姜黄素通过抑制乙酰转移酶P300表达促进HeLa细胞凋亡

Curcumin promotes HeLa cell apoptosis by inhibiting expression of acetyltransferase P300

目的:探讨姜黄素(Cur)通过下调HeLa细胞腺病毒E1A相关300 kD蛋白(P300)调控HeLa细胞的活力及凋亡。方法:将对数生长期的HeLa细胞分别采用20、40、60和80μmol/L Cur处理,并以未处理组细胞作为对照,用CCK-8法检测细胞活力;选取20和40μmol/L Cur处理细胞,以流式细胞术检测细胞凋亡,RT-qPCR、Western blot法检测E6基因的mRNA和蛋白表达及凋亡相关蛋白、P300和组蛋白的表达;构建沉默质粒shE6和阴性对照质粒shNC转染HeLa细胞,设置shNC,shNC+Cur(40μmol/L),shE6以及shE6+Cur(40μmol/L)四组,以CCK-8、流式细胞术以及Western blot法分别检测细胞活力、凋亡以及凋亡相关蛋白的表达;构建沉默质粒siP300和阴性对照质粒siNC转染HeLa细胞,RT-PCR、Western blot法分别检测P300的mRNA和蛋白表达、E6的蛋白表达。结果:与未处理组相比,用不同浓度Cur处理HeLa细胞后能显著抑制其活力并可使早期凋亡率增高(P<0.05);Cur处理HeLa细胞后,E6的mRNA及蛋白表达均下调,而敲减E6与未敲减E6的Cur处理组相比,敲减组的早期凋亡率以及促凋亡相关蛋白表达均低于未敲减组(P<0.05);敲减P300后,HeLa细胞中E6蛋白表达降低;而单以Cur处理HeLa细胞后,P300及相关组蛋白表达均下调(P<0.01)。结论:Cur抑制HPV18阳性宫颈癌细胞的活力并促进其凋亡,机制可能与其下调P300而抑制E6蛋白乙酰化有关。

AIM:To investigate the effect of curcumin on the viability and apoptosis of human papillomavirus(HPV)-positive cervical cancer cells via down-regulating the expression of adenovirus E1A-associated 300 kD protein(P300).METHODS:HeLa cells in logarithmic phase were divided into four experimental groups(20,40,60 and 80μmol/L Cur treatment groups),and the untreated group was used as control.Cell apoptosis was detected by flow cytometry,and the expression of E6 gene was detected by RT-qPCR and Western blot.Silencing plasmid(shE6)and negative control shRNA(shNC)were constructed and transfected into HeLa cells,the cells were randomly divided into four groups including shNC,shNC+Cur(40μmol/L),shE6 and shE6+Cur(40μmol/L).The cell viability,apoptosis and expression of apoptosis-related proteins were detected by CCK-8,flow cytometry and Western blot,respectively.Then silencing plasmid(siP300)and negative control siRNA(siNC)were constructed and transfected into HeLa cells.The mRNA and protein expression of P300 was detected by RT-qPCR and Western blot.The protein expression of E6 and P300 and histone was detected by Western blot after treated with 20 and 40μmol/L curcumin.RESULTS:Compared with the untreated group,the HeLa cells treated with different concentrations of curcumin could significantly inhibit the viability and increase the early apoptosis rate(P<0.05).The results showed that the mRNA and protein expression of E6 were down-regulated in HeLa cells after treated with different concentration curcumin,while the early apoptosis and the expression of pro-apoptosis-related proteins in the shE6 group were lower than those in the non-knockdown group after treated with 40μmol/L curcumin(P<0.05).And the expression of E6 protein was decreased after knockdown of P300,while the expression of P300 and histone was down-regulated after treatment with 20 and 40μmol/L curcumin.CONCLUSION:Curcumin can inhibit the viability and promote the apoptosis of HPV18 positive cervical cancer cells,and the mechanism may be that it can inhibit E6 acetylation by down-regulating P300 expression.