长链非编码RNA FGD5-AS1在肺癌组织中的表达及其对细胞增殖的影响

Expression of long non-coding RNA FGD5-AS1 in lung cancer tissue and its effect on cell proliferation

目的:研究长链非编码RNA(LncRNA)FGD5-AS1在肺癌组织中的表达及其对肺癌细胞增殖的影响。方法:RNA-seq分析肺癌组织中LncRNA及微小RNA(miRNA)的表达改变,根据表达量的改变确定候选LncRNA FGD5-AS1;RT-qPCR检测分析肺癌组织及肺癌细胞中FGD5-AS1的表达;免疫荧光检测分析肺癌组织中细胞增殖和抗原Ki-67的表达;通过线性回归分析肺癌组织中FGD5-AS1与Ki-67的表达相关性。生物在线软件联合miRNA测序结果,并通过荧光素酶检测分析FGD5-AS1与miR-129-5p结合可能性。将A549细胞分成空白对照组(Control)、si-FGD5-AS1组、miR-mimics组(miR-129-5p mimics)、si-FGD5-AS1与miR-mimics联合组,利用脂质体法将si-FGD5-AS1、miR-129-5p mimics分别或联合转染入A549细胞,24 h后,利用CCK-8检测细胞活力、Annexin V-FITC/PI双染检测细胞凋亡、蛋白印迹(Western blot)检测细胞凋亡相关蛋白(Bad、Bcl-2、Cleaved Caspase-3)的表达。结果:FGD5-AS1在肺癌组织及肺癌细胞中的表达均显著升高(P<0.01),其表达量与Ki-67的表达呈正相关(r=0.641 8,P=0.000)。miR-129-5p在肺癌组织及细胞中的表达均显著降低(P<0.01),生物在线软件及荧光素酶检测验证,FGD5-AS1与miR-129-5p存在结合位点;在肺癌组织中,miR-129-5p的表达与Ki-67的表达呈负相关(r=-0.752 8,P=0.000)。与Control组比较,si-FGD5-AS1可显著抑制A549细胞的增殖(P<0.01)、促进其凋亡(P<0.01),Bad及Cleaved caspase-3的表达均显著升高(均P<0.01),Bcl-2的表达显著降低(P<0.05);与si-FGD5-AS1组比较,si-FGD5-AS1与miR-mimics联合组可进一步抑制细胞增殖(P<0.01),促进细胞凋亡(P<0.01),Bad及Cleaved caspase-3的表达均显著提高(均P<0.01),Bcl-2的表达显著降低(P<0.01)。结论:肺癌组织中FGD5-AS1的表达显著上调,可能通过抑制hsa-miR-129-5p的表达而促进肺癌细胞增殖,参与肺癌的发生及进展。

Objective:To research the expression of long non-coding RNA(lncRNA)FGD5-AS1 in lung cancer and its effect on cell proliferation of lung cancer.Methods:RNA-seq was used to analyze the expression changes of lncRNA and microRNA(miRNA)in lung cancer tissues,and the candidate lncRNA FGD5-AS1 was determined according to the expression changes.RT-qPCR was used to detect and analyze the expression of candidate FGD5-AS1 in lung cancer tissues and lung cancer cells.Immunofluorescence assay was used to analyze the expression of Ki-67 in lung cancer tissues.The correlation between FGD5-AS1 and Ki-67 expression in lung cancer tissues was analyzed by linear regression.Bioinformatics prediction was combined with miRNA sequencing and luciferase detection was used to analyze the binding possibility of FGD5-AS1 and miR-129-5p.A549 cells were divided into blank control group(Control),si-FGD5-AS1 group,miR-mimics group(miR-129-5p mimics),si-FGD5-AS1 and miR-mimics combined group.si-FGD5-AS1 and miR-129-5p mimics were respectively or jointly transfected into A549 cells by Lipofectamine 3000.24 h later,Cell viability was detected by CCK-8,and apoptosis was detected by Annexin V-FITC/PI double staining,and the expression of apoptosis related proteins(Bad,Bcl-2,Cleaved caspase-3) was detected by Western blot. Results: The expression of FGD5-AS1 in lung cancer tissues and lung cancer cells was significantly increased( P <0.01),and there was a positive correlation between FGD5-AS1 and Ki-67 expression( r =0.641 8, P =0.000).The expression of miR-129-5p in lung cancer tissues and cells was significantly decreased( P <0.01).Bioinformatics prediction and luciferase detection confirmed that there was a binding site between FGD5-AS1 and miR-129-5p.In lung cancer tissues,the expression of miR-129-5p was negatively correlated with that of Ki-67( r =-0.752 8, P =0.000).Compared with the Control group,si-FGD5-AS1 significantly inhibited the proliferation of A549 cells( P <0.01) and promoted apoptosis( P <0.01),and significantly increased the expression of Bad and Cleaved caspase-3(all P <0.01),and significantly decreased the expression of Bcl-2( P <0.05).Compared with si-FGD5-AS1 group,si-FGD5-AS1 and miR-mimics combined group could further inhibit cell proliferation( P <0.01),promote cell apoptosis( P <0.01).The expression of Bad and Cleaved caspase-3 was significantly increased(all P <0.01).The expression of Bcl-2 was significantly decreased( P <0.01). Conclusion: The expression of FGD5-AS1 is significantly up-regulated in lung cancer,which may promote the proliferation of lung cancer cells by inhibiting the expression of hsa-miR-129-5p and participate in the occurrence and progression of lung cancer.