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

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-...

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

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 downregulated， 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.