LncRNA NORAD通过miR-199a-3p调控ZNF217对非小细胞肺癌细胞增殖、凋亡及化疗敏感性的影响

Impacts of LncRNA NORAD on the Proliferation,Apoptosis,and Chemosensitivity of Non-small Cell Lung Cancer Cells by Regulating ZNF217 through MiR-199a-3p

背景与目的针对非小细胞肺癌(non-small cell lung cancer,NSCLC)的治疗和诊断仍然是医学界的难题,而探究NSCLC发生发展的分子机制是当前研究的热点。长链非编码RNA(long non-coding RNA,lncRNA)NORAD在多种癌细胞中高表达,可能是促进NSCLC发生的分子靶点。探讨LncRNA NORAD通过miR-199a-3p调控锌指蛋白217(zinc finger protein 217,ZNF217)对NSCLC细胞增殖、凋亡及化疗敏感性的影响。方法实时定量聚合酶链式反应(real-time quantitative polymerase chain reaction,qRT-PCR)法检测正常肺上皮细胞BEAS-2B、肺癌H460细胞和顺铂(Cisplatin,DDP)耐药细胞株H460/DDP细胞中NORAD、miR-199a-3p、ZNF217基因表达;将H460/DDP细胞分为control组、si-NC组、si-NORAD组、miR-NC组、miR-199a-3p mimic组、si-NORAD+inhibitor NC组、si-NORAD+miR-199a-3p inhibitor组。检测细胞增殖、细胞凋亡情况,各组细胞NORAD、miR-199a-3p、ZNF217基因表达,Ki-67、caspase-9、ZNF217蛋白表达量;验证miR-199a-3p与NORAD和ZNF217的关系。结果与BEAS-2B细胞相比,H460和H460/DDP细胞中NORAD、ZNF217 mRNA表达显著升高(P<0.05),miR-199a-3p表达显著降低(P<0.05)。与H460细胞相比,H460/DDP细胞中NORAD、ZNF217 mRNA表达显著升高(P<0.05),miR-199a-3p表达显著降低(P<0.05)。与control组和si-NC组相比,si-NORAD组H460/DDP细胞增殖率、NORAD、ZNF217 mRNA表达、Ki-67、ZNF217蛋白表达显著降低(P<0.05),凋亡率、miR-199a-3p表达、caspase-9表达显著升高(P<0.05)。与miR-NC组相比,miR-199a-3p mimic组H460/DDP细胞增殖率、NORAD、ZNF217 mRNA表达、Ki-67、ZNF217蛋白表达显著降低(P<0.05),凋亡率、miR-199a-3p、caspase-9表达显著升高(P<0.05)。与si-NORAD+inhibitor NC组相比,si-NORAD+miR-199a-3p inhibitor组H460/DDP细胞增殖率、ZNF217 mRNA表达、Ki-67、ZNF217蛋白表达显著升高,凋亡率、miR-199a-3p表达、caspase-9表达显著降低(P<0.05)。结论下调NORAD表达可增强miR-199a-3p表达并抑制ZNF217表达,进而抑制H460/DDP细胞增殖,促进其凋亡,增强其DDP化疗敏感性。

Background and objective The treatment and diagnosis of non-small cell lung cancer(NSCLC)is still a difficult problem in the medical community,and exploring the molecular mechanism of the occurrence and development of NSCLC is a hot topic of the current research.Long non-coding RNA(lncRNA)NORAD is highly expressed in a variety of cancer cells.It may be a molecular target that promotes NSCLC.The aim of this study was to investigate the impacts of lncRNA NORAD on the proliferation,apoptosis,and chemosensitivity of NSCLC by regulating zinc finger protein 217(ZNF217)through miR-199a-3p.Methods Real-time quantitative polymerase chain reaction(qRT-PCR)method was applied to detect the expressions of NORAD,miR-199a-3p and ZNF217 genes in normal lung epithelial cells BEAS-2B,lung cancer H460 cells,and Cisplatin(DDP)resistant cell lines H460/DDP.H460/DDP cells were devided into control group,si-NC group,si-NORAD group,miR-NC group,miR-199a-3p mimic group,si-NORAD+inhibitor NC group and si-NORAD+miR-199a-3p inhibitor group.Cell proliferation,apoptosis,the expression of NORAD,miR-199a-3p and ZNF217 genes of cells in each group were detected and the expression levels of Ki-67,caspase-9 and ZNF217 proteins in different cells were also observed.The relationship between miR-199a-3p,NORAD and ZNF217 was vefified.Results Compared with BEAS-2B cells,the expressions of NORAD,ZNF217 mRNA were significantly increased in H460 and H460/DDP cells(P<0.05)but the expression of miR-199a-3p was significantly reduced(P<0.05).Compared with H460 cells,the expression of NORAD and ZNF217 mRNA in H460/DDP cells was significantly increased(P<0.05)and the expression of miR-199a-3p was significantly reduced(P<0.05).Compared with the control group and si-NC group,the proliferation rate,NORAD and ZNF217 mRNA expression,Ki-67 and ZNF217 protein expression of H460/DDP cells in the si-NORAD group were significantly reduced(P<0.05),but the apoptosis rate,miR-199a-3p expression and caspase-9 expression were significantly increased(P<0.05).Compared with the miR-NC group,the proliferation rate,NORAD and ZNF217 mRNA expression,Ki-67 and ZNF217 protein expression of H460/DDP cells in the miR-199a-3p mimic group were significantly reduced(P<0.05),but the apoptosis rate,miR-199a-3p expression and caspase-9 expression were significantly increased(P<0.05).Compared with the si-NORAD+inhibitor NC group,the proliferation rate,ZNF217 mRNA expression,Ki-67 and ZNF217 protein expression of H460/DDP cells in the si-NORAD+miR-199a-3p inhibitor group were significantly increased(P<0.05),the apoptosis rate,miR-199a-3p expression and caspase-9 expression were obviously increased reduced(P<0.05).Conclusion Down-regulating NORAD expression can enhance miR-199a-3p expression and inhibit ZNF217 expression,thereby inhibiting H460/DDP cell proliferation,promoting apoptosis and enhancing its DDP chemotherapy sensitivity.