miR-758-3p通过靶向核仁纺锤体相关蛋白1抑制非小细胞肺癌细胞增殖和侵袭

miR-758-3p inhibits the proliferation and invasion of non-small cell lung cancer cells by targeting NUSAP1

目的探讨miR-758-3p在非小细胞肺癌(NSCLC)中的生物学功能及其作用机制。方法采用脂质体法转染miR-758-3p模拟物(miR-758-3p模拟物组)、miRNA对照(对照组)、核仁纺锤体相关蛋白1(NUSAP1)过表达质粒(NUSAP1过表达组)、miR-758-3p模拟物+NUSAP1(miR-758-3p模拟物+NUSAP1组)至人NSCLC细胞系A549细胞中。采用CCK-8法和Transwell小室法检测A549细胞增殖、迁移和侵袭能力,结合生物信息学预测网站和双荧光素酶报告基因实验验证miR-758-3p及其靶基因的靶向关系。结果转染24 h后,miR-758-3p模拟物组和对照组A549细胞中miR-758-3p基因的相对表达水平分别为3.02±0.16和1.00±0.08,NUSAP1基因的相对表达水平分别为0.04±0.02和1.00±0.03,差异均有统计学意义(均P<0.05)。转染72 h后,miR-758-3p模拟物组和对照组细胞的吸光度值分别为0.78±0.06和1.15±0.06,差异有统计学意义(P<0.05)。miR-758-3p模拟物组细胞的迁移数和侵袭数分别为[(119.04±11.49)个]和[(71.33±5.36)个],均低于对照组[分别为(271.38±19.05)个和(164.30±8.11)个;均P<0.05]。miR-758-3p模拟物转染野生型NUSAP1报告基因的荧光素酶活性(0.37±0.04)低于对照组(1.00±0.03,P<0.05)。突变型NUSAP1报告基因和对照组的荧光素酶活性分别为0.96±0.02和0.95±0.02,差异无统计学意义(P>0.05)。结论miR-758-3p通过调控NUSAP1基因的表达抑制NSCLC细胞的增殖、迁移和侵袭。

Objective To investigate the biological function of miR-758-3p and the underlying mechanism in non-small cell lung cancer(NSCLC).Methods miR-758-3p mimics was transfected to A549 NSCLC cells,miRNA control was used as a negative control,cells transfected with nucleolar and spindle associated protein 1(NUSAP1)-overexpression vector indicated as NUSAP1 group,cells co-transfected with miR-758-3p mimics and NUSAP1-overexpression vector indicated as miR-758-3p mimics+NUSAP1 group.The effects of miR-758-3p on the proliferation,migration and invasion abilities of A549 cells were detected by cell counting kit-8(CCK-8)and Transwell assays,respectively.Bioinformatics and dual luciferase reporter assay were used to predict and verify the target gene of miR-758-3p.Results The expressions of miR-758-3p and NUSAP1 in A549 cells were significantly up-regulated at 24 hours after transfection with miR-758-3p mimics(P<0.05).Compared with the miRNA control group(1.15±0.06),the OD value of miR-758-3p mimic group(0.78±0.06)was significantly decreased at 72 hours after transfection(P<0.05).The number of migrated cells of miR-758-3p mimic group(119.04±11.49)was significantly lower than that of the control group(271.38±19.05)(P<0.05).The number of invaded cells of miR-758-3p mimic group(71.33±5.36)was significantly lower than the control group(164.30±8.11)(P<0.05).The result of dual-luciferase reporter assay showed that NUSAP1 was a direct target of miR-758-3p.Moreover,up-regulation of NUSAP1 abolished the inhibitory effects of miR-758-3p on the proliferation,migration and invasion abilities of A549 cells.Conclusion miR-758-3p inhibits the proliferation,migration and invasion of NSCLC cells by targeting NUSAP1.