长链非编码RNA MEG3对内质网应激诱导的结肠癌细胞凋亡及人结肠癌裸鼠移植瘤模型生长的影响

The effects of long non-coding RNA MEG3 on endoplasmic reticulum stress-induced colon cancer cell apoptosis and the growth of transplanted human colonic carcinoma in nude mice

目的探究人母系表达基因(MEG3)对内质网应激诱导的结肠癌细胞凋亡及人结肠癌裸鼠移植瘤模型生长的作用及其机制。方法通过RT-PCR筛选MEG3表达水平最低的结肠癌细胞系,构建了MEG3过表达及阴性对照细胞系并筛选了稳转株,RT-PCR检测各组细胞MEG3基因表达水平,CCK8检测细胞增殖,Hoechst染色检测细胞凋亡,Western blot检测Bcl-2、Bax、cleaved Caspase-9、p-PERK、ATF-4、p-EIF2α、CHOP、cleaved Caspase-12、p-AMPK、AMPK、p-mTOR、mTOR的蛋白表达。通过si-CHOP沉默结肠癌细胞中CHOP的表达,Hoechst染色检测细胞凋亡。构建人结肠癌裸鼠移植瘤模型并测量不同时期的肿瘤体积,IHC Ki67检测肿瘤生长,TUNNEL检测细胞凋亡,Western blot检测CHOP、cleaved Caspase-12、cleaved Caspase-3的蛋白表达。结果SW480细胞系MEG3基因表达水平最低,被用于后续实验。在细胞实验中,与Control组比较,pc-MEG3组结肠癌细胞增殖、Bcl-2蛋白表达量、p-mTOR/mTOR比率降低,细胞凋亡率、Bax、cleaved Caspase-9、p-PERK、ATF-4、p-eIF2α、CHOP、cleaved Caspase-12蛋白表达量及p-AMPK/AMPK比率升高;沉默结肠癌细胞中CHOP表达可降低pc-MEG3引起的细胞凋亡率升高。在动物实验中,与Control组比较,pc-MEG3组肿瘤体积、Ki67细胞阳性率降低,细胞凋亡率、CHOP、cleaved Caspase-12、cleaved Caspase-3蛋白表达量升高。结论MEG3可能通过内质网应激诱导结肠癌细胞凋亡,CHOP在其中起着关键作用;此外,AMPK/mTOR也参与了MEG3调控细胞凋亡的进程。

Objective To investigate the effects of maternally expressed gene 3(MEG3)on endoplasmic reticulum stress-induced colon cancer cell apoptosis and the growth of transplanted human colonic carcinoma in nude mice.Methods Colon cancer cell lines with the lowest expression level of MEG3 were screened by RT-PCR;negative control cell lines were constructed and stable transfectants were screened.RT-PCR was performed for measuring the lncRNA level of MEG3,cell growth was measured by CCK8,cell apoptosis was determined by Hoechst.Western blot was used to determine the protein levels of Bcl-2,Bax,cleaved Caspase-9,p-PERK,ATF-4,p-EIF2α,CHOP,cleaved Caspase-12,p-AMPK,AMPK,p-mTOR,and mTOR.The expression of CHOP was silenced by si-CHOP,and then cell apoptosis was determined by Hoechst.Transplanted human colonic carcinoma in nude mice was established and tumor volume was measured,tumor growth was measured by IHC Ki67,cell apoptosis was determined by TUNNEL,Western blot was used to determine the protein levels of CHOP,cleaved Caspase-12,and cleaved Caspase-3.Results The SW480 cell line had the lowest expression level of MEG3 and was used in subsequent experiments.In cytological experiments,compared with those in control group,the cell growth,protein levels of Bcl-2 and ratio of p-mTOR/mTOR were decreased significantly;the apoptosis rate,protein levels of Bax,cleaved Caspase-9,p-PERK,ATF-4,p-eIFα,CHOP and cleaved Caspase-12 and ratio of p-AMPK/AMPK increased notably;the expression of CHOP was silenced;the apoptosis induced by pc-MEG3 could be significantly decreased.In animal experiments,compared with control group,the tumor volume and number of positive cells of Ki67 decreased significantly;the apoptosis rate,protein levels of CHOP,cleaved Caspase-12 and cleaved Caspase-3 increased markedly.Conclusion MEG3 can promote colon cancer cell apoptosis by endoplasmic reticulum stress,and CHOP may play a crucial role in the process.Furthermore,AMPK/mTOR is also involved in the regulation of apoptosis by MEG3.