摘要
目的 :探究长链非编码RNA KCNQ1重叠转录物1(LncRNA KCNQ1OT1)对胶质瘤细胞增殖及放疗敏感性的影响以及对miR-181a-5p/TRIM71通路的调控机制。方法 :将人胶质瘤细胞U251分为NC组(未转染质粒)、si-NC组(转染KCNQ1OT1阴性对照)、si-KCNQ1OT1组(转染si-KCNQ1OT1)、si-KCNQ1OT1+miRNC组(共转染si-KCNQ1OT1和miR-181a-5p阴性对照)、si-KCNQ1OT1+miR-181a-5p inhibitor组(共转染siKCNQ1OT1和miR-181a-5p inhibitor),转染培养48 h后,利用6 Gy射线照射各组部分细胞。采用RT-qPCR检测各组细胞中KCNQ1OT1、miR-181a-5p和TRIM71 mRNA水平;采用CKK-8和克隆形成实验检测各组转染细胞的增殖活性,采用流式细胞术检测各组转染细胞的凋亡情况;采用免疫印迹检测各组转染细胞中TRIM71蛋白表达相对水平;采用双荧光素酶报告基因实验验证miR-181a-5p与KCNQ1OT1、TRIM71的靶向关系。结果 :与si-NC组相比,si-KCNQ1OT1组KCNQ1OT1和TRIM71 mRNA表达水平、细胞存活率以及克隆形成数量均降低,miR-181a-5p表达水平升高;而经6 Gy射线垂直照射后,各组转染细胞存活率均降低,细胞凋亡率均升高,其中si-KCNQ1OT1+6Gy组细胞存活率低于si-NC+6Gy组,细胞凋亡率则高于si-NC+6Gy组;回补实验显示,miR-181a-5p inhibitor逆转了敲低KCNQ1OT1对细胞增殖及放疗敏感性所造成的影响;双荧光素酶活性验证了miR-181a-5p与KCNQ1OT1及TRIM71均存在靶向关系。结论 :通过敲低KCNQ1OT1能够引起上调miR-181a-5p、下调TRIM71的表达,抑制胶质瘤细胞的增殖,提高其放疗敏感性。
Objective:To investigate the impacts of long non-coding RNA KCNQ1 overlapping transcript 1(LncRNA KCNQ1OT1)on proliferation and radiation sensitivity of glioma cells,and its regulatory mechanism on the miR-181a-5p/TRIM71 pathway.Methods:Human glioma cells U251 were divided into NC group(untransfected plasmid),si-NC group(transfected with KCNQ1OT1 negative control),si-KCNQ1OT1 group(transfected with si-KCNQ1OT1),si-KCNQ1OT1+miR-NC group(co-transfected with si-KCNQ1OT1 and miR-181a-5p negative control),si-KCNQ1OT1+miR-181a-5p inhibitor group(co-transfected with si-KCNQ1OT1 and miR-181a-5p inhibitor).After 48 hours of transfection,cells selected from each group were irradiated with 6 Gy radiation.RT-qPCR method was applied to detect the levels of KCNQ1OT1,miR-181a-5p,and TRIM71 mRNA of cells in each group.CCK-8 method and colony formation assay were applied to assess the proliferation activity of transfected cells in each group.Flow cytometry was adopted to detect the apoptosis of transfected cells in each group.Western blotting was performed to measure the relative expression levels of TRIM71 protein in transfected cells of each group.Dual luciferase reporter gene assay was applied to verify the targeting relationship between miR-181a-5p and KCNQ1OT1,TRIM71.Results:Compared with the si-NC group,the expression levels of KCNQ1OT1 and TRIM71 mRNA,cell survival rate,and number of clones formed in the si-KCNQ1OT1 group decreased,while the expression 第1作者level of miR-181a-5p increased.After vertical irradiation with 6 Gy of radiation,the survival rate of transfected cells in all groups decreased,while the apoptosis rate increased.Among them,the survival rate of cells in the si-KCNQ1OT1+6Gy group was lower than that in the si-NC+6Gy group,while the apoptosis rate was higher in the si-NC+6Gy group.The rescue experiment demonstrated that miR-181a-5p inhibitor reversed the effects of KCNQ1OT1 knockdown on cell proliferation and radiotherapy sensitivity.Dual luciferase activity confirmed the targeting relationship between miR-181a-5p and KCNQ1OT1,or TRIM71.Conclusion:KCNQ1OT1 knockdown can inhibit the proliferation of glioma cells and improve their radiotherapy sensitivity by up-regulating miR-181a-5p and down-regulating TRIM71 expression.
作者
卑源琪
钱霞
Bei Yuanqi;Qian Xia(Department of Radiotherapy,Affiliated Tumor Hospital of Nantong University,Nantong 226361,China)
出处
《解剖学杂志》
2025年第3期215-220,共6页
Chinese Journal of Anatomy
