基因间长链非编码RNA 113靶向微小RNA-493-3p对肺癌细胞侵袭和迁移的影响

Effects of long intergenic non-coding RNA 113 on invasion and migration of lung cancer cells by targeting microRNA-493-3p

目的探讨基因间长链非编码RNA 113(LINC00113)调控微小RNA-493-3p(miR-493-3p)在非小细胞肺癌(NSCLC)细胞侵袭迁移过程中的分子功能。方法采用实时定量PCR(qPCR)检测LINC00113和miR-493-3p在50对NSCLC癌组织和癌旁组织及4株NSCLC细胞株(A549、NCI-H1975、HCC827和NCI-H1299)的水平,双荧光素酶报告系统验证LINC00113与miR-493-3p的相互关系;将NCI-H1299细胞分为4组:对照组(空白对照)、LINC00113干扰(si-LINC00113)组(转染si-LINC00113+miR-493-3p无关序列miR-NC)、miR-493-3p抑制物inhibitor(miR-inhibitor)组(转染无关序列si-NC+miR-493-3p inhibitor)和共转染组(转染si-LINC00113+miR-493-3p inhibitor)。通过MTT比色法、划痕实验和Transwell小室实验分别检测LINC00113和miR-493-3p对NCI-H1299细胞增殖、迁移和侵袭的影响。结果与癌旁组织相比,NSCLC组织的LINC00113水平升高至2.125±0.119,而miR-493-3p水平降低至0.592±0.040,且两者水平呈负相关(r=-0.760,P<0.05)。与正常肺上皮细胞BEAS-2B相比,NSCLC细胞的LINC00113水平升高,而miR-493-3p水平降低,差异有统计学意义(P<0.05)。野生型LINC00113序列与miR-493-3p模拟物共转染细胞的荧光素酶活性降低(P<0.05)。对照组、si-LINC00113组、miR-inhibitor组和共转染组的细胞活力分别为1.381±0.076、0.863±0.070、1.824±0.102和1.452±0.105,划痕愈合率分别为(54.370±5.366)%、(26.203±1.817)%、(73.067±2.846)%和(51.587±4.288)%,穿膜细胞数分别为(339.265±12.032)、(183.619±9.387)、(467.113±8.950)和(325.667±6.036)个,与对照组相比,si-LINC00113组的细胞活力、划痕愈合率和穿膜细胞数均降低(P<0.05),miR-inhibitor组均升高(P<0.05),而共转染组的无明显变化(P>0.05)。结论NSCLC组织和细胞中LINC00113高表达且miR-493-3p低表达,下调LINC00113可抑制NSCLC细胞的增殖、迁移和侵袭能力,可能通过靶向miR-493-3p来发挥促癌作用,LINC00113/miR-493-3p轴有望成为NSCLC治疗的潜在靶点。

Objective To investigate the biological role of long intergenic non-coding RNA 113(LINC00113)on invasion and migration of non-small cell lung cancer(NSCLC)cells by targeting microRNA-493-3p(miR-493-3p).Methods Levels of LINC00113 and miR-493-3p in 50 pairs of NSCLC tissues and adjacent tissues along with 4 NSCLC cell lines(A549,NCI-H1975,HCC827 and NCI-H1299)were detected by real-time quantitative PCR(qPCR).Interaction between LINC00113 and miR-493-3p was verified by dual luciferase reporter system.NCI-H1299 cells were divided into four groups including control group(blank control),LINC00113 interference(si-LINC00113)group(cells transfected with si-LINC00113 and miR-493-3p unrelated sequence miR-NC),miR-493-3p inhibitor(miR-inhibitor)group(cells transfected with unrelated sequence si-NC and miR-493-3p inhibitor)and co-transfection group(cells transfected with si-LINC00113 and miR-493-3p inhibitor).Effects of LINC00113 and miR-493-3p on the proliferation,migration and invasion of NCI-H1299 cells were detected by MTT colorimetry,scratch assay and Transwell chamber assay.Results Compared with adjacent tissues,LINC00113 level in NCSLC tissues increased to 2.125±0.119,while miR-493-3p level decreased to 0.592±0.040,and LINC00113 level was negatively correlated with miR-493-3p level(r=-0.760,P<0.05).The luciferase activity of cells co-transfected with wild-type LINC00113 sequence and miR-493-3p mimics decreased(P<0.05).For control group,si-LINC00113 group,miR-inhibitor group and co-transfection group,the cell viability were 1.381±0.076,0.863±0.070,1.824±0.102 and 1.452±0.105,scratch healing rates were(54.370±5.366)%,(26.203±1.817)%,(73.067±2.846)%and(51.587±4.288)%,and number of transmembrane cells were 339.265±12.032,183.619±9.387,467.113±8.950 and 325.667±6.036,respectively.Compared with control group,cell viability,scratch healing rate and number of transmembrane cells in si-LINC00113 group were decreased(P<0.05),while those in miR-inhibitor group were increased(P<0.05),and no significant changes were observed in co-transfection group(P>0.05).Conclusion LINC00113 is highly expressed and miR-493-3p is low expressed in NSCLC tissues and cells.Down-regulation of LINC00113 can inhibit the proliferation,migration and invasion of NSCLC cells,which may play a role in promoting cancer by targeting miR-493-3p.LINC00113/miR-493-3p axis is expected to become a potential target for the treatment of NSCLC.