论文部分内容阅读
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。
AIM: To investigate the effects of miR-575 on the proliferation and invasion of non-small cell lung cancer (NSCLC) cells in vitro and its related mechanisms. Methods: The expression of miR-575 and BLID in different non-small cell lung cancer cell lines was detected by real-time quantitative PCR. The proliferation of A549 cells transfected with miR-575 mimics was detected by CCK-8 assay. Transwell Method to detect the invasion of A549 cells; Targetcan method and dual luciferase detection of miR-575 on BLID 3’UTR end of the role; Western blot detection of BLID protein expression. Results: The expression of miR-575 in A549, SPC-A1, H1299, H1650 and other human non-small cell lung cancer cell lines was significantly higher than that of the immortalized human bronchial epithelial cell line 16HBE (P <0.001). MiR-575 mimics transfected A549 cells miR-575 expression was significantly higher than the control group (P <0.001), while cell proliferation and invasiveness increased (P <0.05); the contrary, miR-575 inhibitor transfected The expression of miR-575 in A549 cells was significantly decreased, and the cell proliferation and invasion were significantly decreased (P <0.01). Targetscan predicts that BLID may be the downstream target of miR-575, and luciferase results show that miR-575 can not only effectively inhibit the luciferase reaction at the 3 ’UTR of wild-type BLID (P <0.01), but also decrease the protein expression of BLID Amount (P <0.01). The results of real-time quantitative PCR showed that the expression of BLID was significantly lower in NSCLC cell lines (P <0.001), and the proliferation and invasion of NSCLC cells were significantly inhibited (P <0.05) When co-transfected with BLID, miR-575 reversed the cell proliferation and invasion inhibited by BLID (P <0.01). Conclusion: The expression of miR-575 is upregulated in NSCLC cell lines and it can promote the proliferation and invasion of non-small cell lung cancer cells by directly acting on the downstream tumor suppressor gene BLID.