LMP2A对胃癌细胞增殖及凋亡的影响

The effects of LMP2A on the proliferation and apoptosis of gastric cancer cells

目的探讨膜潜伏蛋白2A(LMP2A)对胃癌细胞增殖、凋亡的影响以及可能的调控机制。方法选取2017年11月-2019年11月于郑州煤炭工业(集团)有限责任公司总医院确诊的105例胃癌组织,以及胃癌细胞SGC7901、MGC-803、AGS为研究对象。采用Western blot检测LMP2A蛋白表达;将LMP2A的敲低质粒Sh-LMP2A-1慢病毒、LMP2A的敲低质粒Sh-LMP2A-2慢病毒,及其空载质粒慢病毒分别感染SGC7901细胞,并分别命名为ShLMP2A-1组、Sh-LMP2A-2组、Sh-NC组,另取未感染的SGC7901细胞作为空白组,Western blot检测各组细胞中LMP2A、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白及核因子κB(NF-κB)信号通路相关蛋白的表达;CCK-8法、克隆形成实验检测各组细胞增殖情况;流式细胞术检测各组细胞凋亡情况。结果胃癌组织中LMP2A蛋白表达水平为(0.86±0.08),显著高于癌旁组织(0.11±0.01),差异有统计学意义(P<0.05)。与人胃正常上皮细胞株GES-1比较,胃癌细胞系SGC7901、MGC-803、AGS中LMP2A蛋白表达水平显著升高(P<0.05),且LMP2A蛋白在SGC7901细胞中表达水平最高,因此,选择SGC7901细胞为研究对象进行后续实验。与空白组和Sh-NC组比较,ShLMP2A-1组、Sh-LMP2A-2组SGC7901细胞OD450值(24、48、72 h)、细胞克隆数、LMP2A、Bcl-2、磷酸化核因子κB抑制蛋白α(p-IκBα)及磷酸化核因子κB(p-NF-κB)蛋白表达水平显著降低,细胞凋亡率及Bax蛋白表达水平显著升高(P<0.05),NF-κB抑制蛋白α(IκBα)及NF-κB蛋白表达差异无统计学意义(P>0.05);与空白组比较,Sh-NC组SGC7901细胞上述指标差异无统计学意义(P>0.05)。结论LMP2A在胃癌中高表达,沉默LMP2A后可能通过抑制NF-κB信号通路来抑制SGC7901细胞增殖、促进细胞凋亡。

Objective To investigate the effects of latent membrane protein 2A(LMP2A)on the proliferation and apoptosis of gastric cancer cells and the possible regulatory mechanism.Methods 105 cases of gastric cancer tissues collected from November 2017 to November 2019,as well as gastric cancer cells SGC7901,MGC-803,and AGS were used as the research objects.Western Blot was used to detect the expression of LMP2A protein;the SGC7901 cells were infected with LMP2A knockdown plasmid Sh-LMP2A-1 lentivirus,the LMP2A knockdown plasmid Sh-LMP2A-2 lentivirus or the empty plasmid lentivirus,and respectively named as Sh-LMP2A-1 group,Sh-LMP2A-2 group,Sh-NC group.In addition,uninfected SGC7901 cells were taken as the blank group.Western Blot was used to detect the expression of LMP2A,B lymphoma-2(Bcl-2),Bcl-2 related X protein(Bax)protein and nuclear factor kappa B(NF-κB)signaling pathway related proteins in each group.CCK-8 method and clone formation experiment were used to detect cell proliferation;flow cytometry was used to detect cell apoptosis.Results The expression level of LMP2A protein in gastric cancer tissue was significantly higher than that in adjacent tissues(0.86±0.08)vs.(0.11±0.01)(P<0.05).Compared with human gastric normal epithelial cell line GES-1,the expression level of LMP2A protein in gastric cancer cell lines SGC7901,MGC-803,and AGS was significantly increased(P<0.05),and the expression level of LMP2A protein was the highest in SGC7901 cells,therefore,SGC7901 cells were selected as the research object for follow-up experiments.Compared with blank group and Sh-NC group,the OD450value(24,48,72 h),number of cell clones,the expression levels of LMP2A,Bcl-2,phosphorylation inhibitor of Nf-κB(p-IκBα)and phosphorylation NF-κB(p-NF-κB)proteins in SGC7901 cells of Sh-LMP2A-1 group and Sh-LMP2A-2 group were significantly reduced;the apoptosis rate and Bax protein expression were significantly increased(P<0.05),and the expression of NF-κB inhibitory of NF-κBα(IκBα)and NF-κB protein did not change significantly(P>0.05);compared with the blank group,the above indicators of SGC7901 cells in the Sh-NC group did not change significantly(P>0.05).Conclusions LMP2A was highly expressed in gastric cancer.Silencing LMP2A might inhibit SGC7901 cell proliferation and promote cell apoptosis by inhibiting NF-κB signaling pathway.