环状RNA circ_0040646靶向抑制miR-188-3p调控膝骨关节炎软骨细胞的增殖、分化和凋亡

A circular RNA,circ_0040646,regulates the proliferation,differentiation,and apoptosis of knee osteoarthritis chondrocytes by targeted inhibition of microRNA-188-3p

背景:环状RNA在越来越多的疾病中发挥重要的调控作用,近期有研究显示环状RNA在膝骨关节炎中存在异常表达,因此研究环状RNA在骨关节炎中的作用对阐明膝骨关节炎的发病机制具有重要作用。目的:探讨circ_0040646调控miR-188-3p对膝骨关节炎软骨细胞增殖、分化和凋亡的影响。方法:采用实时荧光定量PCR检测circ_0040646在膝骨关节炎患者和膝骨关节炎软骨细胞中的表达水平,并以半月板损伤患者的正常软骨细胞为对照。在膝骨关节炎软骨细胞中分别转染si-NC(低表达circ_0040646对照组质粒)、si-circ_0040646、oe-NC(过表达circ_0040646对照组质粒)、oe-circ_0040646、miR-NC(对照组)和miR-188-3p抑制剂后,采用实时荧光定量PCR和Western blot法检测增殖细胞核抗原、骨形成蛋白2和runt相关转录因子2的表达水平;流式细胞术检测细胞凋亡情况;双荧光素酶报告检测circ_0040646和miR-188-3p的靶点结合情况。结果与结论:①与对照组相比,circ_0040646在膝骨关节炎患者和膝骨关节炎软骨细胞中明显高表达;②在膝骨关节炎软骨细胞中低表达circ_0040646能够促进细胞增殖和分化,抑制细胞凋亡;过表达circ_0040646能够抑制细胞增殖和分化,促进细胞凋亡;③circ_0040646在膝骨关节炎软骨细胞中能够靶向结合miR-188-3p,在膝骨关节炎软骨细胞中共转染si-circ_0040646和miR-188-3p inhibitors能够反转单独转染si-circ_0040646对膝骨关节炎软骨细胞增殖、分化和凋亡的影响;④结果显示circ_0040646通过靶向抑制miR-188-3p调控膝骨关节炎软骨细胞的增殖、凋亡和分化,circ_0040646可能成为治疗膝骨关节炎的干预靶点。

BACKGROUND:Circular RNAs play an important regulatory role in various diseases.Recent studies have shown that circular RNAs are abnormally expressed in knee osteoarthritis.Therefore,it is very essential to study the effect of circular RNAs on the pathogenesis of knee osteoarthritis.OBJECTIVE:To investigate the effect of circ_0040646 regulating microRNA-188-3p(miR-188-3p)on the proliferation,differentiation and apoptosis of knee osteoarthritis chondrocytes.METHODS:Quantitative real-time fluorescence PCR was used to detect the expression level of circ_0040646 in knee osteoarthritis patients and knee osteoarthritis chondrocytes,in comparison with normal chondrocytes from meniscal injury patients.Chondrocytes from knee osteoarthritis patients were transfected with small-interfering negative control(lowly expressed circ_0040646 control plasmid),small-interfering circ_0040646,overexpressed negative control(overexpressed circ_0040646 control plasmid),overexpressed circ_0040646,miRNA negative control(control group),or microRNA-188-3p inhibitor.Quantitative real-time fluorescence PCR and western blot were used to detect the expression levels of proliferating cell nuclear antigen,bone morphogenetic protein 2 and runt-related transcription factor 2.Flow cytometry was used to detect cell apoptosis.Dual luciferase reporter assay was used to detect the targeted binding of circ_0040646 and miR-188-3p.RESULTS AND CONCLUSION:Compared with the control group,circ_0040646 was highly expressed in knee osteoarthritis patients and knee osteoarthritis chondrocytes.In knee osteoarthritis chondrocytes,the low expression of circular RNA-0040646 could promote cell proliferation and differentiation,and inhibit cell apoptosis,while overexpression of circ_0040646 could inhibit cell proliferation and differentiation,and promote cell apoptosis.circ_0040646 could target miR-188-3p in knee osteoarthritis chondrocytes.Co-transfection of small-interfering circ_0040646 and miR-188-3p inhibitors could reverse the effect of smallinterfering circ_0040646 alone on the proliferation,differentiation and apoptosis of knee osteoarthritis chondrocytes.These findings reveal that circ_0040646 can regulate the proliferation,apoptosis and differentiation of knee osteoarthritis chondrocytes by targeted inhibition of miR-188-3p,and therefore,circ_0040646 may become a target for the treatment of knee osteoarthritis.