MicroRNA-575靶向抑制BLID促进非小细胞肺癌细胞的增殖和侵袭

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(P0.001)。MiR-575模拟物转染的A549细胞miR-575的表达明显高于对照组(P0.001),同时细胞的增殖和侵袭力增强(P0.05);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(P0.01)。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(P0.01),而且能够降低BLID的蛋白表达量(P0.01)。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(P0.001),且转染BLID后,NSCLC细胞的增殖和细胞侵袭被明显抑制(P0.05),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(P0.01)。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。

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

ABSTRACT 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 of miR-575 and BLID in differ- ent 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 detect- ed 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 of miR-575 in A549 cells (P<0.001), and strengthened the proliferation and in- vasion ability of NSCLC cells (P<0.05), while, transfection of miR-575 inhibitor could down-regulate the expression of miR-575, and al- so 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 of BLID (P<0.01). Real-time PCR results showed that NSCLC cell lines had lower level of BLID 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 of miR - 575 could promote cell proliferation and invasion ability by directly regulating downstream target tumor-suppressor gene BLID expression.