Down-regulating Of UBC9 Increasing The Sensitivity Of HCC To Doxorubicin

ObjectiveTo investigate expression of UBC9 in hepatocellular carcinoma and its clinical significance.Then detect the expression of UBC9 in human hepatoma cells and explore the effects of down-regulating of UBC9 expression via short hairpin RNA on the sensitivity of HepG2 to Doxorubicin in HCC and analyze its possible mechanism.Methods1.IHC and Western blot analysis were used to detect the expressions of UBC9 in HCC.2.Western blot analysis were used to detect the expressions of UBC9 in HepG2、Huh7、Hep3B、 SMMC-7721、 LM3 and LO2 cells.3.After transfection of UBC9 shRNA into HepG2 cells,the expression levels of UBC9 mRNA and UBC9 protein were examined.4.The effects of apoptosis and drug sensitivity were respectively determined by FACS analysis of Annexin-V expression and CCK8 assay..Results1.The IHC results showed that the UBC9 protein was highly expressed in69.92%(72 of 103)of the HCC tissue samples and in only 17.47%(18 of 103)of the adjacent tissues(P < 0.001).2.In addition,the Western blotting results showed that the UBC9 protein levels were significantly elevated in the HCC tissues which was consistent with the IHC results.3.By compared the UBC9 protein overexpression with HCC clinicopathologic parameters.The result found that UBC9 overexpression correlated closely with tumor size,tumor microsatellite formation,and vascular invasion(P < 0.001).However,the expression of UBC9 protein in tumor tissues is not related with sex(P=0.239),age(P=0.095),HBsAg(P=0.216)and AFP(P=0.914).3.The amounts of UBC9 mRNA and protein were significantly reduced in HepG2 cells transfected with UBC9 shRNA(shUBC9-a).4.To investigate UBC9 expression in HCC cells,we examined the levels of UBC9 in a variety of HCC cells by Western blotting.The results indicated that the expression of UBC9 in HCC cells was higher than that in normal liver cells.5.UBC9 knockdown enhanced sensitivity to DOX in HepG2 cells.CCK8 experiment found that the HepG2 cells vitality reduced,drug inhibition rate increased in shUBC9-a grounp cells.We also can see the IC50 significantly reduce in shUBC9-a cells.6.We found that HepG2 shUBC9-a in combination with Doxorubicin treatment had a stronger effect on the increase of apoptosis than these cells alone and HepG2,HepG2-negative with doxorubicin.ConclusionOur research demonstrated that the expression of UBC9 was overexpressed in Hepatocellular carcinoma.Knockdown the expression of UBC9 in HepG2 cells can inhibit the growth of cancer cells and increase the chemosensitivity to DOX.It was suggested that Ubc9 may be a promising target for the therapy of Hepatocellular carcinoma.