miR-21靶向调控PTEN/PI3K/AKT通路对骨肉瘤细胞增殖、侵袭和凋亡的影响

EEffect of miR-21 Targeted Regulation of PTEN/PI3K/AKT Pathway on the Proliferation,Invasion and Apoptosis of Osteosarcoma Cells

目的检测微小核糖核酸(micro RNA,miR)-21在人正常骨细胞hFOB1.19与人骨肉瘤细胞系U2OS,Saos-2和MG-63中的表达,并探讨miR-21对骨肉瘤细胞增殖、侵袭和凋亡的影响。方法实时荧光定量PCR法(real-time fluorescent quantitative PCR,qRT-PCR)检测人正常骨细胞hFOB1.19与人骨肉瘤细胞系U2OS,Saos-2和MG-63中miR-21的表达。选择MG-63细胞随机分为3组,利用脂质体2000分别转染空白对照(control)、阴性对照miR-21-NC及抑制剂组(miR-21-inhibitor),再通过qRT-PCR法验证转染后MG-63细胞中miR-21表达。CCK-8法检测MG-63细胞增殖能力变化;流式细胞技术检测抑制miR-21表达对MG-63细胞凋亡的影响;Transwell实验检测对MG-63细胞侵袭能力的影响以及采用Western blot检测抑制miR-21表达对MG-63细胞中PTEN,PI3K,AKT及p-AKT蛋白表达的影响。结果人骨肉瘤细胞系Saos-2,U2OS和MG-63中miR-21相对表达水平分别为1.29±0.14,1.75±0.21和2.12±0.25,较人正常骨细胞hFOB 1.19中miR-21表达水平(0.75±0.12)显著升高,差异具有统计学意义(F=38.037,P=0.002)。转染抑制剂培养48h后,与Control组miR-21表达水平(2.07±0.28)和miR-21-NC组miR-21表达水平(2.02±0.33)相比,miR-21-inhibitor组miR-21表达水平(1.07±0.15)显著下降,差异具有统计学意义(F=33.357,P=0.005)。CCK-8结果表明,与Control组和miR-21-NC组相比,miR-21-inhibitor组各个时间点A值均显著下降,差异具有统计学意义(F=71.409~378.281,均P<0.001)。流式细胞检测结果表明,miR-21-inhibitor组凋亡数占比为45.0%,较Control组(8.65%)和miR-21-NC组(10.60%)均有增加,差异有统计学意义(F=56.134,P<0.001)。Transwell实验结果显示,miR-21-inhibitor组细胞穿膜数(102±9个)较Control组细胞穿膜数(189±12个)及miR-21-NC组细胞穿膜数(177±16个)明显减少,差异有统计学意义(F=158.781,F<0.001)。Western blot结果表明,miR-21-inhibitor组PTEN表达上调,PI3K和p-AKT表达下调,差异均有统计学意义(F=86.309~138.615,均P<0.001),但对AKT蛋白表达无明显影响,差异无统计学意义(F=14.527,P=0.152)。结论miR-21在人骨肉瘤细胞系中呈高表达,抑制miR-21表达可以抑制骨肉瘤细胞增殖和侵袭,促进其凋亡,作用机制可能与PTEN/PI3K/AKT通路有关。

Objective To detect the expression of miR-21 in human normal bone cell hFOB1.19 and human osteosarcoma cell lines U2OS,Saos-2 and MG-63,and explore the effect of miR-21 on the proliferation,invasion and apoptosis of osteosarcoma cells.Methods Real-time fluorescent quantitative PCR(qRT-PCR)method was used to detect the expression of miR-21 in human normal bone cell hFOB1.19 and human osteosarcoma cell lines U2OS,Saos-2 and MG-63.MG-63 cells were randomly divided into 3 groups,and used liposome 2000 to transfect with control,miR-21-NC and miR-21-inhibitor,respectively.Then verified the expression of miR-21 in MG-63 cells after transfection by qRT-PCR.The proliferation of MG-63 cells was detected by CCK-8 method.The effect of inhibiting miR-21 expression on MG-63 cell apoptosis was detected by flow cytometry.Transwell assay was used to detect the invasion ability of MG-63 cells.Western blot was further used to detect the effect of inhibiting the expression of miR-21 on the protein expression of PTEN,PI3K,AKT and p-AKT in MG-63 cells.Results The relative expression levels of miR-21 in the human osteosarcoma cell lines Saos-2,U2OS and MG-63 were 1.29±0.14,1.75±0.21 and 2.12±0.25 compared with the miR-21 in human normal bone cells hFOB 1.19.The expression level(0.75±0.12)increased significantly,the difference was statistically significant(F=38.037,P=0.002).After the transfection inhibitor was cultured for 48 hours,compared with the expression level of miR-21 in the control group(2.07±0.28)and the expression level of miR-21-NC(2.02±0.33),the expression level of miR-21 in the miR-21-inhibitor group(1.07±0.15)significantly decreased,and the difference was statistically significant(F=33.357,P=0.005).The results of CCK-8 showed that compared with the control group and the miR-21-NC group,the A value of the miR-21-inhibitor group decreased significantly at each time point,and the difference were more statistically significant(F=71.409~378.281,all P<0.001).Flow cytometry results showed that the proportion of apoptotic number in the miR-21-inhibitor group was 45.0%,which was an increase compared with 8.65%in the control group and 10.60%in the miR-21-NC group,the difference was statistically significant(F=56.134,P<0.001).Transwell experiment results showed that the number of cells penetrating the membrane in the miR-21-inhibitor group(102±9)was higher than the number of cells penetrating the control group(189±12)and the number of cells penetrating the miR-21-NC group(177±16)significantly reduced,the difference was statistically significant(F=158.781,P<0.001).Western blot results showed that the expression of PTEN in the miR-21-inhibitor group was up-regulated,and the expression of PI3K and p-AKT was down-regulated,the difference were statistically significant(F=86.309~138.615,all P<0.001),but it had no significant effect on the expression of AKT protein,the difference was not statistically significant(F=14.527,P=0.152).Conclusion MiR-21 was highly expressed in human osteosarcoma cell lines,and inhibition of miR-21 expression can inhibit the proliferation and invasion of osteosarcoma cells and promote their apoptosis.The mechanism of action may be related to the PTEN/PI3K/AKT pathway.