miR-218靶向LASP1调控TGF-β/Smad信号通路调节肺癌细胞的侵袭和转移

miR-218 targets LASP1 in regulation of TGF-β/Smad signaling pathway regulating invasion and metastasis of lung cancer cells

目的:观察miR-218对肺癌细胞生物学行为的影响并探讨其可能的作用机制。方法:收集62例肺癌患者癌组织及癌旁正常组织,qRT-PCR和免疫组化方法检测miRNA-218、LASP1和TGF-β的表达情况;通过脂质体转染法转染肺癌细胞系A549,从而构建miRNA-218过表达细胞模型及空载体组,并以A549细胞作为空白对照组;观察过表达miRNA-218对A549细胞生物学行为的影响,采用CCK-8法检测过表达miRNA-218对A549细胞增殖的影响;通过划痕实验及Transwell侵袭实验检测过表达miRNA-218对A549细胞侵袭和迁移能力的影响;StarBase数据库及荧光素酶报告基因实验检测miRNA-218与LASP1的靶向关系;Western blot法检测细胞转染前后LASP1、TGF-β及Smad蛋白的表达。结果:qRT-PCR结果显示,肺癌组织中miRNA-218的表达水平低于正常组织,且与患者肿瘤浸润深度(P=0.004)、肿瘤大小(P=0.005)、淋巴结转移(P=0.014)以及TNM分期(P=0.021)密切相关,肺癌组织LASP1和TGF-β阳性表达率高于正常组织(P<0.05);相比于未转染的空白对照组,转染miRNA-218的细胞增殖、侵袭、迁移能力显著降低(P<0.05),空载体组与空白对照组比较无显著差异。StarBase数据库及荧光素酶报告基因实验结果显示miRNA-218与LASP1存在靶向关系。与空白对照组相比,转染miRNA-218的A549肺癌细胞LASP1、TGF-β及Smad蛋白的表达水平明显降低(P<0.05),空载体组的表达水平无明显变化。结论:miRNA-218可能通过靶向抑制LASP1蛋白的表达,进而调控TGF-β/Smad信号通路,从而抑制肺癌细胞的侵袭和迁移。

Objective:To observe the effect of miR-218 on the biological behavior of lung cancer cells and explore its possible mechanism.Methods:The expressions of miRNA-218,LASP1 and TGF-βwere detected by qRT-PCR and immunohistochemistry.A549 lung cancer cells were transfected by liposome transfection method,so as to construct miRNA-218 overexpression cell model and empty vector group,and A549 cells were used as blank control group.The effects of overexpression of miRNA-218 on biological behavior of A549 cells were observed,and the effects of overexpression of miRNA-218 on proliferation of A549 cells were detected by CCK-8 method.Scratch test and Transwell invasion test were used to detect the effect of overexpression of miRNA-218 on invasion and migration ability of A549 cells.The targeting relationship between miRNA-218 and LASP1 was detected by StarBase database and lu-ciferase reporter gene experiment.The expression of LASP1,TGF-βand Smad protein before and after transfection was detected by Western blot.Results:The qRT-PCR results showed that the expression level of miRNA-218 in lung cancer tissues was lower than that in normal tissues,and was closely related to the depth of tumor invasion(P=0.004),tumor size(P=0.005),lymph node metastasis(P=0.014)and TNM stage(P=0.021).The positive expression rates of LASP1 and TGF-βin lung cancer tissues were higher than that in normal tissues(P<0.05).Compared with the blank control group without transfection,the proliferation,invasion and migration abilities of the transfected miRNA-218 cells were significantly reduced compared with the blank control group(P<0.05),and there was no significant difference between the empty vector group and the blank control group.StarBase database and luciferase reporter gene ex-periment results showed that there was a targeted relationship between miRNA-218 and LASP1.Compared with the blank control group,the expression levels of LASP1,TGF-βand Smad proteins in the A549 lung cancer cells transfected with miRNA-218 were significantly decreased(P<0.05),while the expression levels in the empty vector group were not significantly changed.Conclusion:The miR-218 can regulate the TGF-β/Smad signaling pathway by targeting the expression of LASP1 protein,thus inhibiting the invasion and migration of lung cancer cells.