【摘要】 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-490-3p mimics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。更多还原

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