LINC01270竞争性抑制微小RNA-770-5p表达对肺癌细胞增殖、侵袭和迁移的影响

Effects of competitive inhibition of microRNA-770-5p by LINC01270 on proliferation,migration and invasion of lung cancer cells

目的阐明长基因间非蛋白质编码RNA 1270(LINC01270)在肺癌细胞增殖、迁移和侵袭中的分子作用。方法经实时荧光定量PCR(qPCR)检测肺癌组织的LINC01270和微小RNA-770-5p(miR-770-5p)及肺癌细胞的LINC01270水平。转染LINC01270干扰序列siRNA-LINC01270或空白质粒siRNA-NC至A549细胞并分为3组:空白对照组、阴性对照组和LINC01270干扰组;MTT法、划痕实验和Transwell小室实验分别检测A549细胞的增殖、迁移和侵袭能力变化情况,qPCR和Western blotting检测基质金属蛋白酶9(MMP-9)、N-钙黏蛋白(N-cad)和E-钙黏蛋白(E-cad)水平。双荧光素酶报告系统验证LINC01270与miR-770-5p的靶向关系。结果与癌旁组织相比,肺癌组织的LINC01270水平升高而miR-770-5p水平降低(P<0.05),且肺癌组织中LINC01270水平与miR-770-5p水平呈负相关性(r=-0.716,P<0.05)。双荧光素酶报告基因实验显示LINC01270通过直接结合与miR-770-5p靶向作用并负调节miR-770-5p的表达。肺癌细胞H1299、SPC-A-1、SK-MES-1、H1650和A549的LINC01270水平分别为1.403±0.057、1.474±0.059、1.603±0.067、1.725±0.060和1.940±0.058,高于16HBE细胞的1.000±0.066(P<0.05)。LINC01270干扰组A549细胞48、72 h的细胞活力较阴性对照组减弱,划痕愈合率和穿膜细胞数分别为(21.537±2.205)%和(71.000±6.429)个,少于阴性对照组的(47.767±3.180)%和(284.667±7.623)个,且MMP-9、N-cad和E-cad水平低于阴性对照组,上述差异均有统计学意义(P<0.05)。结论LINC01270促进NSCLC的增殖、迁移和侵袭,至少部分通过负性调节miR-770-5p表达,在肺癌中发挥促癌功效。

Objective To illustrate the role of long intergenic non-protein coding RNA 1270(LINC01270)in proliferation,migration and invasion of lung cancer cells.Methods Quantitative real-time polymerase chain reaction(qPCR)was employed to detect levels of LINC01270 and microRNA-770-5p(miR-770-5p)in lung cancer tissues as well as levels of LINC01270 in lung cancer cells.A549 cells were transfected with siRNA-LINC01270 or siRNA-NC and allocated into 3 groups:Blank control group,Negative control group and LINC01270 interference group.MTT,Wound-healing and Transwell chamber assays were performed to determine the proliferation,migration and invasion abilities of A549 cells.Levels of matrix metallopeptidase(MMP)-9,N-cadherin(N-cad)and E-cadherin(E-cad)were tested by qPCR and Western blotting assays.The targeted binding effect between miR-770-5p and LINC01270 was confirmed by a Dual-Luciferase reporter assay.Results Compared with adjacent tissues,the LINC01270 level in lung cancer tissues increased while the miR-770-5p level decreased(P<0.05),and there was a negative correlation between LINC01270 level and miR-770-5p level in lung cancer tissues(r=-0.716,P<0.05).Dual-Luciferase reporter assay showed that LINC01270 negatively regulated the expression of miR-770-5p by directly binding to miR-770-5p.LINC01270 levels in lung cancer H1299,SPC-A-1,SK-MES-1,H1650,and A549 cells were 1.403±0.057,1.474±0.059,1.603±0.067,1.725±0.060 and 1.940±0.058,respectively,higher than 1.000±0.066 in 16HBE cells(P<0.05).Cell viabilities of A549 cells in the LINC01270 interference group at 48 and 72 h were weaker than those in the Negative control group(P<0.05).The scratch healing rate and number of penetrating cells were(21.537±2.205)%and 71.000±6.429 in LINC01270 interference group,lower than(47.767±3.180)%and 284.667±7.623 in the Negative control group(P<0.05).Levels of MMP-9,N-cad and E-cad were significantly reduced in the LINC01270 interference group as compared with Negative control group(P<0.05).Conclusion LINC01270 promotes the proliferation,migration,and invasion of NSCLC,at least in part by negatively regulating the expression of miR-770-5p,and plays a pro-cancer role in lung cancer.