lncRNA MALAT1通过靶向miR-200a对非小细胞肺癌细胞增殖的影响

Effects of lncRNA MALAT1 on the proliferation of non-small cell lung cancer cells by targeting miR-200a

目的研究lncRNA MALAT1通过靶向miR-200a对非小细胞肺癌细胞增殖的影响。方法选取2016年1月至2019年6月在咸宁市中心医院手术切除的90例非小细胞肺癌患者进行前瞻性研究。将肺癌细胞A549进行传代培养、转染,根据转染的方式不同分为sh-Ctrl组、sh-MALAT1组、miR-200a-mimic组、miR-200a-inhibitor组、sh-MALAT1+miR-200a-inhibitor组和sh-MALAT1+miR-200a-mimic组。采用实时荧光定量PCR检测肺癌组织和细胞中MALAT1、miR-200a表达;MTT法检测A549细胞增殖,流式细胞仪检测A549细胞凋亡;双荧光素酶报告基因法检测MALAT1与miR-200a。结果非小细胞肺癌组织MALAT1相对表达水平高于癌旁组织(t=4.589,P<0.001)。与sh-Ctrl组相比,sh-MALAT1组MALAT1表达水平降低(t=4.954,P<0.001);sh-MALAT1组miR-200a表达水平升高(t=7.908,P<0.001),miR-200a-inhibitor组miR-200a表达水平降低(t=5.187,P<0.001),sh-MALAT1+miR-200a-inhibitor组sh-MALAT1表达水平降低(t=8.571,P<0.001),miR-200a表达水平显著升高(t=5.284,P<0.001)。荧光素酶实验结果显示:与sh-Ctrl组相比,miR-200a-mimic组的荧光素酶活性明显下降(t=4.536,P<0.001);而sh-MALAT1组和sh-MALAT+miR-200a-mimic组的荧光素酶活性无明显变化(t=0.247,P=0.834)。与sh-Ctrl组相比,sh-MALAT1组细胞增殖率降低(t=4.158,P<0.001);与sh-MALAT1组相比,sh-MALAT1+miR-200a-inhibitor组细胞增殖率增加(t=3.895,P=0.001)。与sh-Ctrl组相比,sh-MALAT1组细胞凋亡率升高(t=5.012,P<0.001),miR-200a-inhibitor组细胞凋亡率降低(t=3.861,P=0.001);与sh-MALAT1+miR-200a-inhibitor组相比,sh-MALAT1组细胞凋亡率升高(t=3.748,P=0.001),miR-200a-inhibitor组细胞凋亡率下降(t=4.703,P<0.001)。结论抑制lncRNA MALAT1的表达,可靶向miR-200a发挥抑制非小细胞肺癌增殖、促进凋亡的生物学作用。

Objective To study the effect of lncRNA MALAT1 on the proliferation of non-small cell lung cancer(NSCLC)cells by targeting miR-200a.Methods The study enrolled 90 cases of NSCLC who underwent surgical resection in Xianning Central Hospital and the First Affiliated Hospital of Hubei Science and Technology College enrolled in the prospective study during January 2016 and June 2019.The lung cancer cells A549 were subcultured,transfected and divided into sh-Ctrl group,sh-MALAT1 group,miR-200a-mimic group,miR-200a-inhibitor group,sh-MALAT1+miR-200a-inhibitor group and sh-MALAT1+miR-200a-mimic group.The level of MALATA1 and miR-200a of the lung cancer tissues and cells were measured with RT-PCR.The proliferation of A549 was measured by MTT and apoptosis was measured by flow cytometry.The relationship between MALATA1 and miR-200a was detected with dual luciferase reporter gene assay.Results MALAT1 in NSCLC tissues was significantly higher than in adjacent tissues(t=4.589,P<0.001).Compared with sh-Ctrl group,MALAT1 in sh-MALAT1 group was significantly lower(t=4.954,P<0.001).miR-200a in sh-MALAT1 group was significantly higher(t=7.908,P<0.001).miR-200a in miR-200a-inhibitor group was significantly lower(t=5.187,P<0.001).sh-MALAT1 was significantly lower(t=8.571,P<0.001)and miR-200a was significantly higher(t=5.284,P<0.001).Luciferase assay showed that activity of luciferase of miR-200a-mimic group was significantly decreased compared with sh-Ctrl group(t=4.536,P<0.001),while there was no significant change of the activity of luciferase between sh-MALAT1 group and sh-MALAT1+miR-200a-mimic group(t=0.247,P=0.834).The proliferation rate of sh-MALAT1 group decreased significantly compared with sh-Ctrl group(t=4.158,P<0.001).The proliferation rate of sh-MALAT+miR-200a-inhibitor group increased significantly compared with sh-MALAT1 group(t=3.895,P=0.001).The apoptosis rate of sh-MALAT1+miR-200a-inhibitor group increased significantly compared with sh-MALAT1 group(t=5.012,P<0.001),while that decreased significantly of miR-200a-inhibitor group compared with sh-MALAT1 group(t=3.861,P=0.001).The apoptosis rate of sh-MALAT1 group increased significantly compared with sh-MALAT1+miR-200a-inhibitor group(t=3.748,P=0.001),but that of miR-200a-inhibitor group decreased significantly compared with sh-MALAT1+miR-200a-inhibitor group(t=4.703,P<0.001).Conclusions Inhibition of lncRNA MALAT1 expression can inhibit the proliferation and promote apoptosis of NSCLC through targeting miR-200a.