摘要
目的 本研究通过体外培养胃癌细胞HGC-27,探究lncRNA MIAT能否通过靶向miR-203调控神经元PAS结构域蛋白4(NPAS4)促进胃癌细胞增殖、侵袭、迁移。方法 体外培养HGC-27与GES-1细胞,qRT-PCR法检测各细胞中lncRNA MIAT、miR-203、NAPS4 mRNA表达。双荧光素酶报告基因实验验证lncRNA MIAT与miR-203、miR-203与NPAS4之间的靶向关系。将对数生长期HGC-27细胞分组并进行转染:对照组、si-NC组(转染si-NC)、si-MIAT组(转染si-MIAT)、miR-NC组(转染miR-NC)、miR-203组(转染miR-203)、anti-miR-NC组(转染anti-miR-NC)、anti-miR-203组(转染anti-miR-203)、si-MIAT+anti-miR-NC组(转染si-MIAT+anti-miR-NC)、si-MIAT+anti-miR-203组(转染si-MIAT+anti-miR-203)、pc-NC组(转染pc-NC)、pc-NAPS4组(转染pc-NAPS4)、pc-NAPS4+miR-NC组(转染pc-NAPS4+miR-NC)、pc-NAPS4+miR-203组(转染pc-NAPS4+miR-203),qRT-PCR法检测lncRNA MIAT、miR-203、NPAS4 mRNA表达;MTT法检测细胞增殖活力;Transwell实验检测细胞迁移与侵袭能力;Western blot法检测MMP-3、MMP-9、CyclinD1、NPAS4蛋白表达。结果 与GES-1细胞比较,胃癌细胞HGC-27中lncRNA MIAT、NAPS4 mRNA表达显著升高,miR-203表达显著降低(P<0.05)。双荧光素酶报告基因实验证实,lncRNA MIAT能够靶向miR-203调控NAPS4(P<0.05)。沉默lncRNA MIAT可显著降低细胞活力以及迁移与侵袭能力,下调MMP-3、MMP-9、CyclinD1蛋白表达,降低NAPS4 mRNA和蛋白表达(P<0.05);过表达miR-203可显著降低NAPS4 mRNA和蛋白表达,降低细胞活力以及迁移与侵袭能力,下调MMP-3、MMP-9、CyclinD1蛋白表达(P<0.05);而抑制miR-203表达则与miR-203过表达作用相反,且抑制miR-203表达可显著逆转沉默lncRNA MIAT对细胞活力以及迁移与侵袭能力的抑制作用(P<0.05);过表达NAPS4可显著促进HGC-27细胞增殖、迁移与侵袭,上调MMP-3、MMP-9、CyclinD1蛋白表达(P<0.05),同时其对细胞恶性行为的促进作用可被miR-203过表达削弱(P<0.05)。结论 沉默lncRNA MIAT可通过靶向miR-203调控NAPS4表达,减弱胃癌细胞增殖、侵袭、迁移等细胞生物学行为过程。
Objective In this study, gastric cancer cells HGC-27 were cultured in vitro to explore whether lncRNA MIAT can promote the proliferation, invasion and migration of gastric cancer cells by targeting miR-203 to regulate neuronal PAS domain protein 4(NPAS4). Methods HGC-27 and GES-1 cells were cultured in vitro, and the expressions of lncRNA MIAT, miR-203 and NAPS4 mRNA in each cell were detected by qRT-PCR. Dual-luciferase reporter gene experiment was used to verify the targeting relationship between lncRNA MIAT and miR-203, miR-203 and NPAS4. HGC-27 cells in logarithmic growth phase were grouped and transfected: control group, si-NC group(transfected with si-NC), si-MIAT group(transfected with si-MIAT), miR-NC group(transfected with miR-NC), miR-203 group(transfected with miR-203), anti-miR-NC group(transfected with anti-miR-NC), anti-miR-203 group(transfected with anti-miR-203), si-MIAT+anti-miR-NC group(transfected with si-MIAT+anti-miR-NC), si-MIAT+anti-miR-203 group(transfected with si-MIAT+anti-miR-203), pc-NC group(transfected with pc-NC), pc-NAPS4 group(transfected with pc-NAPS4), pc-NAPS4+miR-NC group(transfected with pc-NAPS4+miR-NC), and pc-NAPS4+miR-203 group(transfected with pc-NAPS4+miR-203). QRT-PCR was used to detect the expression of lncRNA MIAT, miR-203 and NPAS4mRNA;MTT assay was used to detect the cell proliferation activity;Transwell assay was used to detect cell migration and invasion abilities;the protein expressions of MMP-3, MMP-9, CyclinD1 and NPAS4 were detected by Western blot. Results Compared with GES-1 cells, the expressions of lncRNA MIAT and NAPS4 mRNA in gastric cancer cells HGC-27 were obviously increased, and the expression of miR-203 was obviously decreased(P<0.05). Dual-luciferase reporter gene experiments confirmed that lncRNA MIAT could target miR-203 to regulate NAPS4(P<0.05). Silencing lncRNA MIAT could obviously reduce cell viability, migration and invasion abilities, down-regulate MMP-3, MMP-9, CyclinD1 protein expression and NAPS4 mRNA and protein expression(P<0.05);overexpression of miR-203 could obviously reduce NAPS4 mRNA and protein expression, reduce cell viability, migration and invasion abilities, and down-regulate MMP-3, MMP-9, CyclinD1 protein expression(P<0.05);however, inhibiting the expression of miR-203 was opposite to the overexpression of miR-203, and inhibiting the expression of miR-203 could obviously reverse the inhibitory effects of silencing lncRNA MIAT on cell viability, migration and invasion(P<0.05);overexpression of NAPS4 could obviously promote the proliferation, migration and invasion of HGC-27 cells, and up-regulate the protein expressions of MMP-3, MMP-9 and CyclinD1(P<0.05), meantime, its promoting effect on cell malignant behavior could be weakened by overexpression of miR-203(P<0.05). Conclusion Silencing lncRNA MIAT can regulate the expression of NAPS4 by targeting miR-203, and attenuate the cellular biological behaviors such as proliferation, invasion and migration of gastric cancer cells.
作者
刘丹
吴瑾
周红凤
刘东辉
胡晓薇
LIU Dan;WU Jin;ZHOU Hong-feng;LIU Dong-hui;HU Xiao-wei(Department of Head and Neck and Genito-Urinary Oncology,Harbin Medical University Cancer Hospital,Harbin,Heilongjiang,150081;Department of oncology,Heilongjiang Provincial Hospital,Harbin,Heilongjiang,150036)
出处
《现代消化及介入诊疗》
2022年第9期1122-1128,共7页
Modern Interventional Diagnosis and Treatment in Gastroenterology
基金
黑龙江省卫生健康委科研课题(2017-117)。
