lncRNA PCGEM1对肺癌A549细胞恶性生物学行为的影响及其作用机制

目的:探讨长链非编码RNA(long-chain noncoding,lncRNA)PCGEM1对肺癌A549细胞恶性生物学行为的影响及其作用机制。方法:收集2016年3月至2018年5月湖北医药学院附属太和医院胸外科接受手术治疗的62例肺癌(lung cancer,LC)患者癌组织及相应的癌旁组织标本,并用以上组织构建LC组织芯片。用qPCR检测lncRNA PCGEM1及miR-148a在LC组织相应的癌旁组织及LC细胞株中的表达。构建lncRNA PCGEM1沉默细胞系A549-siPCGEM1和阴性对照A549-NC,并以A549细胞作为空白对照(Control组),用MTT和平板克隆实验检测敲减PCGEM1对A549细胞增殖能力的影响,Transwell和划痕实验检测敲减PCGEM1对A549细胞侵袭和迁移能力的影响。使用生物信息学网站StarBase预测可互补结合PCGEM1的miRNA,再根据Targetscan网站预测相应可靶向结合miRNA的基因;Western blotting实验检测TGF-β2/Smad2信号通路蛋白表达情况。结果:在LC组织中PCGEM1的表达水平高于癌旁组织而miR-148a的表达量明显低于癌旁组织(均P<0.05),PCGEM1在5种LC细胞株中的表达明显高于人肺成纤维细胞HLF-02(均P<0.05),且以A549细胞中表达最高。敲减PCGEM1后,与Control组和A549-NC组比较,A549-siPCGEM1组A549细胞增殖、侵袭和迁移能力显著降低(均P<0.05)。StarBase和Targetscan网站预测结果显示,PCGEM1可与miR-148a互补结合,miR-148a与TGF-β2存在靶向结合位点。与Control组和A549-NC组比较,A549-siPCGEM1组中miR-148a表达明显升高,TGF-β2及p-Smad2蛋白的表达明显降低(均P<0.05)。结论:lncRNA PCGEM1在LC组织和细胞株中高表达,高表达的PCGEM1可通过下调miR-148a水平强化TGFβ2/Smad2信号通路,从而促进A549细胞恶性生物学行为的进展。

Effect of lncRNA PCGEM1 on malignant biological behavior of lung cancer A549 cells and its mechanism

Objective:To investigate the long-chain noncoding RNA(Lnc RNA)PCGEM1 regulating the lung cancer(LC)cell invasion and metastasis through the TGF-β/Smad signaling pathways.Methods:From March 2016 to May 2018,total 62 cases of LC patients receiving surgical treatment in our hospital were collected,including cancer tissues and normal tissues more than 2 cm away from the cancer tissues.qRT-PCR was used to detect the expression of lncRNA PCGEM1 and miR-148a in LC,corresponding para-cancer tissues and different LC cell strains.LncRNA PCGEM1 silenced cell line A549-siPCGEM1 and negative control A549-NC were constructed,and A549 was used as blank control.MTT and plate cloning assay were used to detect the effect of PCGEM1 on the proliferation of A549 cells.Transwell and scratch assay were used to detect the effect of PCGEM1 on the invasion and migration of A549 cells.The bioinformatics website StarBase was used to predict the complementary binding miRNA of PCGEM1.Furthermore,according to the website Targetscan,the genes that the corresponding miRNAs could target and bind were predicted.Results:qRT-PCR results showed that the expression of PCGEM1 in LC tissues and lung cancer cell lines was higher than that in normal tissues,and the expression level of miR-148a was lower than that in normal tissues(all P<0.05).The expression level of PCGEM1 in A549 cells was the highest,and the difference was statistically significant compared with other cell lines(P<0.05).After successful construction of PCGEM1 silenced cells,compared with the blank control group and A549-NC group,the cell OD492nm value of A549-siPCGEM1 group was significantly decreased,the number of cell clones and the number of matrigel matrix gels was significantly reduced,the cell migration rate was significantly reduced,the differences were statistically significant(P<0.05).According to the prediction results of StarBase website,PCGEM1 could be complementary to miR-148a,and the prediction analysis on microRNA.org website shows that miR-148a had a targeted binding site with TGF-β2.qRT-PCR and Western blotting results showed that the expression of miR-148a was significantly increased in the A549-siPCGEM1 group compared with the blank control group and A549-NC group,and the expression of TGF-β2 and p-Smad 2 was significantly decreased(P<0.05),while the expression of the above indicators in the blank control group and A549-NC group was not statistically significant(P>0.05).Conclusion:Lnc RNA PCGEM1 is highly expressed in lung cancer.High expression of PCGEM1 may enhance the TGF-β2/Smad2 signaling pathway by downregulation of miR-148a,thus promoting the development of LC and the malignant biological behavior.