The Study Of Establishment And Proliferation And DNA Methylation Level Of Sorafenib-Induced Human Hepatocellular Carcinoma Resistant Cells

Backgrounds: The incidence of hepatocellular carcinoma(HCC)ranks fifth among the world's cancers and the mortality rate ranks third in the world.Because a significant number of patients have been diagnosed with advanced HCC or have a combination of underlying liver disease.Such patients have lost the chance of surgical removal of the tumor or undergo liver transplantation.In this case,molecular targeted therapy with sorafenib as an important treatment can effectively prolong the median survival time of HCC patients,but the emergence of drug resistance has become a major obstacle to its application.Related studies have shown that DNA methylation can promote the occurrence of tumor resistance to chemotherapy drugs.Objectives: In vitro application of sorafenib induces hepatocellular carcinoma cell lines Hep3 B and Huh7 to establish a drug resistance model and detects proliferation characteristics of sorafenib-resistant cells;detection of DNA methylation levels in sorafenib-resistant cell lines includes DNMTs and total DNA Methylation level.Methods: In this study,the Sorafenib intermittent induction method was used to establish Hep3 B and Huh7 sorafenib-resistant cell lines.Western Blot,Dot Blot,CCK8 and wound healing tests and data analysis were used to detect and evaluate differences in proliferation and DNA methylation of sorafenib parental cells and drug resistant cells.Differences were analyzed using Graph Pad Prism5 software.The t/t' test was used between the two groups(P<0.05).The differences in the data and statistical significance of the parental cells and drug resistant cells of sorafenib were determined.Results: The experiment established a sorafenib-resistant model of Hep3 B and Huh7 liver cancer in vitro.CCK-8 and data analysis showed that the proliferation of parental cells could be significantly inhibited by Sorafenib in comparison with sorafenib-resistant cells.Statistical significance.The tumor spheronization experiments showed that the tumor-bearing ability of drug-resistant cells was stronger,and the difference was statistically significant.Wound-healing showed that the drug-resistant cells showed a stronger migration ability,and the difference was statistically significant;The results showed that the expression of DNMTs in drug-resistant cells was up-regulated,and the difference was statistically significant.Dot blot showed that the total DNA 5mc levels of drug-resistant cells were higher,and the difference was statistically significant.Conclusion: We successfully established a sorafenib-resistant model of liver cancer,and drug-resistant cells have a higher proliferative potential;compared with the parental cells,changes in DNA methylation levels of drug-resistant cells include up-regulation of DNMTs and increased levels of total DNA 5mc.