[1]姜富祥,阿尔宾,高 飞,等.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[J].现代检验医学杂志,2022,37(04):18-22.[doi:10.3969/j.issn.1671-7414.2022.04.004]
　JIANG Fu-xiang,A Er-bin,GAO Fei,et al.EEffect of miR-21 Targeted Regulation of PTEN/PI3K/AKT Pathway on the Proliferation, Invasion and Apoptosis of Osteosarcoma Cells[J].Journal of Modern Laboratory Medicine,2022,37(04):18-22.[doi:10.3969/j.issn.1671-7414.2022.04.004]
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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()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第37卷
期数:
2022年04期
页码:
18-22
栏目:
论著
出版日期:
2022-07-15

文章信息/Info

Title:
EEffect of miR-21 Targeted Regulation of PTEN/PI3K/AKT Pathway on the Proliferation, Invasion and Apoptosis of Osteosarcoma Cells
文章编号:
1671-7414（2022）04-018-05
作者:
姜富祥; 阿尔宾; 高 飞; 王 兴
内蒙古自治区巴彦淖尔市医院脊柱外科，内蒙古巴彦淖尔 015000
Author(s):
JIANG Fu-xiang;  A Er-bin;  GAO Fei;  WANG Xing
Department of Spine Surgery ，Bayannur City Hospital, Inner Mongolia Autonomous Region ，Inner Mongolia Bayannur 015000，China
关键词:
骨瘤细胞; 微小核糖核酸-21; PTEN/PI3K/AKT 通路; 增殖和侵袭; 凋亡
分类号:
R738.1；R730.43
DOI:
10.3969/j.issn.1671-7414.2022.04.004
文献标志码:
A
摘要:
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-timefluorescent 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。
Abstract:
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.

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备注/Memo

备注/Memo:
基金项目：内蒙古自治区自然科学基金项目（20190203MS1571）。
作者简介：姜富祥（1978-），男，硕士，主任医师，研究方向：擅长脊柱与关节疾病、损伤的诊治，E-mail：kg8xbt1026@126.com。
更新日期/Last Update: 1900-01-01