| 焉春华,史晓东,于方飞,王宏伟,张晓飞,刘雅欣,于 瑶,杨原荻,邵玉霞.MicroRNA-575靶向抑制BLID促进非小细胞肺癌细胞的增殖和侵袭[J].,2017,17(23):4432-4436 |
| 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 |
| 投稿时间：2017-02-23 修订日期：2017-03-19 |
| DOI：10.13241/j.cnki.pmb.2017.23.007 |
| 中文关键词: 非小细胞肺癌 microRNA-575 BLID |
| 英文关键词: Non-small cell lung cancer cell MicroRNA-575 BLID |
| 基金项目:黑龙江省卫生计生委科研课题(2016-039) |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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. |
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