Abstract: 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 1 283 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.

Key words: steroid-induced necrosis of the femoral head, autophagy, circCDR1as, RAF1, signaling pathway, target regulation