miRNA-382靶向LMO3对非小细胞肺癌上皮间质转化的影响

Effect of miRNA-382 Targeting LMO3 on Epithelial-mesenchymal Transition in Non-small Cell Lung Cancer

目的探究miRNA-382靶向LMO3对非小细胞肺癌(NSCLC)上皮间质转化及侵袭的影响。方法收集2017年11月至2018年7月于福建医科大学附属第二医院手术切除的NSCLC组织和癌旁组织标本各60例,采用荧光定量PCR检测NSCLC和癌旁组织中miRNA-382的表达水平。构建miRNA-382过表达(miRNA-382 OE)和质粒对照(miRNA-382 NC)慢病毒稳定感染细胞系,通过生物信息学和荧光素酶基因检测验证miRNA-382与LMO3的靶向关系。采用Western blot分析miRNA-382过表达对LMO3、Vimentin、E-cadherin蛋白水平的影响。采用Transwell实验检测miRNA-382过表达对A549细胞侵袭能力的影响。结果与癌旁组织(1.36±0.27)相比,NSCLC组织中miRNA-382的相对表达水平(0.49±0.10)显著下调,差异有统计学意义(P<0.05)。生物信息学预测LMO3可能是miRNA-382的一个靶基因;荧光素酶报告检测显示,与转染WT-LMO3的miRNA-382 NC细胞比较,转染WT-LMO3的miRNA-382 OE细胞荧光素酶活性显著下降,差异有统计学意义(P<0.05)。与对照组和miRNA-382 NC组比较,miRNA-382 OE组细胞中LMO3和Vimentin的蛋白表达水平显著降低,E-cadherin的蛋白表达水平显著增强,差异有统计学意义(均P<0.05);与miRNA-382 OE组比较,miRNA-382 inhibitor组细胞中LMO3和Vimentin的蛋白表达水平明显上调,E-cadherin的蛋白表达水平则明显降低,差异有统计学意义(均P<0.05)。Transwell小室检测结果表明,miRNA-382 OE组细胞的侵袭能力[(34.72±7.29)个]较对照组[(77.83±9.71)个]和miRNA-382 NC组[(81.16±10.53)个]显著下调,miRNA-382 OE+LMO3组细胞的侵袭能力[(60.77±7.31)个]较miRNA-382 OE组明显上调,差异均有统计学意义(均P<0.05)。结论 miRNA-382可能通过下调LMO3的表达抑制NSCLC细胞的侵袭和上皮间质转化。

Objective To investigate the effect of miRNA-382 targeting LMO3 on epithelial-mesenchymal transition and tumor invasion in non-small cell lung cancer(NSCLC).Methods NSCLC tissue and adjacent tissue from 60 cases were collected in Second Affiliated Hospital of Fujian Medical University from November 2017 to July 2018,and the mRNA expression of miRNA-382 in NSCLC and adjacent tissues were detected by real-time PCR.miRNA-382 OE and miRNA-382 NC lentiviral stable cell lines were constructed,and the targeting relationship between miRNA-382 and LMO3 was verified by bioinformatics and luciferase gene detection.The effect of miRNA-382 overexpression on LMO3,vimentin and E-cadherin protein was analyzed by Western blot.Transwell was used to detect the effect of miRNA-382 overexpression on the invasive ability of A549 cells.Results Compared with adjacent tissues(1.36±0.27),the relative mRNA expression of miRNA-382 in NSCLC tissues(0.49±0.10)was significantly down-regulated(P<0.05).Bioinformatics predicted that LMO3 might be a target gene of miRNA-382.Dual-luciferase reporter gene assay showed that the luciferase activity of miRNA-382 OE cells transfected with WT-LMO3 was significantly decreased compared with miRNA-382 NC cells transfected with WT-LMO3(P<0.05).Compared with the control group and the miRNA-382 NC group,the protein expression levels of LMO3 and Vimentin were significantly decreased in the miRNA-382 OE group,while the expression of E-cadherin was significantly increased(P<0.05).The protein expression levels of LMO3 and Vimentin were significantly up-regulated in the miRNA-382 inhibitor group compared with the miRNA-382 OE group,while the protein expression levels of E-cadherin was significantly decreased(P<0.05).Transwell assay showed that the invasive ability of miRNA-382 OE group(34.72±7.29)was significantly lower than that of control group(77.83±9.71)and miRNA-382 NC group(81.16±10.53),whereas the invasive ability of miRNA-382 OE+LMO3 group(60.77±7.31)was significantly greater than that of miRNA-382 OE group(P<0.05).Conclusion miRNA-382 may inhibit invasion and epithelial-mesenchymal transition of NSCLC cells by down-regulating the expression of LMO3.