miR-424-5p靶向NLRP1抑制非小细胞肺癌增殖迁移和侵袭

miR-424-5p Targets NLRP1 to Inhibit Proliferation,Migra⁃tion and Invasion of Non-Small Cell Lung Cancer

目的:揭示miR-424-5P及NAcht富含亮氨酸重复蛋白1(NLRP1)在非小细胞肺癌细胞系增殖迁移和侵袭过程的作用机制。方法:利用qRT-PCR检测来自医院的15名非小细胞肺癌患者肿瘤组织和癌旁组织中NLRP1的mRNA水平;通过qRT-PCR检测2种非小细胞肺癌细胞HCC827、A549和正常肺上皮细胞BEAS-2B中NLRP1的mRNA水平;利用CCK-8检测下调表达NLRP1对A549细胞增殖能力的影响,Transwell检测下调表达NLRP1对A549细胞迁移和侵袭的影响;通过Targetscan软件预测,miR-424-5p是NLRP1的调控miRNA之一,将检测细胞分为miRNC组、miR-424-5p mimics组及miR-424-5p inhibitor组,利用qRT-PCR检测3组细胞中miR-424-5p及NLRP1的mRNA水平,Western印迹检测NLRP1蛋白表达水平,双萤光素酶报告基因实验检测miR-424-5p与NLRP1之间靶向调控关系;将肺癌细胞A549分为miR-NC组、miR-424-5p inhibitor组及miR-424-5p inhibitor+si-NLRP1组,利用CCK-8法检测3组细胞的增殖能力,Transwell检测3组细胞的迁移和侵袭能力。结果:非小细胞肺癌患者肿瘤组织中NLRP1表达量明显高于癌旁组织;A549细胞中NLRP1表达量最高(P<0.01),而BEAS-2B中NLRP1表达量最低(P=0.157);下调表达NLRP1抑制A549细胞增殖迁移和侵袭能力;过表达miR-424-5p的A549细胞中NLRP1蛋白表达量降低(P<0.01),而敲低miR-424-5p增加NLRP1的蛋白表达(P<0.01);双萤光素酶报告基因实验验证miR-424-5p能与NLRP1的3'非翻译区结合抑制其表达(P<0.01);敲低miR-424-5p能够促进A549细胞的增殖迁移和侵袭能力(P<0.01),同时敲低miR-424-5p和NLRP1的表达量则可以使A549细胞的增殖迁移和侵袭能力降低到miR-NC的水平。结论:miR-424-5p能够靶向NLRP1抑制非小细胞肺癌A549细胞增殖迁移和侵袭能力。

Objective:To study the mechanism of miR-424-5P and NAcht leucine-rich repeat protein 1(NLRP1)in the proliferation,migration and invasion of non-small cell lung cancer(NSCLC)cell lines.Methods:qRT-PCR was used to detect the mRNA expression level of NLRP1 in tumor tissues and adjacent tissues of 15 non-small cell lung cancer patients from the hospital.qRT-PCR was used to detect the mRNA expression level of NLRP1 in two types of NSCLC cell lines HCC827 and A549,and normal lung epithelial cells BEAS-2B.CCK-8 detected the effect of the proliferation of siNLRP1 A549 cells,detected the effect of migration and invasion of siNLRP1 A549 cells by Transwell.Predicted that NLRP1 was the target gene of miR-424-5p using Targetscan software,qRT-PCR was used to detect mRNA expression level of miR-424-5p and NLRP1 in miR-NC group,miR-424-5p mimics group and miR-424-5p inhibitor group,and Western blotting was used to detect protein expression level of NLRP1 in the three groups.The interaction between miR-424-5p and NLRP1 was detected by double luciferase reporter gene assay.Divided into miR-NC group,miR-424-5p inhibitor group and miR-424-5p inhibitor+siNLRP1 group,CCK-8 was used to detect the proliferation ability of the three groups,and Transwell was used to detect the migration and invasion ability of the three groups.Results:The expression of NLRP1 in NSCLC tumor tissues was significantly higher than that of adjacent tissues.The expression level of NLRP1 was the highest in A549 cells(P<0.01),and the expression level of NLRP1 was the lowest in BEAS-2B cells(P=0.157).Downregulated NLRP1 inhibited the ability of proliferation,migration and invasion in A549 cells.The protein expression level of NLRP1 in A549 cells overexpressed miR-424-5p was reduced(P<0.01),while downregulated the expression level of miR-424-5p increased the protein expression of NLRP1(P<0.01).miR-424-5p could inhibit the expression of NLRP1 by binding 3'-UTR of NLRP1(P<0.01).Down-regulated miR-424-5p promoted the ability of proliferation,migration and invasion in A549 cells(P<0.01).Down-regulated the expression levels of both miR-424-5p and NLRP1 could restore the proliferation,migration and invasion ability of A549 cells.Conclusion:miR-424-5p can target NLRP1 to inhibit the proliferation,migration and invasion of non-small cell lung cancer A549 cells.