临床肿瘤学杂志

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miR-134靶向EGFR对非小细胞肺癌增殖的影响

刘东利,贺小龙   

  1. 716000 陕西延安 延安大学附属医院呼吸内科
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2016-12-31 发布日期:2016-12-31

The influence of miR-134 on cell growth by targeting epidermal growth factor receptor in non-small cell lung cancer

LIU Dongli,HE Xiaolong   

  1. Department of Breath Internal Medicine,Yan'an University Affiliated Hospital,Yan'an 716000,China
  • Received:1900-01-01 Revised:1900-01-01 Online:2016-12-31 Published:2016-12-31

摘要:

目的 探讨微小RNA-134(miR-134)对非小细胞肺癌(NSCLC)细胞增殖和凋亡的影响及可能的机制。方法 采用实时荧光定量PCR(QPCR)检测miR134在NSCLC细胞株(A549、H252)和正常胚肺细胞株WI38中的表达情况;将A549和H252细胞分为3组,分别为空白对照组(不转染)、miR-NC组(转染不相关siRNA)和miR-134组(转染miR-134 mimics)。分别于转染后24、48、72、96h收集细胞,采用MTS法检测细胞的增殖情况;转染后96h用流式细胞仪检测细胞的凋亡情况;双荧光素酶报告基因实验检测miR-134与表皮生长因子受体(EGFR)3’UTR的结合情况,QPCR检测过表达miR-134的A549和H252细胞中EGFR的表达情况。结果 与WI38 细胞相比,miR-134在A549细胞中的表达下调85.91%,在H252细胞中下调78.13%(P<0.05)。MTS检测显示,miR-134能显著降低A549和H252细胞的增殖能力,并呈时间依赖性。流式细胞仪检测显示,与空白对照组比较,miR-134组A549细胞的凋亡比例提高226.31%,H252细胞提高47.85%(P<0.05)。双荧光素酶报告基因实验显示miR134能与EGFR3’UTR结合,显著降低荧光值(P<0.05)。QPCR检测显示,与空白对照组比较,转染miR-134 mimics 后,EGFR在A549细胞中的相对表达量下调57.0%,在H252中下调35.0%,差异均有统计学意义(P<0.01)。结论 miR-134在NSCLC中低表达,能通过靶向EGFR抑制NSCLC细胞增殖,并诱导凋亡。

Abstract:

Objective To investigate the effect of microRNA-134 (miR-134) on suppressing the proliferation of non-small cell lung cancer(NSCLC)and the possible mechanism. Methods The expression levels of miR-134 in NSCLC cell lines(A549,H252)and human embryo lung WI38 cells were detected by real-time quantitative PCR(QPCR). A549 and H252 cell lines were divided into miR-NC group,miR-134 group and blank control group,which were transfected with control-siRNA,miR-134 mimics and none,respectively. The proliferation in the three groups of A549 and H252 cells was detected by MTS assays 24,48,72,96 h after transfect. Cell apoptosis were detected by flow cytometry after 96 h. A luciferase reporter assay was performed to confirm whether epidermal growth factor receptor(EGFR was a direct target of miR-134. The expression level of EGFR was detected by QPCR. Results The level of miR-134 was found down-regulated 85.91% in A549 cells and 78.13% in H252 cells comparing with it in WI38 cells(P<0.05). MTS assays showed that miR-134 suppressed A549 and H252 cells proliferation significantly in a time-depended manner. Flow cytometry showed that the apoptotic rate of A549 and H252 cells increased to 226.31% and 47.85% respectively compared with blank control group(P<0.05). A dual-luciferase reporter assay confirmed that EGFR was a direct target of miR-134 by reducing the activity of luciferase(P<0.05). QPCR showed that after the transfect of miR-134 mimics,the expression of EGFR down-regulated 57.0% in A549 cells and 35.0% in H252 cells compared with blank control group(P<0.01). Conclusion miR-134 is down-regulated in NSCLC and inhibites cells growth,as well as induces cell apoptosis by targeting EGFR in NSCLC.

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