circCDR1as/miR-7-5p/RAF1轴参与调控激素性股骨头坏死中的自噬

CircCDR1as/miR-7-5p/RAF1 axis promotes autophagy levels in steroid-induced necrosis of the femoral head

背景:自噬可能参与激素性股骨头坏死的病理过程。有研究证实环状RNA(circular RNA,circRNA)在激素性股骨头坏死中具有调控机制,然而,circCDR1as是否在激素性股骨头坏死中影响自噬尚未被研究。目的:探讨激素性股骨头坏死中自噬水平及circCDR1as的调控机制。方法:①从GSE26316数据集中获取激素性股骨头坏死及对照组大鼠的基因表达谱并进行差异表达分析,随后分析差异表达基因的生物学功能。通过数据库预测circCDR1as的靶标miRNAs及靶标基因。比较靶标基因与差异表达基因,构建circCDR1as的调控网络。②收集激素性股骨头坏死患者及健康对照人群的股骨头样本;同时应用骨髓间充质干细胞进行细胞实验:随机分为骨髓间充质干细胞组、模型组(甲泼尼龙处理)、模型+si-NC组、模型+si-CDR1as组。利用RT-qPCR检测组织和细胞circCDR1as及靶标基因的表达,Western blot检测自噬相关蛋白的表达。③构建荧光素酶报告基因载体:pmirGLO-CDR1as(WT)、pmirGLO-RAF1(WT)、pmirGLO-CDR1as(MUT)和pmirGLO-RAF1(MUT),转染到细胞中,并设miR-7-5p mimic和mimic NC组,检测circCDR1as网络的靶向调控关系。结果与结论:①大鼠激素性股骨头坏死组与对照组之间鉴定了1283个差异表达基因,主要参与细胞凋亡和自噬信号通路。预测发现circCDR1as靶向调控6个miRNAs,这些miRNAs靶向调控305个靶标基因,其中有31个靶标基因在激素性股骨头坏死中差异表达,RAF1参与自噬被选择为关键基因,并构建了circCDR1as/miR-7-5p/RAF1的调控网络。②与对照组比较,circCDR1as,RAF1和自噬水平在激素性股骨头坏死患者及激素诱导的骨髓间充质干细胞中表达上调(P<0.05),miR-7-5p表达下调(P<0.05);敲降circCDR1as后细胞自噬水平显著下降(P<0.05)。③双荧光素酶报告检测证实了circCDR1as与miR-7-5p以及miR-7-5p与RAF1的靶向调控关系。④结论:CircCDR1as/miR-7-5p/RAF1可能通过自噬信号促进激素性股骨头坏死,靶向circCDR1as是通过部分自噬修复治疗激素性股骨头坏死的潜在策略。

BACKGROUND:Autophagy may be involved in the pathological process of steroid-induced necrosis of the femoral head(SINFH).Some studies have confirmed that circular RNAs(circRNAs)have a regulatory mechanism in SINFH;however,whether circCDR1as affects autophagy in SINFH has not been investigated.OBJECTIVE:To explore the level of autophagy and the regulatory mechanism of circCDR1as in SINFH.METHODS:Gene expression profiles of SINFH and control rats were extracted from the GSE26316 dataset and differential expression analysis was performed.Subsequently,the biological functions of differentially expressed genes were analyzed.Then,the target miRNAs of circCDR1as and the target genes of target miRNAs were predicted.Further,the target genes were compared with the differentially expressed genes to construct the regulatory network of circCDR1as.In addition,femoral head samples from patients with SINFH and healthy control individuals were collected.Bone marrow mesenchymal stem cells were also applied for cellular experiments and randomly divided into bone marrow mesenchymal stem cell group,model group(methylprednisolone-treated),model+si-NC group,and model+si-CDR1as group.RT-qPCR was used to detect the expression of circCDR1as and target genes in cells and tissue samples.Western blot was used to examine the expression of autophagy proteins.The luciferase reporter gene vectors,pmirGLO-CDR1as(WT),pmirGLO-RAF1(WT),pmirGLO-CDR1as(MUT),and pmirGLO-RAF1(MUT),were constructed and transfected into the cells.miR-7-5p mimic and mimic NC groups were established.The target-regulatory relationship of the circCDR1as network was detected.RESULTS AND CONCLUSION:A total of 1283 differentially expressed genes were identified between the SINFH and control groups,which were mainly involved in apoptotic and autophagic signaling pathways.Prediction analysis revealed that circCDR1as targeted 6 miRNAs,which in turn regulated 305 target genes.Among these target genes,31 showed differential expression in SINFH.Among the differentially expressed target genes,RAF1,involved in autophagy,was selected as a key gene,leading to the construction of the circCDR1as/miR-7-5p/RAF1 regulatory network.Compared with the control group,circCDR1as,RAF1,and autophagy levels were upregulated in patients with SINFH and in hormone-induced bone marrow mesenchymal stem cells(P<0.05),while miR-7-5p expression was downregulated(P<0.05).Knockdown of circCDR1as significantly decreased cellular autophagy levels(P<0.05).Dual-luciferase reporter assays confirmed the targeting relationships between circCDR1as and miR-7-5p,as well as between miR-7-5p and RAF1.To conclude,the CircCDR1as/miR-7-5p/RAF1 may potentially promote SINFH through autophagy.Targeting circCDR1as is a potential strategy for partial autophagic repair in the treatment of SINFH.