低氧诱导因子-1α介导的微小RNA-210对胃癌细胞增殖与转移功能的影响及机制

Effect of microRNA-210 induced by hypoxia inducible factor-1αon proliferation and metastasis of gastric cancer cells and the underlying mechanism

目的观察低氧微环境对胃癌细胞增殖、转移功能的影响, 并探讨其相关的分子学机制。方法细胞增殖检测试剂盒(CCK-8)、Transwell法检测SGC-7901细胞经低氧处理后的增殖、迁移及侵袭能力, 应用高通量测序(Illumina Hiseq)对低氧处理后的微小RNA(miRNA, miR)差异基因进行检测;实时定量聚合酶链反应(RT-PCR)法、干扰低氧诱导因子-1α(HIF-1α)表达法验证低氧环境下HIF-1α诱导miR-210的能力;后在正常胃黏膜组织、胃癌及癌周组织中检测miR-210的表达, 分析其表达量与患者临床病理学特征之间相关性;接下来进一步在体外上调/抑制miR-210后, 应用CCK-8、Transwell检测miR-210对胃癌细胞株AGS、SGC-7901增殖、迁移及侵袭能力的影响;后应用生物信息分析、报告基因实验、蛋白质免疫印迹(Western blot)及靶基因干扰实验探讨其发挥功能的具体分子学机制。两组间比较采用t检验。结果与常氧组比较, 低氧可明显促进SGC-7901细胞的增殖(0.43±0.13比0.56±0.17, t=5.43, P<0.05)、迁移(250±28比390±30, t=10.28, P<0.05)及侵袭能力(220±40比310±45, t=9.74, P<0.05);miR-210低氧处理后显著上调的差异miRNA之一;低氧在胃癌细胞中可通过HIF-1α诱导miR-210的表达;胃癌组织中异常表达的miR-210与肿瘤的大小及临床分期相关(P<0.05);与对照组比较, 过表达miR-210可明显促进AGS与SGC-7901细胞的增殖(0.58±0.16比0.73±0.21, t=6.58, P<0.05)、(0.53±0.11比0.63±0.25, t=7.18, P<0.05), 迁移(200±20比295±35, t=8.36, P<0.05)、(185±20比260±10, t=7.54, P<0.05), 及侵袭(150±25比215±30, t=5.87, P<0.05)、(145±25比190±30, t=4.37, P<0.05)能力, 而其拮抗剂却显著抑制胃癌细胞的上述恶性能力;最后Western blot等研究表明磷脂酶和张力蛋白同源物(PTEN)是miR-210在胃癌细胞中发挥促癌功能的靶基因。结论低氧环境下HIF-1α介导miR-210可通过靶向调控PTEN的表达促进胃癌细胞的增殖及转移能力。

Objective To observe the effect of hypoxia on the proliferation and metastasis of gastric cancer cells,and explore its related mechanism.Methods Cell counting kit-8(CCK-8)and Transwell assays were used to detect the proliferation,migration and invasion ability of SGC-7901 cells after treatment with hypoxia,and high throughput sequencing(Illumina)was used to detect the differential miRNA.Reverse transcription polymerase chain reaction(RT-PCR)and gene silencing were used to verify the ability of hypoxia inducible factor-1α(HIF-1α)to induce microRNA-210(miR-210)under the hypoxia.The correlation between miRA-210 expression and the clinicopathological characteristics was analyzed.After up-regulating/down-regulating miR-210 in vitro,CCK-8 and Transwell assays were used to detect the effects of miR-210 on the proliferation,migration and invasion of AGS and SGC-7901 cells.Bioinformatics analysis,reporter gene experiment,Western blotting and target gene interference were performed to explore the related molecular mechanisms.The t-test was used for comparison between two groups,and the analysis of variance was used for comparison between multiple groups.Results Compared with normoxia,hypoxia could significantly promote the proliferation,migration(250±28 vs.390±30,t=10.28,P<0.05)and invasion ability(220±40 vs.310±45,t=9.74,P<0.05)of SGC-7901 cells.miR-210 was one of the differential miRNAs that were significantly up-regulated after hypoxia.The miR-210 expression was related to tumor size and the clinical stage(P<0.05).Overexpression of miR-210 could significantly promote the proliferation and migration(200±20 vs.295±35,t=8.36,P<0.05;185±20 vs.260±10,t=7.54,P<0.05)and invasion(150±25 vs.215±30,t=5.87,P<0.05;145±25 vs.190±30,t=4.37,P<0.05)of AGS and SGC-7901 cells,and its antagonist obviously inhibited the above-mentioned malignant behaviors.The phosphate and tension homology(PTEN)was the target gene of miR-210 in gastric cancer cells.Conclusion Hypoxia-induced miR-210 regulates proliferation and metastasis of gastric cancer cells through targeting PTEN.