Expression Relationship Between CT23 And MT1 And Its Effect On The Malignant Biological Behavior Of Hepatoma Cells

Objective:to explore the expressive relationship of CT23 gene and MT1 gene in hepatocellular carcinoma(HCC)cell,SMMC-7721 and Hep G2,and in normal liver cell,L02 and CCL13.To investigate the effect of expression of CT23 gene and MT1 gene on biological behavior change of HCC cell.Methods:(1)Transfection of lentivirus to construct stable transfected cell line:stable transfected cells constructed by lentivirus,successfully transfected cells were pinked by puromycin,and screened by fluorescent labeling.(2)Detection of mRNA:The mRNA of CT23 and MT1 were detected by RT-qPCR.(3)Detection of biological behavior:①to evaluate the effect of CT23 gene and MT1 gene on normal liver cells and liver cancer proliferation ability by using cell proliferation curve detection.②Cell migration assay:Cell scratch assay and Transwell assay were used to detect the effect expression of CT23 gene and MT1 gene on the migration of normal hepatocytes and HCC cells.③Apoptosis and cell cycle detection:Flow cytometry was used to detect the effect of CT23 gene and MT1 gene on cell cycle and apoptosis of normal liver cells and HCC cells.(4)The expression detection of CT23 gene and MT1 gene in primary HCC tissues and its corresponding adjacent tissues:Combined with clinical data,the expression of CT23 and MT1 gene in HCC and adjacent non-carcinomatous(NC)tissues was detected by immunohistochemistry.Result:(1)Expression of CT23 gene and MT1 gene:RT-qPCR detection found that Knockin of CT23 gene caused an expression decrease of MT1 gene in normal liver cells L02 and CCL13;Knock-down of CT23 gene caused an expression increase of MT1 gene in HCC cell SMMC-7721.In the well-differentiated HCC cell lines Hep G2,the relative expression level of the CT23 gene is very low,transfection of the lentivirus could up-regulate the expression of the CT23 gene in Hep G2.(2)The effect of CT23 gene and MT1 gene on the proliferation of hepatoma cells:The proliferation rate of CCL13 cells and L02 cells transfected with LV8-CT23 knockin was higher than that of wild-type cells and empty vector transfected cells(P<0.05).The proliferation rate of HCC SMMC-7721 and Hep G2 transfected with LV3-CT23 knockdown was lower than that of wild type and transfected with empty vector(P<0.05),and the transfection of LV8-MT1F Cell proliferation rate compared with wild-type cells showed no significant difference(P>0.05).(3)Effect of CT23 gene and MT1 gene on migration of HCC cell lines:The wound healing rates of CCL13 cells and L02 cells transfected with LV8-CT23 knockin were higher than that of wild-type cells and empty vector transfected cells(P<0.05).The wound healing rate of SMMC-7721 and Hep G2 transfected with LV3-CT23 knockdown was lower than that of wild type and transfected with empty vector(P<0.05).The proliferation rate of MT1F knockin Hep G2 comparing with wild-type cells,no significant difference was found(P>0.05),scratch healing rate of LV8-MT1F transfected SMMC-7721 was lower than those of wild type cells and empty vector transfected cells(P<0.05).(4)The expression of MT1 and CT23 in HCC tissues and their corresponding adjacent NC tissues:The expression of MT1 in HCC tissues was lower than that in NC tissues(P<0.05).There was no significant difference in the expression of CT23 between HCC tissues and adjacent NC tissues(P>0.05).In HCC tissues,the expression of MT1 was higher in lower than that in higher Edmondson grade tissues(P<0.05),and was higher in single than that in multiple HCC tissues(P<0.05).There was no significant relationship between MT1 and age,sex,HBsAg,tumor size,AFP,cirrhosis,steatosis and metastasis in HCC tissues(P>0.05).The expression of MT1 in adjacent NC tissues had no significant relationship with age,sex,HBsAg,tumor size,Edmondson grade,AFP,cirrhosis,steatosis,metastasis and tumor number(P>0.05).The expression of CT23 in HCC tissues and adjacent NC tissues had no significant correlation with age,gender,HBsAg,tumor size,Edmondson grade,AFP,cirrhosis,steatosis,metastasis and tumor number(P>0.05).Conclusion:(1)Increased expression of CT23 gene leads to decreased expression of MT1 and MT1F genes;decreased expression of CT23 gene leads to increased expression of MT1 and MT1F genes;increased MTIF gene expression leads to decreased expression of CT23 gene.(2)Increased expression of CT23 gene leads to increased proliferation and migration of hepatocytes and decreased apoptosis.Decreased gene expression of CT23 gene leads to reduced proliferation and migration,and increased apoptosis of poorly differentiated HCC cell,but no effect on those of highly differentiated HCC cells.(3)The expression of MT1 in HCC tissue was lower than paracancerous tissue and that is negative related to Edmondson and positive related HCC number.There was no significant difference in the expression of CT23 gene in HCC tissues and adjacent tissues.