摘要
[目的]研究微小RNA-1271(miR-1271)对胶质瘤细胞增殖及凋亡的影响,并探讨其可能作用机制。[方法]体外培养胶质瘤细胞U251、A172、SHG139与人脑正常胶质细胞HEB,将胶质瘤U251细胞分为miR-con组、miRNA-1271组、anti-miR-con组、anti-miR-1271组、si-con组、si-DDI2组、miRNA-1271+pcDNA组、miRNA-1271+pcDNA-DDI2组。qRT-PCR与蛋白印迹(Western Blot)检测细胞中miRNA-1271与DNA损伤诱导蛋白2(DDI2)的表达。应用MTT实验评估转染后胶质瘤U251细胞的增殖能力;应用流式细胞术检测细胞凋亡;双荧光素酶报告基因检测miRNA-1271与DDI2的靶标关系。[结果]与HEB细胞相比,miRNA-1271在胶质瘤U251、A172、SHG139细胞中的表达水平显著降低[(0.24±0.04)/(0.314±0.04)/(0.29±0.03)vs(1.00±0.12)](P<0.05),而DDI2 mRNA[(5.25±0.55)/(4.08±0.29)/(4.58±0.36)vs(1.00±0.11)]及蛋白表达水平[(0.64±0.07)/(0.51±0.06)/(0.59±0.07)vs(0.41±0.05)]均显著升高(P<0.05);与阴性对照组相比,miRNA-1271组胶质瘤U251细胞增殖活性显著降低[(1.24±0.15)vs(0.88±0.09)](P<0.05),而细胞凋亡率[(7.95±0.45)%vs(19.23±1.25)%]显著升高(P<0.05),Bax蛋白表达明显上调(P<0.05);与阴性对照组相比,si-DDI2组胶质瘤U251细胞增殖活性显著降低[(1.31±0.14)vs(0.89±0.09)](P<0.05),而细胞凋亡率[(4.73±0.58)%vs(18.67±1.08)%]显著升高(P<0.05);双荧光素酶报告实验证实DDI2是miRNA-1271的靶基因;与miRNA-1271+pcDNA组相比,miRNA-1271+pcDNA-DDI2组胶质瘤U251细胞增殖活性显著升高[(0.81±0.11)vs(0.97±0.13)](P<0.05),而细胞凋亡率显著降低[(19.28±1.19)%vs(12.67±1.11)%](P<0.05)。[结论]miRNA-1271过表达可通过靶向DDI2基因进而抑制脑胶质瘤细胞增殖[(1.24±0.15)vs(0.88±0.09)]并诱导细胞凋亡[(7.95±0.45)%vs(19.23±1.25)%]。
[Objective]To investigate the effects of microRNA-1271(miR-1271)on proliferation and apoptosis of glioma cells,and to explore its possible mechanism.[Method]The glioma cells U251,A172,SHG139 and human brain normal glial cells HEB were cultured in vitro,and the glioma U251 cells were divided into miR-con group,miRNA-1271 group,anti-miR-con group and anti-miR-1271 group,si-con group,si-DDI2 group,miRNA-1271+pcDNA group,miRNA-1271+pcDNA-DDI2 group.The expression of miRNA-1271 and DNA damage-inducing protein 2(DDI2)was detected by qRT-PCR and Western Blot.The purpose of the MTT assay was to evaluate the proliferative capacity of glioma U251 cells after transfection.Flow cytometry was used to detect apoptosis.Dual luciferase reporter gene was used to detect target relationship between miRNA-1271 and DDI2.[Result]Compared with HEB cells,the expression level of miRNA-1271 in glioma U251,A172,SHG139 cells was significantly reduced[(0.24±0.04)/(0.314±0.04)/(0.29±0.03)vs(1.00±0.12)](P<0.05),and DDI2 mRNA[(5.25±0.55)/(4.08±0.29)/(4.58±0.36)vs(1.00±0.11)]and protein expression level[(0.64±0.07)/(0.51±0.06)/(0.59±0.07)vs(0.41±0.05)]were significantly increased(P<0.05).Compared with the negative control group,the proliferation activity of glioma U251 cells in the miRNA-1271 group was significantly reduced[(1.24±0.15)vs(0.88±0.09)](P<0.05),while the apoptosis rate[(7.95±0.45)%vs(19.23±1.25)%]was significantly increased(P<0.05).Compared with the negative control group,the proliferation activity of glioma U251 cells in the si-DDI2 group was significantly reduced[(1.31±0.14)vs(0.89±0.09)](P<0.05),while the apoptosis rate[(4.73±0.58)%vs(18.67±1.08)%]was significantly increased(P<0.05).The dual luciferase report experiment confirmed that DDI2 was the target gene of miRNA-1271.Compared with the miRNA-1271+pcDNA group,the proliferation activity of glioma U251 cells in the miRNA-1271+pcDNA-DDI2 group was significantly increased[(0.81±0.11)vs(0.97±0.13)](P<0.05),while the cell apoptosis was significantly reduced[(19.28±1.19)%vs(12.67±1.11)%](P<0.05).[Conclusion]Overexpression of miRNA-1271 can inhibit glioma cell proliferation[(1.24±0.15)vs(0.88±0.09)]and induce apoptosis[(7.95±0.45)%vs(19.23±1.25)%]by targeting the DDI2 gene.
作者
杨明环
张波
汤祥军
黄晓东
罗杰
YANG Ming-huan;ZHANG Bo;TANG Xiang-jun;HUANG Xiao-dong;LUO Jie(Taihe Hospital of Shiyan City(Affiliated Hospital of Hubei Medical College),Shiyan 442000,China)
出处
《生物技术》
CAS
2022年第3期325-331,393,共8页
Biotechnology
