethodsWe performed bioinformatics analysis on the TCGA database of hepatocellular carcinoma data and used RT-qPCR to detect the expression of GCNT3 mRNA in clinical specimens of hepatocellular carcinoma to obtain the differential expression and prognosis of GCNT3 in hepatocellular carcinoma.Lentivirus and small interfering RNA were also transfected in Hep3 B and HCCLM3 to overexpress and knock down the expression of GCNT3.The effects of GCNT3 on the biological functions of hepatocellular carcinoma cells were investigated using the CCK-8 assay,scratch assay,Transwell assay and clone formation assay in cell function assays.The specific mechanism by which GCNT3 promotes the proliferation and metastasis of hepatocellular carcinoma at the protein level was then investigated by Western Blot assay.Results(1)The analysis of GCNT3 mRNA expression in TCGA database and clinical specimens revealed that GCNT3 was highly expressed in hepatocellular carcinoma with poor prognosis.GCNT3,as one of the predictors in hepatocellular carcinoma,had good predictive ability with AUC=0.757.(2)In HCCLM3 and Hep3 B cell lines,overexpression and knockdown of GCNT3 expression using lentiviral and small interfering RNA were demonstrated by CCK-8 assay and clone formation assay that overexpression of GCNT3 promoted the proliferation ability of hepatocellular carcinoma cells,while knockdown of GCNT3 expression reduced the proliferation level of hepatocellular carcinoma cells.It was confirmed that high expression level of GCNT3 was enhancing the growth and proliferation ability of hepatocellular carcinoma cells.(3)The enhanced expression of GCNT3 was found to promote the invasive and metastatic ability of hepatocellular carcinoma cells by scratch assay and Transwell assay,while decreasing the expression of GCNT3 gave the opposite result in the invasive metastasis assay.This demonstrates that GCNT3 is associated with the invasive and metastatic ability of hepatocellular carcinoma cells.(4)Overexpression of GCNT3 promoted the expression of N-cadherin and Vimentin and inhibited the expression of E-cadherin in EMT by protein blotting.The expression of E-cadherin was significantly increased in the knockdown group,while the expression of N-cadherin and Vimentin was decreased.Overexpression of GCNT3 promoted the expression of molecules that can promote the GSK3β/β-catenin protein signaling pathway,while the function of GSK3β/β-catenin protein signaling pathway was inhibited in the knockdown group.This demonstrated that GCNT3 could promote the development of hepatocellular carcinoma through the GSK3β/β-catenin protein signaling pathway.(5)Analysis of GCNT3 function identified the downstream regulated mucin MUC13 and demonstrated by protein blotting experiments that GCNT3 promotes MUC13 expression and that a decrease in GCNT3 expression is accompanied by a decrease in MUC13 expression.The knockdown of MUC13 while overexpressing GCNT3 revealed that the promotion of GSK3β/β-catenin protein signaling pathway by GCNT3 was inhibited.The regulatory role of GCNT3 on the MUC13/GSK3β/β-catenin protein axis was demonstrated.(6)A cell derived xenograft model in nude mice was constructed using a stable transitional cell line overexpressing GCNT3,and the tumours in the group overexpressing GCNT3 were significantly larger than those in the control group.This demonstrates that GCNT3 affects the development of hepatocellular carcinoma in vivo.Conclusion:Upregulation of GCNT3 expression in hepatocellular carcinoma also predicted a poorer prognosis.Enhanced expression levels of GCNT3 in HCC promoted the MUC13/GSK3-β/β-catenin signal pathway to regulate tumour development in HCC.