Study On The Effect Of Hic-5 In Hepatic Stellate Cells On Biological Behavior Of Hepatocellular Carcinoma

Objective: To investigate the effect of Hic-5 in hepatic stellate cells on the biological behavior of hepatocellular carcinoma(HCC)at the histological and cytological levels,and to provide new therapeutic targets and theoretical basis for the treatment of HCC.Methods: Liver tissues that were diagnosed as HCC or liver trauma in our hospital from July2015 to October 2015 were collected.For this part of the liver tissue: 1)using Western Blot(WB)method to detect the degree of Hic-5 expression in liver tissue: cancer tissue,adjacent liver tissue(distance from cancer tissue <1.0cm),distant liver tissue(distance from cancer tissue> 1.0cm);well-differentiated,moderately differentiated,poorly differentiated HCC patients;HCC with hepatitis B cirrhosis,HCC without hepatitis B liver cirrhosis patients.2)Immunohistochemical method was used to detect the expression of Hic-5 in high,middle and poorly differentiated hepatocellular carcinoma and normal liver tissues.3)16 cases HCC patients were randomly selected and the expression of Hic-5 was detected by immunohistochemistry.The median of photo-sensitivity was used as the critical value in order to distinguish the level of its expression.The relationship between the level of Hic-5 and the postoperative survival time was analyzed by the product-limit estimat.4)the Si RNA method was used to silence the Hic-5 gene of human hepatic stellate cell line(LX-2),and Western blot was used to detect the silence of Hic-5.5)Human hepatic stellate cell line(LX-2)and human hepatocellular carcinoma cell line(Hep G2)were co-cultured in three groups: Group A(normal control group): normal LX-2 cell line and Hep G2 cell line co-cultured group Group B(blank control group): SiRNA-NC transfected LX-2 cell line and Hep G2 cell line co-cultured group,C group(experience group):SiRNA-Hic-5 transfected LX-2 Cell line and Hep G2 cell line co-culture group.And cells or cell culture fluid was collected at 0,24,48,and 72 hours,which was detected by western blot method.The expression of Hic-5,Collagen 1a1,and Collagen 3a1 were detected at the time points.The expression of Collagen 1a1 and Collagen 3a1 was detected by reverse transcription-PCR.6)the cell scrach test was performed on conditioned medium that we got after 24 hours of co-culture to test the invasiveness of HCC cells.Results.1.Western bolt results showed that Hic-5 was highly expressed in the cancer tissue group(P<0.05)in cancer tissue,adjacent liver tissues,and adjacent liver tissue.Hic-5 is highly expressed in poorly differentiated hepatocellular carcinoma in hepatocellular carcinoma;Hic-5 is highly expressed in hepatocellular carcinoma with hepatic cirrhosis in hepatocellular carcinoma with hepatitis B cirrhosis and liver cancer without hepatitis B cirrhosis(P<0.05).2.The results of immunohistochemistry showed that Hic-5 was highly expressed in poorly differentiated group(P<0.05)in high,moderate,poorly differentiated HCC and normal liver tissue.3.The survival time of Hic-5 low expression group was significantly longer than that of high expression group(P<0.05).4.The expression of Hic-5 in SiRNA-Hic-5 transfected human stellate cell(LX-2)cell line was significantly lower than that of the control group,and the difference was statistically significant(P<0.05).5.When human stellate cells were co-cultured with hepatoma cells,the expression levels of Hic-5,Collagen1a1,and Collagen 3a1 in group A,group B,and group C increased with the extension of culture time,and they were set at the same time point(0h,24 h,48 h or 72 h),the protein expression of Collagen 1a1 and Collagen 3a1 in group C was significantly lower than that in group B(P<0.05),but the difference between group B and group A was not statistically significant(P>0.05);6.Cell scratch test: The migration distance of liver cancer cells in group C was significantly decreased compared with group A and B.Conclusion: 1.The degree of Hic-5expression depends on the distance from cancerous tissue and its degree of differentiation.With the increase in distance and differentiation from cancerous tissue,the expression of Hic-5 gradually decreases;2.The level of Hic-5 expression and postoperative The survival time is closely related,and its high expression indicates a poor prognosis;3.Hepatoma cells(Hep G2)can promote the up-regulation of Hic-5 expression in hepatic stellate cells(LX-2),and suppress the Hic in hepatic stellate cellsthrough SiRNA silencing technology.The expression of-5 can inhibit the expression of cell matrix proteins Collagen 1a1 and Collagen 3a1,and reduce the invasion and migration of hepatocellular carcinoma.