ABSTRACT
Aim To investigate the effect of nitidine chloride (NC) on the apoptosis of cervical cancer cells and its mechanism. Methods Cervical cancer cell lines HeLa and SiHa were selected as subjects. The cytotoxicity of NC and its inhibitory effect on cell growth were detected by CCK-8 assay and clone formation assay. The effect of NC on the apoptosis of cervical cancer cells was detected by TUNEL assay, and the expression of apoptosis-related proteins was detected by Western blot. The effects of NC on the interaction between p53 and E6AP protein, the level of p53 ubiquitination modification and the stability of p53 protein in cervical cancer cells were analyzed by immunoprecipi-tation assay, ubiquitination assay and Western blot assay. Results NC could significantly inhibit the proliferation and induce apoptosis of cervical cancer cells. NC could inhibit the interaction between tumor suppressor protein p53 and E6AP in cervical cancer cells, reduce the level of p53 ubiquitination modification, delay the degradation of p53 and increase the expression level of p53 protein. Conclusions NC can inhibit the ubiquitination and degradation of p53, improve the expression level of p53 protein, restore its tumor suppressor function, and thus play an anti -cervical cancer role.
ABSTRACT
@#Objective To investigate the effect of silencing E6-associated protein(E6AP)on the level of p53 protein in human papilloma virus(HPV)negative cervical cancer cells(C33A cells).Methods The siRNA sequence silencing E6AP(siE6AP)and silencing control disordered siRNA sequence(siControl)were transfected into C33A cells with the mediation of LipofectamineTM2000 transfection reagent respectively.The silencing effect of siRNA on E6AP and the expression of p53and cleaved-caspase-3 proteins were detected by Western blot.Results The levels of E6AP protein in C33A cells of siE6AP group were significantly lower(t =-4.597,P<0.05),while the levels of p53 and cleaved-caspase-3 proteins were significantly higher than those of siControl group(t = 4.533 and 7.099 respectively,each P<0.05).Conclusion Silencing of E6AP significantly increased the expression of p53 protein in C33A cells,indicating that silencing of E6AP may restore the activity and function of p53 protein in C33A cells.
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Objective To detect the effect of E6AP on gastric cancer cell proliferation and migration.Methods The expression of E6AP in different gastric cancer cell lines and normal gastric mucosa epithelial cell lines was detected by Western blotting.Gastric cancer cells BGC-823 stably expressing E6AP short hairpan RNA(shRNA) were obtained by lentiviral vector of E6AP.The effect of E6AP on BGC-823 cell growth and migration was determined by CCK-8 kit, Tran-swell and wound healing assay.Results Gastric cancer cell line BGC-823 in which E6AP was stably knocked down was established.Knockdown of E6AP inhibited the proliferation and migration of BGC-823 cells.Conclusion E6AP plays a key role in gastric cancer proliferation and migration.
ABSTRACT
Objective To construct the lentiviral vector (pSIH-H1) for E6AP small-interfering RNA(siRNA) and to detect its effect on breast cancer ZR 75-1cell growth.Methods E6AP siRNA was designed and constructed based on hu-man papillomavirus E6-associated protein ( E6AP) cDNA sequence.The expression of E6AP was examined by real-time quantitative PCR(qRT-PCR) and Western blotting.The effect of E6AP on ZR75-1 cell growth was determined by cck-8 kit.Results DNA sequencing indicated that E 6AP siRNA expression vector was constructed successfully .qRT-PCR and Western blotting experiments showed that pSIH-H1-E6AP siRNA could suppress the E6AP gene expression.Suppression of E6AP could markedly inhibit the growth of ZR 75-1.Conclusion A lentivirus RNA interference ( RNAi) vector targeting E6AP gene is successfully constructed ,which inhibits the cell growth of ZR 75-1.