摘要
目的:探讨长链非编码RNA(Long non-coding RNA,LncRNA)人母系表达基因(Maternally expressed gene 3,MEG3)对骨关节炎(osteoarthritis,OA)软骨细胞增殖和凋亡的影响及作用机制。方法:选取我院住院的OA患者和半月板损伤患者各40例,采用RT-PCR检测两组患者软骨组织和细胞中MEG3的表达。在OA软骨细胞中,转染si RNA-MEG3(si-MEG3)或过表达MEG3的慢病毒载体(LV-MEG3),采用CCK8法检测细胞增殖情况,流式细胞仪检测细胞周期和凋亡情况,RT-PCR和western blot检测PCNA、Dvl2、GSK-3β、cyclinD1和β-catenin m RNA和蛋白表达。结果:OA患者膝关节软骨组织中MEG3的表达水平明显低于半月板损伤患者软骨组织(P<0.05),同时OA软骨细胞中MEG3的表达水平明显低于正常软骨细胞(P<0.05)。OA软骨细胞转染siMEG3后,细胞增殖能力和PCNA表达明显下降(P均<0.05),G0/G1期细胞比例明显升高(P<0.05),S期细胞比例明显下降(P<0.05),细胞凋亡率明显增加(P均<0.05)。低表达MEG3能够显著降低Dvl2、GSK-3β、cyclinD1和β-catenin m RNA和蛋白表达水平(P均<0.05),增加GSK-3βm RNA和蛋白表达水平(P均<0.05)。在OA软骨细胞转染LV-MEG3后,细胞增殖能力和PCNA表达明显升高(P均<0.05),G0/G1期细胞比例明显下降(P<0.05),S期细胞比例明显增加(P<0.05),细胞凋亡率明显减少(P均<0.05)。高表达MEG3能够显著增加OA软骨细胞中Dvl2、GSK-3β、cyclinD1和β-catenin m RNA和蛋白表达水平(P均<0.05),降低GSK-3βm RNA和蛋白表达(P均<0.05)。同时,采用XAV939阻滞Wnt/β-catenin信号通路能够显著逆转过表达MEG3对OA软骨细胞增殖和凋亡的影响。结论:MEG3在OA患者和软骨细胞中均显著低表达,并能够通过阻滞Wnt/β-catenin信号通路激活影响OA软骨细胞增殖和凋亡。MEG3可能成为OA治疗的重要靶分子。
Objective:To investigate the effects of Long non-coding RNA(lncRNA)Maternally expressed gene 3(MEG3)on osteoarthritis(OA)chondrocyte proliferation and apoptosis,and the underlying mechanisms.Methods:40 patients with OA and 40 patients with meniscus injury in our hospital were selected.MEG3 expression in OA cartilage tissues and cells were analyzed by real-time polymerase chain reaction(RT-PCR).After transfecting OA chondrocytes with si-MEG3 or LV-MEG3,cell proliferation was detected by the cell counting kit-8(CCK8)assay,cell cycle distribution and apoptosis were measured by flow cytometry analysis,and PCNA,Dvl2,GSK-3β,cyclinD1 andβ-catenin were detected by RT-PCR and western blotting.Results:The expression level of MEG3 in OA cartilage tissues was lower than that in patients with meniscus injury(P<0.05)and the expression level MEG3 in OA chondrocytes was lower than that in normal chondrocytes(P<0.05).Cell proliferation,and PCNA expression decreased significantly(P<0.05),the percentage of OA chondrocytes in G0/G1 significantly increased(P<0.05),the percentage of OA chondrocytes in the S phase significantly decreased(P<0.05),and apoptosis significantly increased(P<0.05)in OA chondrocytes transfected with si-MEG3 compared to those in the si-NC group.Down regulation of MEG3 decreased DVL2,cyclin D1,andβ-catenin expression(P<0.05)and increased GSK-3βexpression(P<0.05).Cell proliferation,and PCNA expression increased significantly(P<0.05),the percentage of OA chondrocytes in G0/G1 significantly decreased(P<0.05),the percentage of OA chondrocytes in the S phase significantly increased(P<0.05),and apoptosis significantly decreased(P<0.05)in OA chondrocytes transfected with LV-MEG3 compared to those in the LV-NC group.Up-regulation of MEG3 increased DVL2,cyclin D1,andβ-catenin expression(P<0.05)and decreased GSK-3βexpression(P<0.05).Meanwhile,the effects of overexpression MEG3 on OA chondrocyte proliferation and apoptosis were reversed by inhibition of the Wnt/β-catenin signal pathway by XAV939.Conclusions:MEG3 expression was down-regulated in both OA patients and chondrocytes,which could affect the proliferation and apoptosis in OA chondrocytes by blocking the activation of Wnt/β-catenin signaling pathway.MEG3 may become an important target molecule for OA treatment.
作者
赵轶男
袁志
毕龙
郝赋
牛志霞
孙强
ZHAO Yi-nan;YUAN Zhi;BI Long;HAO Fu;NIU Zhi-xia;SUN Qiang(Department of Orthopaedics,Xijing Hospital,Xi'an,Shaanxi,710000,China)
出处
《现代生物医学进展》
CAS
2019年第24期4617-4623,共7页
Progress in Modern Biomedicine
基金
国家自然科学基金项目(2016YFC1100304).
