miR-490-3p通过靶向调控TGFBR1抑制非小细胞肺癌细胞的增殖和侵袭

miR-490-3p Inhibits the Proliferation and Invasion of NonSmall Cell Lung Cancer Cells by Targeting TGFBR1

目的:以非小细胞肺癌A549细胞为模型,探讨miR-490-3p在肺癌发生发展过程中的作用及其调控机制。方法:通过miRBase数据库获得miR-490-3p序列,设计miR-490-3pmimics并转染A549细胞,CCK8、细胞划痕及Transwell实验分别检测miR-490-3p过表达对A549细胞增殖、迁移和侵袭能力的影响;使用miRwalk在线工具预测miR-490-3p可能的调控基因,通过实时荧光定量PCR及Western印迹对候选调控基因进行筛选,最后通过双萤光素酶报告基因实验验证miR-490-3p与调控基因之间的靶向关系。结果:过表达miR-490-3p可显著抑制A549细胞的增殖、侵袭和迁移能力;在预测的miR-490-3p候选靶基因中,选择与细胞增殖、迁移等表型相关的RASAL2、TGFBR1、PAPPA、HMGA2、TGFA靶基因进行实时荧光定量PCR及Western印迹筛选,结果仅TGFBR1基因在mRNA和蛋白水平的表达与miR-490-3p水平呈负相关,且双萤光素酶报告实验证实miR-490-3p可直接与TGFBR1的3'-UTR结合并抑制其表达。结论:miR-490-3p通过靶向调控TGFBR1的表达抑制非小细胞肺癌A549细胞的增殖和侵袭。

Objective: The effects and regulatory mechanism of miR-490-3p in the oncogenesis and progression of lung cancer were studied using A549 model cells. Methods: The miR-490-3p sequence was obtained from miRBase, miR-490-3p mimics was designed and transfected into A549 cells. CCK8, wound healing and transwell assay were used to detect the effect of overexpression of miR-490-3p on proliferation, migration and invasion phenotypes of A549 cells, respectively. The online miRwalk tool was used to predict the possible target genes of miR- 490-3p, and candidate regulatory genes were screened by quantitative real-time PCR and Western blot. Finally, the targeted relationship between miR-490-3p and regulatory genes was validated by double-luciferase reporter gene system. Results: Overexpression of miR-490-3p significantly inhibited the proliferation, invasion and migration of A549 cells. Among the proliferation and migration related genes, the RASAL2, TGFBR1, PAPPA, HMGA2, TGFA were chosen as candidate target genes of miR-490-3p. Quantitative real-time PCR and Western blot assay showed that only TGFBR1 gene revealed negatively correlation with miR-490-3p both in mRNA and protein level,and double-luciferase reporter gene experiment confirmed that miR-490-3p could directly bind TGFBR1 3'-UTR and inhibit its expression. Conclusion: miR-490-3p inhibits the proliferation and invasion phenotypes of non- small cell lung cancer A549 cells by targeting and regulating the expression of TGFBR1.