LncRNA LINC01194在非小细胞肺癌中对miR-302e的影响及机制研究

The Impacts and Mechanism of lncRNA LINC01194 on miR-302e in Non-Small Cell Lung Cancer

目的:初步探索长链非编码RNA(lncRNA LINC01194)在非小细胞肺癌(NSCLC)中对miR-302e的影响及机制。方法:实时荧光定量PCR(qRT-PCR)分别检测NSCLC组织、癌旁组织和人肺正常上皮细胞BEAS-2B及NSCLC细胞株中lncRNA LINC01194、miR-302e表达,筛选最佳干预细胞系;将NCI-H1975细胞分为:control组(正常培养)、si-NC组(转染si-NC)、si-LINC01194组(转染si-LINC01194)、si-LINC01194+anti-miR-NC组(si-LINC01194和anti-miR-NC共转染)、si-LINC01194+anti-miR-302e组(si-LINC01194和anti-miR-302e共转染);qRT-PCR检测细胞中lncRNA LINC01194、miR-302e的表达;MTT法、平板克隆实验、Transwell小室、流式细胞仪分别检测细胞增殖、迁移及侵袭、凋亡;Western blot方法检测增殖细胞核抗原(PCNA)、基质金属蛋白酶-2(MMP-2)、B细胞淋巴瘤蛋白2(Bcl-2)、Bcl-2相关X蛋白(Bax)的表达;双荧光素酶报告基因实验验证lncRNA LINC01194与miR-302e靶向调控关系。结果:在NSCLC组织和细胞中,lncRNA LINC01194表达水平升高,miR-302e表达水平降低(P<0.05);抑制lncRNA LINC01194表达可显著抑制NCI-H1975细胞增殖、迁移与侵袭,促进细胞凋亡(P<0.05);抑制miR-302e表达可逆转抑制lncRNA LINC01194表达对NCI-H1975细胞增殖、迁移、侵袭的抑制作用及对细胞凋亡的促进作用(P<0.05);双荧光素酶报告基因实验证实lncRNA LINC01194与miR-302e存在靶向调控关系。结论:在NSCLC组织和细胞中lncRNA LINC01194上调表达,miR-302e下调表达,抑制lncRNA LINC01194可通过上调miR-302e表达,从而抑制NSCLC细胞增殖、迁移与侵袭,促进其凋亡。

Objective:To explore the impacts and mechanism of long non-coding RNA(lncRNA LINC01194)on miR-302e in non-small cell lung cancer(NSCLC).Methods:Real-time fluorescence quantitative PCR(qRT-PCR)was applied to detect the expression of lncRNA LINC01194 and miR-302e in NSCLC tissue,paracancerous tissue,human lung normal epithelial cells BEAS-2B,and NSCLC cell lines,respectively,to screen the best intervention cell line;NCI-H1975 cells were divided into:control group(normal culture),si-NC group(transfected with si-NC),si-LINC01194 group(transfected with si-LINC01194),si-LINC01194+anti miR-NC group(co-transfected with si-LINC01194 and anti miR-NC),and si-LINC01194+anti miR-302e group(co-transfected with si-LINC01194 and anti miR-302e);qRT-PCR was applied to detect the expression of lncRNA LINC01194 and miR-302e in cells;MTT assay,plate cloning assay,Transwell chamber flow cytometry were applied to detect cell proliferation,migration,invasion and apoptosis,respectively;Western blot was applied to detect the expression of proliferating cell nuclear anti-gen(PCNA),matrix metalloproteinase-2(MMP-2),B-cell lymphoma protein 2(Bcl-2),and Bcl-2 as-sociated X protein(Bax);and double Luciferase reporter gene experiment was applied to verify the targeting regulation relationship between lncRNA LINC01194 and miR-302e.Results:In NSCLC tissues and cells,the expression level of lncRNA LINC01194 increased,while the expression level of miR-302e decreased(P<0.05);inhibiting the expression of lncRNA LINC01194 was able to obviously inhibit the proliferation,migra-tion,and invasion of NCI-H1975 cells,and promote cell apoptosis(P<0.05);inhibiting miR-302e expres-sion was able to reverse the inhibitory effect of lncRNA LINC01194 expression on NCI-H1975 cell prolifera-tion,migration,invasion,and promote cell apoptosis(P<0.05);double Luciferase reporter gene experiment confirmed that there was a targeted regulatory relationship between lncRNA LINC01194 and miR-302e.Con-clusion:lncRNA LINC01194 is up-regulated and miR-302e is down-regulated in NSCLC tissues and cells,inhibiting lncRNA LINC01194 can up-regulate the expression of miR-302e,thereby inhibiting the prolifera-tion,migration,and invasion of NSCLC cells and promoting their apoptosis.