TWIST调控miR-145表达促进胶质瘤干细胞侵袭、迁移的实验研究

Experimental study of TWIST regulating the expression of miR-145 to promote the invasion and migration of glioma stem cells

目的:探究转录因子TWIST对胶质瘤干细胞侵袭、迁移的影响,并探讨与微小RNA(microRNA,miR)-145之间的关系。方法:以人神经胶质瘤细胞U251为研究对象,提取干细胞并经免疫荧光染色CD133、胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)鉴定。实验分组:对照组、过表达对照组、TWIST过表达组、沉默对照组、TWIST沉默组,分别用pcDNA3、pcDNA3-TWIST、pGPH1-shNC、pGP-TWIST-shRNA转染提取并鉴定后的干细胞。实时荧光定量PCR(RT-qPCR)法检测各细胞中TWIST mRNA和miR-145水平;蛋白质免疫印迹实验检测细胞中TWIST、基质金属蛋白酶(matrix metalloproteinase, MMP)-2、MMP-9蛋白水平;Transwell检测细胞侵袭和迁移情况;划痕实验检测细胞迁移情况;双荧光素酶鉴定miR-145与TWIST的靶向关系。结果:干细胞分选与鉴定:CD133阳性干细胞约占5.6%左右;分选后细胞用CD133和GFAP染色显示双阳性。与对照组相比,TWIST过表达组细胞中TWIST mRNA和蛋白水平,细胞侵袭、迁移数量,愈合率,细胞中MMP-2、MMP-9蛋白水平升高(P<0.05),miR-145水平降低(P<0.05);TWIST沉默组细胞中TWIST mRNA和蛋白水平,细胞侵袭、迁移数量,愈合率,细胞中MMP-2、MMP-9蛋白水平降低(P<0.05),miR-145水平升高(P<0.05);过表达对照组、沉默对照组上述指标差异均无统计学意义(P>0.05)。miR-145与TWIST存在结合位点并经双荧光素酶靶向关系验证。结论:升高TWIST能够促进胶质瘤干细胞侵袭、迁移,且能靶向miR-145发挥作用。

Objective:To explore the effects of transcription factor TWIST on the invasion and migration of glioma stem cells,and to explore its relationship with microRNA(miR)-145.Methods:The human glioma cell U251 was used as the research objects.The stem cells were extracted,and the CD133 and glial fibrillary acidic protein(GFAP)were identified with immunofluorescence staining.The experiment was divided into control group,overexpression control group,TWIST overexpression group,silence control group and TWIST silence group.The extracted and identified stem cells were transfected with pcDNA3,pcDNA3-TWIST,pGPH1-shNC and pGP-TWIST-shRNA,respectively.Real-time fluorescent quantitative PCR(RT-qPCR)method was used to detect the levels of TWIST mRNA and miR-145 in each cell.Western blotting was used to detect the protein levels of TWIST,matrix metalloproteinase(MMP)-2 and MMP-9 in cells.Transwell was used to detect cell invasion and migration.Scratch test was used to detect cell migration.Dual luciferase was used to identify the targeting relationship between miR-145 and TWIST.Results:Stem cell sorting and identification result showed CD133 positive stem cells accounted for about 5.6%.After sorting,the cells were stained with CD133 and GFAP and showed double positive.Compared with the control group,the levels of TWIST mRNA and protein,the numbers of cell invasion and migration,the healing rate,and the protein levels of MMP-2 and MMP-9 in the cells of the TWIST overexpression group increased(P<0.05),the level of miR-145 decreased(P<0.05).The levels of TWIST mRNA and protein,the numbers of cell invasion and migration,the healing rate,and the protein levels of MMP-2 and MMP-9 in the cells of the TWIST silence group decreased(P<0.05),the level of miR-145 increased(P<0.05).There was no statistically significant difference in the above indicators between the overexpression control group and the silence control group(P>0.05).There was a binding site between miR-145 and TWIST,which was verified by the dual luciferase targeting relationship.Conclusion:Increasing TWIST can promote the invasion and migration of glioma stem cells,and can target miR-145 to play a role.