MicroRNA-575靶向抑制BLID促进非小细胞肺癌细胞的增殖和侵袭  

miR-575 Promotes the Proliferation and Invasion of Non-small Cell Lung Cancer Cell by Negatively Regulating BLID Expression

作　　者：焉春华[1] 史晓东[2] 于方飞[1] 王宏伟[1] 张晓飞[1] 刘雅欣[1] 于瑶[1] 杨原荻[1] 邵玉霞[3] 

机构地区：[1]哈尔滨医科大学附属第二医院呼吸科,黑龙江哈尔滨150081 [2]哈尔滨医科大学附属第二医院神经科,黑龙江哈尔滨150081 [3]哈尔滨医科大学附属第二医院体检中心,黑龙江哈尔滨150081

出　　处：《现代生物医学进展》2017年第23期4432-4436,4524,共6页Progress in Modern Biomedicine

基　　金：黑龙江省卫生计生委科研课题(2016-039)

摘　　要：目的:通过体外实验探讨miR-575对非小细胞肺癌(NSCLC)细胞增殖与侵袭能力的影响及相关机制。方法:采用实时定量PCR法检测不同非小细胞肺癌细胞系中miR-575、BLID的表达;CCK-8法检测转染miR-575模拟物、抑制因子后不同时间A549细胞增殖情况的变化;Transwell法检测A549细胞的侵袭情况;Targetcan法及双荧光素酶检测miR-575对BLID 3'UTR端的靶向作用;Western blot法检测BLID蛋白的表达。结果:A549、SPC-A1、H1299、H1650等人非小细胞肺癌细胞系中miR-575的表达均显著高于永生化的人支气管上皮细胞系16HBE(P<0.001)。MiR-575模拟物转染的A549细胞miR-575的表达明显高于对照组(P<0.001),同时细胞的增殖和侵袭力增强(P<0.05);反之,miR-575抑制因子转染的A549细胞miR-575的表达显著降低,且细胞的增殖和侵袭力明显降低(P<0.01)。Targetscan法预测BLID可能是miR-575的下游靶基因,荧光素酶结果显示miR-575不仅能够有效抑制野生型BLID 3'UTR端的荧光素酶反应(P<0.01),而且能够降低BLID的蛋白表达量(P<0.01)。实时定量PCR结果显示BLID在NSCLC细胞系中均呈现显著的低表达(P<0.001),且转染BLID后,NSCLC细胞的增殖和细胞侵袭被明显抑制(P<0.05),而当miR-575与BLID共转染时,miR-575能够逆转BLID所抑制的细胞增殖和侵袭(P<0.01)。结论:在NSCLC细胞系中,miR-575的表达上调,且能够通过直接作用于下游靶点抑癌基因BLID从而促非小细胞肺癌细胞增殖及侵袭。Objective： To explore the mechanisms of regulation of miR-575 on the proliferation and invasion properties of non-small cell lung cancer cell （NSCLC）. Methods： Real-time PCR was selected to detect the expression ofmiR-575 and BLID in different NSCLC cell lines. CCK-8 assay was processed to measure the alternations of A549 cell proliferation at different time points after transfection of miR-575 mimic and miR-575 inhibitor. The invasion ability of A549 cells was evaluated by transwell. The targeting of BLID by miR-575 was predicted by Targetcan software and verified by dual- Luciferase assay. BLID protein expression level was detected by western blot. Results： miR-575 highly expressed in NSCLC cell lines, including A549, SPC-A1, H1299, H1650 （P〈0.001）, miR-575 mimic could efficiently elevated the expression ofmiR-575 in A549 cells （P〈0.001）, and strengthened the proliferation and invasion ability of NSCLC cells （P〈0.05）, while, transfection ofmiR-575 inhibitor could down-regulate the expression of miR-575, and also inhibit the proliferation and invasion ability of NSCLC cells （P〈0.01）. Targetscan software predicted that BLID might be the target gene of miR-575, and dual-luciferase assay revealed that miR-575 could obviously decrease the luciferase reaction of wild type BLID 3＇UTR （P〈0.01）, besides, miR-575 could down-regulate the protein expression ofBLID （P〈0.01）. Real-time PCR results showed that NSCLC cell lines had lower level ofBLID mRNA expression compared with 16HBE control cells （P〈0.001）, and restore of BLID could markedly inhibited cell proliferation and invasion ability （P〈0.05）, which could be reversed by miR-575 co-tranfection （P〈0.01）. Conclusion： In NSCLC cells, the expression ofmiR - 575 could promote cell proliferation and invasion ability by directly regulating downstream target tumor-suppressor gene BLID expression.

关 键 词：非小细胞肺癌 microRNA-575 BLID 

分 类 号：R-33[医药卫生] R734.2