LncRNA NEAT1靶向调控miR-582-5p/COL5A1信号通路对肺癌A549细胞生物学功能的影响

Effect of LncRNA NEAT1 on the Biological Function of Lung Cancer A549 Cell through Targeted Regulation of the miR-582-5p/COL5A1 Signaling Pathway

目的:探讨长链非编码RNA富含核的丰富转录物1(LncRNA NEAT1)调控miR-582-5p/COL5A1信号通路对肺癌A549细胞生物学功能的影响。方法:体外培养人肺癌A549细胞并随机分为对照组、si-NEAT1组(转染LncRNA NEAT1 siRNA质粒)、miR-582-5p mimics组(转染miR-582-5p mimics)、si-NC+miR-582-5p-NC组(共转染LncRNA NEAT1 siRNA阴性对照与miR-582-5p阴性对照)、si-NEAT1+miR-582-5p inhibitor组(共转染LncRNA NEAT1 siRNA质粒与miR-582-5p inhibitor),分组转染后以实时荧光定量PCR实验检测细胞LncRNA NEAT1、miR-582-5p、COL5A1表达;构建其裸鼠移植瘤模型,测量裸鼠移植瘤质量、体积。以CCK-8实验、克隆形成实验检测细胞增殖;以免疫印迹实验检测细胞及裸鼠移植瘤组织增殖相关蛋白(cyclin D1、PCNA)表达;以Transwell实验检测细胞迁移及侵袭;以免疫印迹实验检测细胞上皮-间质转化(EMT)相关蛋白(Vimentin、MMP2、E-cadherin)表达;以双荧光素酶报告基因实验检测细胞中LncRNA NEAT1对miR-582-5p、miR-582-5p对COL5A1的靶向调控作用。结果:与对照组相比,si-NEAT1组、miR-582-5p mimics组细胞COL5A1 mRNA表达、细胞活力和克隆形成率、裸鼠移植瘤质量和体积、细胞及裸鼠移植瘤组织cyclin D1、PCNA蛋白表达、细胞迁移和侵袭数目、细胞Vimentin和MMP2蛋白表达均降低(P<0.05),细胞miR-582-5p表达、E-cadherin蛋白表达均升高(P<0.05);si-NC+miR-582-5p-NC组各指标均无显著差异(P>0.05)。与si-NEAT1组相比,si-NEAT1+miR-582-5p inhibitor组细胞COL5A1 mRNA表达、细胞活力和克隆形成率、裸鼠移植瘤质量和体积、细胞及裸鼠移植瘤组织cyclin D1、PCNA蛋白表达、细胞迁移和侵袭数目、细胞Vimentin和MMP2蛋白表达均升高(P<0.05),细胞miR-582-5p表达、E-cadherin蛋白表达均降低(P<0.05)。LncRNA NEAT1可靶向下调A549细胞miR-582-5p,且miR-582-5p可靶向下调A549细胞COL5A1(P<0.05)。结论:敲低LncRNA NEAT1可通过上调miR-582-5p表达而降低COL5A1表达,从而抑制肺癌A549细胞的体内外生长,并可减弱其侵袭与迁移活性。

Objective:To investigate the effect of long non-coding RNA nuclear enriched abundant transcript 1(LncRNA NEAT1)on the biological function of lung cancer A549 cells by regulating the miR-582-5p/COL5A1 signaling pathway.Methods:Human lung cancer A549 cells were cultured in vitro and randomly grouped into a control group,si-NEAT1 group(transfected with LncRNA NEAT1 siRNA plasmid),miR-582-5p mimics group(transfected with miR-582-5p mimics),si-NC+miR-582-5p-NC group(co transfected with LncRNA NEAT1 siRNA negative control and miR-582-5p negative control),and si-NEAT1+miR-582-5p inhibitor group(co transfected with LncRNA NEAT1 siRNA plasmid and miR-582-5p inhibitor).After grouping and transfection,real-time fluorescence quantitative PCR experiment was applied to detect the expression of LncRNA NEAT1,miR-582-5p,and COL5A1 in cells.Nude mouse transplantation tumor model was constructed,and the mass and volume of the nude mouse transplantation tumor were measured.CCK-8 experiment and clone formation experiment were applied to detect cell proliferation.Immunoblotting experiments were applied to detect the expression of proliferation related proteins(cyclin D1,PCNA)in cells and nude mouse transplanted tumor tissues.Transwell experiment was applied to detect cell migration and invasion.Immunoblotting experiment was applied to detect the expression of epithelial mesenchymal transition(EMT)related proteins(Vimentin,MMP2,E-cadherin)in cells.Dual luciferase reporter gene assay was applied to detect the targeted regulatory effects of LncRNA NEAT1 on miR-582-5p and miR-582-5p on COL5A1 in cells.Results:Compared with the control group,the expression of COL5A1 mRNA,cell viability and clone formation rate,weight and volume of nude mouse transplanted tumors,expression of cyclin D1 and PCNA proteins,numbers of cell migration and invasion,and expression of Vimentin and MMP2 proteins in cells and nude mouse transplanted tumor tissues reduced in the si-NEAT1 group and miR-582-5p mimics group(P<0.05),the expression of miR-582-5p and E-cadherin protein in cells increased(P<0.05);there was no great difference in all indicators in the si-NC+miR-582-5p-NC group(P>0.05).Compared with the si-NEAT1 group,the expression of COL5A1 mRNA,cell viability and clone formation rate,weight and volume of nude mouse transplanted tumors,expression of cyclin D1 and PCNA proteins,numbers of cell migration and invasion,and expression of Vimentin and MMP2 proteins in cells and nude mouse transplanted tumor tissues increased in the si-NEAT1+miR-582-5p inhibitor group(P<0.05),the expression of miR-582-5p and E-cadherin protein in cells decreased(P<0.05).LncRNA NEAT1 was able to target downregulation of miR-582-5p in A549 cells,and miR-582-5p was able to target downregulation of COL5A1 in A549 cells(P<0.05).Conclusion:Knocking down LncRNA NEAT1 can reduce COL5A1 expression by upregulating miR-582-5p expression,thereby inhibiting in vitro and in vivo growths of lung cancer A549 cells and weakening their invasion and migration activities.