CircRNA ARHGAP10 promotes osteogenic differentiation through the miR-335-3p/ RUNX2 pathway in aortic valve calcification

Yun Jiang; Jiaqi Zhu; Zhijian Chen; Weixin Wang; Zhenyu Cao; Xingyou Chen; Jianle Chen

doi:10.21037/jtd-23-919

CircRNA ARHGAP10 promotes osteogenic differentiation through the miR-335-3p/ RUNX2 pathway in aortic valve calcification

J Thorac Dis. 2023 Nov 30;15(11):5971-5991. doi: 10.21037/jtd-23-919. Epub 2023 Oct 24.

Authors

Yun Jiang^#¹, Jiaqi Zhu^#², Zhijian Chen^#², Weixin Wang², Zhenyu Cao², Xingyou Chen³, Jianle Chen²

Affiliations

¹ Department of Burn and Plastic Surgery, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong, China.
² Department of Thoracic Surgery, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong, China.
³ Medical School of Nantong University, Nantong, China.

^# Contributed equally.

Abstract

Background: Calcific aortic valve disease (CAVD) is a common cardiovascular disease with high morbidity and mortality, and no effective prevention or treatment is available. In recent years, increasing evidence has shown that noncoding RNAs (ncRNAs) play an important role in the pathogenesis and prognosis of CAVD. Several associated circular RNAs (circRNAs) have been reported to be involved in CAVD, such as circRIC3 and TGFBR2. However, the limited number of circRNAs identified in CAVD warrants further in-depth investigation, and the comprehensive elucidation of their role in the key mechanisms of this disease is needed.

Methods: The expression of circRNAs and microRNAs (miRNAs) were analyzed by RNA sequencing. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to analyze the expression of circRNA ARHGAP10 (circARHGAP10), miR-335-3p, and RUNX2. Luciferase reporter assay, pull-down assay, and RNA binding protein immunoprecipitation (RIP) assay were performed to evaluate the binding of miR-335-3p to circARHGAP10 or RUNX2. Alizarin red S staining showed the formation of calcified nodules in valve interstitial cells (VICs). The expression of circARHGAP10 and miR-335-3p was altered through lentivirus infection. Alkaline phosphatase (ALP) activity was used to verify the correlation between circARHGAP10 and miR-335-3p. The expression of proteins was assessed via Western blot. RNA fluorescence in situ hybridization (FISH) was used to confirm the localization of circARHGAP10 in the cytoplasm of VICs. Immunofluorescence was used to detect the expression level of RUNX2. ApoE^-/- mice were used to construct a CAVD model, circARHGAP10 short hairpin RNA (shRNA) and miR-335-3p inhibitor lentivirus were intraperitoneally injected, and scramble and inhibitor normal control (NC) lentivirus were injected as controls, followed by hematoxylin and eosin (HE) staining.

Results: Through RNA sequencing, we found that circARHGAP10 (hsa_circ_0008975) was highly expressed in calcific aortic valves. CircARHGAP10 knockdown effectively inhibited the extent of osteogenic differentiation of VICs. We then found that circARHGAP10 was a competing endogenous RNA (ceRNA) of miR-355-3p and that miR-355-3p targeted RUNX2. In vitro experiments confirmed that circARHGAP10 regulated the osteogenic differentiation of VICs through the miR-355-3p/RUNX2 pathway, and this was validated in vivo using an ApoE^-/- mouse model.

Conclusions: These findings provide a foundation for circRNA-directed diagnostics and therapeutics for CAVD.

Keywords: Calcific aortic valve disease (CAVD); RUNX2; circRNA ARHGAP10 (circARHGAP10); miR-335-3p; osteogenic differentiation; valve interstitial cells (VICs).