MARCH8在宫颈癌组织中的表达及其沉默后对人宫颈癌细胞增殖、凋亡的影响

Expression of MARCH8 in cervical cancer tissues and effects of silencing on proliferation and apoptosis of human cervical cancer cells

目的探索MARCH8表达量对宫颈癌的影响以及MARCH8沉默后宫颈癌细胞增殖、凋亡的影响,并对其可能存在的机制进行研究。方法选取我院宫颈癌手术切除标本20份作为研究对象,同时选取20例正常宫颈组织人群作为对照。免疫组化检测宫颈癌组织MARCH8表达水平;构建MARCH8的siRNA片段通过脂质体介导转染人宫颈癌Hela细胞(MARCH8-RNAi),Western blot与RT-PCR进行检测验证;MTT法检测MARCH8-RNAi细胞增值率、FCM法检测细胞周期及细胞凋亡率,Hoechst 33342检测凋亡小体形成情况,免疫组化及Western blot检测细胞周期、荧光定量PCR检测凋亡相关蛋白表达水平。结果免疫组化结果显示宫颈癌组织中MARCH8阳性表达率高于正常宫颈组织(P<0.01);Western blot与RT-PCR结果显示MARCH8-RNAi细胞中MARCH8表达量显著低于Ctrl(P<0.01);MARCH8-RNAi细胞增殖率及存活率均降低、处于G 0/G 1期细胞增多、PCNA、CDK2、CyclinD1表达水平降低,与Ctrl差异有统计学意义(P<0.01);FCM及Hoechst 33342结果显示MARCH8-RNAi细胞凋亡率升高、凋亡小体数目增加,Ki67、Bcl-2蛋白水平降低,而Caspase-3、Bax、P53蛋白表达量增加与Ctrl差异有统计学意义(P<0.01)。结论MARCH8在宫颈癌中高表达且MARCH8沉默后可通过调节细胞周期、凋亡相关基因蛋白水平抑制人宫颈癌Hela细胞增殖、促进细胞凋亡。

Objective To observe the expression of MARCH8 in cervical cancer,explore the effects on the proliferation and apoptosis of cervical cancer cells after MARCH8 silencing,and study the possible mechanism.Methods Twenty samples of cervical cancer obtained from resection in our hospital were selected as the research subjects,and 20 normal cervical tissues were selected as the controls.Immunohistochemistry was used to detect the level of MARCH8 in cervical cancer tissues.The siRNA fragments of MARCH8 were constructed and transfected into human cervical cancer Hela cells(MARCH8-RNAi)via liposomes.Western blot and RT-PCR were used to verify the results.MTT was used to detect MARCH8-RNAi cell proliferation rate,FCM method was used to detect cell cycle distribution and apoptotic rate,Hoechst 33342 was used to detect apoptotic body formation,immunohistochemistry and Western blot were used to detect the apoptosis-related protein levels,and RT-PCR was used to detect the cell cycle-related gene expressions.Results The immunohistochemical results showed that the expression of MARCH8 in cervical cancer tissues was higher than that in normal cervical tissues(P<0.01).Western blot and RT-PCR results showed that the expression of MARCH8 in MARCH8-RNAi cells was significantly lower than that in the control cells(P<0.01).MARCH8-RNAi cells had reduced proliferation and survival rates,increased G 0/G 1 cells,and decreased PCNA,CDK2,and CyclinD1 gene expressions,and the differences with the control cells were statistically significant(P<0.01).FCM and Hoechst 33342 results showed MARCH8-RNAi cell apoptosis rate was increased,the number of apoptotic bodies was increased,Ki67,Bcl-2 protein levels were decreased,while Caspase-3,Bax,P53 protein levels were increased significantly(P<0.01).Conclusion MARCH8 is highly expressed in cervical cancer and its silencing can inhibit the proliferation and promote the apoptosis of human cervical cancer Hela cells by regulating cell cycle and apoptosis-related gene or protein levels.