摘要
目的:分析miR-449a对人非小细胞肺癌细胞株A549增殖、侵袭和迁移的影响。方法:通过Real-time PCR检测人肺成纤维细胞MRC-5、人非小细胞肺癌细胞株A549和HCC827中miR-449a和组蛋白去乙酰化酶1(HDAC1)的表达水平。利用CCK-8法和Transwell法分析miR-449a对A549细胞增殖、侵袭和迁移能力的影响。使用生物信息软件分析miR-449a的靶向集合位点,并构建荧光素酶报告基因质粒,通过荧光素酶报告基因法分析miR-449a对HDAC1的靶向调控作用。通过Western blot分析miR-449a和HDAC1表达水平对A549细胞增殖及EMT相关分子表达水平的影响。建立裸鼠皮下瘤模型分析miR-449a和HDAC1对A549细胞体内增殖的影响。结果:miR-449a在MRC-5细胞中表达水平显著高于A549(P<0.0001)和HCC827(P<0.01);HDAC1在MRC-5细胞中表达水平显著低于A549(P<0.0001)和HCC827(P<0.01)。CCK-8法检测显示在A549细胞中转染miR-449a能够显著抑制其增殖。Transwell法检测显示在A549细胞中转染miR-449a能够显著抑制其侵袭和迁移(P<0.0001)。生物信息学预测显示miR-449a能够靶向结合HDAC1的3'-UTR;双荧光素酶报告基因分析显示,miR-449a能够靶向作用于HDAC1的3'-UTR抑制萤光素酶表达,Real-time PCR的结果表明过表达miR-449a能够显著抑制HDAC1的表达。在A549细胞中转染miR-449a或针对HDAC1小干扰RNA(siRNA)均能够下调HDAC1的表达,抑制细胞体内、外增殖;同时E-cadherin和ZO-1的表达水平下降,而N-cadherin、Fibronectin和Vimentin的表达水平增加。结论:miR-449a通过靶向抑制HDAC1的表达,抑制肺癌细胞的增殖、侵袭和迁移。
Objective:To analyze the effects of miR-449a on the proliferation,invasion and migration of human non-small cell lung cancer cell line A549.Methods:The expression levels of miR-449a and histone deacetylase 1(HDAC1)in human lung fibroblasts MRC-5 and human non-small cell lung cancer cell lines A549 and HCC827 were detected by real-time PCR.The effects of miR-449a on proliferation,invasion and migration of A549 cells were analyzed by CCK-8 method and Transwell method.Bioinformatics software was used to analyze the targeted collection site of miR-449a,and luciferase reporter gene plasmid was constructed,and the targeted regulation of miR-449a on HDAC1 was analyzed by luciferase reporter gene method.Western blot was used to analyze the effects of miR-449a and HDAC1 expression levels on A549 cell proliferation and expression levels of epithelial mesenchymal transformation(EMT)-related molecules.The subcutaneous tumor model of nude mice was established to analyze the effects of miR-449a and HDAC1 on A549 cell proliferation in vivo.Results:The expression level of miR-449a in MRC-5 cells was significantly higher than that of A549(P<0.0001)and HCC827(P<0.01).HDAC1 expression level in MRC-5 cells was significantly lower than that of A549(P<0.0001)and HCC827(P<0.01).CCK-8 assay showed that transfection of miR-449a in A549 cells could significantly inhibit its proliferation.Transwell assay showed that transfection of miR-449a in A549 cells significantly inhibited its invasion and migration(P<0.0001). Bioinformatics prediction showed that miR-449a could target the 3'-UTR of HDAC1.Dual luciferase reporter gene analysis showed that miR-449a could target the 3'-UTR of HDAC1 to inhibit luciferase expression,and real-time PCR results showed that overexpression of miR-449a significantly inhibited HDAC1 expression.Transfection of miR-449a or small interfering RNA(siRNA)targeting HDAC1 in A549 cells down-regulated HDAC1 expression and inhibited cell proliferation in vitro and in vivo.Meanwhile,the expression levels of E-cadherin and ZO-1 decreased,while the expression levels of N-cadherin,Fibronectin and Vimentin increased.Conclusion:miR-449a inhibits the proliferation,invasion and migration of lung cancer cells by targeted inhibition of HDAC1 expression.
作者
黄洁
余荣环
王萌萌
王葆青
HUANG Jie;YU Ronghuan;WANG Mengmeng;WANG Baoqing(Department of Respiratory and Critical Care Medicine,Shanghai Xuhui Central Hospital,Zhongshan-Xuhui Hospital,Fudan University,Shanghai 200031,China;Department Respiratory and Critical Care Medicine,Zhongshan Hospital,Fudan University,Shanghai 200032,China)
出处
《现代肿瘤医学》
CAS
北大核心
2022年第22期4046-4051,共6页
Journal of Modern Oncology
基金
上海市医学重点专科项目(编号:ZK2019C14)
上海市徐汇区医学科研项目(编号:SHXH202006)。
