miR-33b-3p通过靶向CDKN1A调控软骨细胞增殖与凋亡及ECM降解
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保定市科技计划项目(18ZF011)


miR-33b-3p regulates chondrocyte proliferation, apoptosis and ECM degradation by targeting CDKN1A
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    摘要:

    目的 探讨miR-33b-3p对软骨细胞增殖、凋亡及ECM降解的影响。方法 将miR-33b-3p mimics和miR-33b-3p inhibitors转染软骨细胞,通过CCK-8法检测软骨细胞的增殖情况,利用流式细胞术检测软骨细胞的凋亡百分数,RTPCR及Western blot实验检测周期相关蛋白CyclinD1、CyclinE1、p21和凋亡相关蛋白Bcl2、caspase9、Bax以及ECM降解相关蛋白TIMPs、MMP-9、EMMPRIN的表达水平。荧光素酶活性分析实验检测CDKN1A是miR-33b-3p的作用靶点,最后将已构建的过表达pcDNA3.1CDKN1A质粒转染软骨细胞,通过Western blot实验检测软骨细胞中CyclinD1、CyclinE1、p21、Bcl-2、caspase9、Bax以及TIMPs、MMP-9、EMMPRIN的表达水平。结果 与软骨细胞相比,miR33b3p mimic组细胞明显减少;细胞凋亡数显著升高;CyclinD1、CyclinE1表达下降,p21表达升高;Bcl-2表达下降,caspase9、Bax表达升高;TIMPs表达升高,MMP9、EMMPRIN表达下降。miR-33b-3p靶向作用于CDKN1A基因的3′非翻译区(3′UTR)。与空白软骨细胞相比,过表达pcDNA3.1CDKN1A质粒后,能够促进软骨细胞的增殖,抑制细胞的凋亡,促进ECM的降解。结论 miR-33b-3p通过靶向作用于CDKN1A基因的3′UTR,抑制软骨细胞的增殖,促进软骨细胞的凋亡,以及抑制软骨细胞ECM的降解。

    Abstract:

    Objective To investigate the effects of miR-33b-3p on chondrocyte cell proliferation, apoptosis, and ECM degradation.Methods MiR33b3p mimics and miR-33b-3p inhibitors were transfected into chondrocytes. The proliferationof chondrocytes was detected by the CCK-8 method, and the percentage of chondrocyte apoptosis was measured by flow cytometry,RTPCR and Western blot experiments were performed to detect the expression of CyclinD1, CyclinE1, p21, and apoptosisrelated proteins Bcl-2, caspase 9,and Bax, and ECM degradationrelated proteins TIMPs, MMP-9,and EMMPRIN. Analysis of dual luciferase activity CDKN1A was an experimental target for miR-33b-3p. The constructed overexpressing pcDNA3.1CDKN1A plasmid was transfected into chondrocytes, and the expression levels of CyclinD1, CyclinE1, p21, Bcl-2, caspase-9, Bax and TIMPs, MMP-9 and EMMPRIN in chondrocytes were detected by Western blot experiments.Results Compared with chondrocytes, miR-33b-3p mimic group cells were significantly reduced. The flow cytometry detected a significantly increased number of apoptotic cells in miR-33b-3p mimic group. CyclinD1,CyclinE1 expression decreased. ,p21 expression increased. Bcl-2 expression decreased. Caspase 9 and Bax expression increased. TIMPs expression increased. MMP9 and EMMPRIN expression decreased. miR33b3p targeted CDKN1A3′UTR. After overexpression of pcDNA3.1CDKN1A plasmid, it can promote chondrocyte proliferation, inhibit cell apoptosis, and promote ECM degradation.Conclusion miR-33b-3p can inhibit the proliferation of chondrocytes, promote the apoptosis of chondrocytes, and inhibit the degradation of ECM of chondrocytes by targeting the 3′UTR of CDKN1A gene.

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  • 在线发布日期: 2020-08-17
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