The Expression Of GRHL3 And MDR1 In Hepatocellular Carcinoma And Its Moudulation MDR1-Mediatd Mutidrug Resisitance

Objective 1.To investigate the expression of Grainyhead-like-3(GRHL3)and multidrug resistance-1(MDR1)in hepatocellular carcinoma(HCC)and the relationship among the clinicopathologic features.2.To have a preliminary exploration of the regulation of GRHL3 on the MDR1-mediated multidrug resistance at the cytological level.Methods 1.Immunohistochemistry was used to detect the expression of GRHL3 protein and MDR1 protein in the normal liver tissues of 132 cases of HCC,60 cases of tissues adjacent to the liver cancer and 20 cases of hepatic hemangioma.The clinicopathological data were detected through various ways.2.The expression of GRHL3 protein and MDR1 protein in human hepatocarcinoma cell line Bel-7402 and drug-resistant cell line Bel-7402/5-FU were detected by Western blot.3.To verify whether GRHL3 could affect the drug resistance of hepatocarcinoma cells by regulating MDR1,the expression vector PCV-2B-GRHL3 that carried GRHL3 cDNA was transiently transferred to the hepatocarcinoma cell line 7402 for overexpression,and the Gr287 vector that carried GRHL3-shRNA was transferred to the drug-resistant cell line7402/5-FU for intervention.4.The Western blot was used to detect the expression of GRHL3 protein and MDR1 protein in the four kinds of cells.Results 1.The positive rates of GRHL3 in hepatocellular carcinoma,tissues adjacent to the cancer and normal tissues were 72%,53% and 0,respectively.The positive expression of GRHL3 was mainly located in the nucleus.The positive rates of MDR1 in hepatocellular carcinoma,tissues adjacent to the cancer and normal tissues were 69%,52% and 0,respectively.The positive expression of MDR1 was mainly located in the cytoplasm and on the cell membrane.The positive rates of GRHL3 andMDR1 in primary hepatocellular carcinoma,tissues adjacent to the cancer and normal tissues were significantly different(P <0.001).The positive rate of GRHL3 protein was not related to patients’ age,sex,clinical stage,portal vein tumor thrombus,tumor size and the presence of HBs infection,and the difference was not statistically significant.The positive rate of GRHL3 protein was related to the level of alpha-fetoprotein,TNM stage and the degree of pathological differentiation,and the difference was statistically significant(P<0.05).in addition,the positive rate of MDR1 protein was related to the level of alpha-fetoprotein,TNM stage and the degree of pathological differentiation,and the difference was statistically significant(P<0.05).There were no statistically significant differences in other pathological data.2.The results of the Western blot analysis showed that the expression of GRHL3 and MDR1 in drug-resistant cell line Bel-7402/5-FU cell line was significantly higher than that in the drug-sensitive cell line Bel-7402,and the expression of GRHL3 and MDR1 was positively correlated.3.The Western blot analysis showed that the expression vector PCMV-2B-GRHL3 that carried FLAG and GRHL3 cDNA was successfully transiently transfected to hepatocarcinoma cell line Bel-7402 for overexpression,and the vector GV287 that carried FLAG and GRHL3-shRNA was also successfully transiently transfected to drug-resistant cell line Bel-7402/5-FU for intervention.4.The expression of MDR1 in the drug-sensitive cells Bel-7402 that overexpressed GRHL3 was significantly higher than that in the normal drug-sensitive cells Bel-7402.The expression of MDR1 protein in drug-resistant cell strain Bel-7402/5-FU was significantly lower than that in the normal drug-resistant cells Bel-7402/5-FU,which further demonstrated that the expression of MDR1 in hepatocarcinoma cells may be positively regulated by the GRHL3 gene.Conclusion 1.GRHL3 and MDR1 were both highly expressed in HCC tissues.The expression had a significant positive correlation and was significantly related to patients’ clinicopathological feature like level of alpha-fetoprotein,TNM stage and the degree of pathological d,which may play a key role in the development of drug resistance and suggested a poor prognosis.2.GRHL3 gene may have a significant effect on the drugresistance of liver cancer by positively regulating MDR1.