miR-335-5p通过靶向Oct4调控胃癌细胞的增殖

miR-335-5p inhibits the proliferation of gastric cancer cells by tagerting Oct4

摘要:

目的：研究微小（miR）-335-5p对胃癌细胞增殖的影响及其机制。方法：采用荧光定量PCR检测miR-335-5p在胃癌细胞系SGC-7901、BGC-823、MKN-28、MKN-45和人正常胃黏膜上皮细胞系GES-1中的表达水平，利用miR-335-5p过表达慢病毒及封闭慢病毒分别感染SGC-7901和BGC-823细胞株，实验分为SGC-7901细胞中miR-335-5p组（转染过表达miR-335-5p组）和阴性对照组（空病毒组，NC），BGC-823细胞中antago-miR-335-5p组（转染封闭miR-335-5p慢病毒）和阴性对照组（空病毒组，NC）。通过CCK-8实验、平板克隆实验、EdU实验检测miR-335-5p对细胞增殖的影响，用生物信息学软件预测miR-335-5p可能的靶基因，通过Western blot验证miR-335-5p对靶基因的调控作用并检测AKT及pAKT的表达。结果：miR-335-5p在胃癌细胞系中表达明显下降。CCK-8实验、平板克隆实验及EdU实验中过表达miR-335-5p组光密度值、克隆形成率、EdU阳性细胞率均低于其阴性对照组，而封闭miR-335-5p组光密度值、克隆形成率、EdU阳性细胞率均高于其阴性对照组。同时用生物信息学软件预测了Oct4作为miR-335-5p的靶基因，Western blot表明过表达miR-335-5p组Oct4的表达量（0.469±0.054）明显低于阴性对照组（0.670±0.045）（P=0.008），封闭miR-335-5p组Oct4的表达（0.804±0.046）高于阴性对照组（0.600±0.073）（P=0.015），同时双荧光素酶报告实验证明miR-335-5p能够靶向Oct4；过表达miR-335-5p组中pAKT的表达量（0.525±0.046）较阴性对照组（0.847±0.053）有所下降（P=0.001），封闭miR-335-5p组（0.841±0.069）中pAKT的表达量较阴性对照组（0.563±0.063）有所升高（P=0.007）。结论：miR-335-5p可以通过靶向Oct4抑制AKT通路从而抑制胃癌细胞的增殖。

Abstract:

Objective：To investigate the effect of miR-335-5p on the proliferation of gastric cancer cells and its underlying mecha－nism. Methods：The expression of miR-335-5p in gastric cancer cell lines BGC-823，MKN-28，MKN-45 and normal gastric mucosal epithelial cell line GES-1 were tested by qRT-PCR. SGC-7901 and BGC-823 cell lines were transfected by overexpression and antagonist lentivus respectively and the experimental groups were divided into：miR-335-5p group（transfected with miR-335-5p overexpression lentivus） and negative control group（transfected with empty lentivus，NC） in SGC-7901 cell line，and antago-miR-335-5p group（transfected with antagonist lentivus） and negative control group（transfected with empty lentivus，NC） in BGC-823 cell line. The effect of miR-335-5p on the proliferation of gastric cancer cells was tested by CCK-8 assay，colony formation assay and EdU assay. The target gene of miR-335-5p was predicted by bioinformatics software. Western blot was selected to test the expression level of Oct4，AKT and pAKT. Results：The expression level of miR-335-5p was downregulated in gastric cancer cell lines compared with that in normal gastric mucosal epithelial cell line. CCK-8 assay，colony formation assay and EdU assay showed that the absorbance（A） value，colony formation number and EdU positive cell in overexpression miR-335-5p group were lower than those in NC group while antago-miR-335-5p group had higher A value，colony formation number and EdU positive cell than NC group. Oct4 was pre－dicted as a target gene of miR-335-5p. The protein expression level of Oct4 was lower in miR-335-5p group（0.469±0.054） than in NC group（0.670±0.045）（P=0.008）；the protein ex－pression level of Oct4 was higher in antago-miR-335-5p group（0.804±0.046） than in NC group（0.600±0.073）（P=0.015），and dual luciferase reporter assay was conducted to prove that miR-335-5p could target Oct4. The expression level of pAKT was lower in overexpression miR-335-5p group（0.525±0.046） than in its NC group（0.847±0.053）（P=0.001）；the expression level of pAKT was higher in antago-miR-335-5p group（0.841±0.069） than in NC group（0.563±0.063）（P=0.007）. Conclusion：miR-335-5p works as a tumor suppressor to inhibit the proliferation of gastric cancer cells by targeting Oct4 to inhibit the AKT pathway.