THOC5-Dependent Posttranscriptional Control Maintains Vascular Smooth Muscle Cells Homeostasis Against Chronic Kidney Disease Related Vascular Calcification [RNA-seq]
Background: Despite transcriptional control mechanisms of VSMC osteogenic transition having been extensively studied, posttranscriptional regulation is still awaiting elucidation. In the present study, we explore the mechanism of THOC5-dependent VSMCs osteochondrogenic switching. Methods: Von Kossa staining and immunohistochemistry staining were used to detect calcification and expression of THOC5 respectively. Thoc5 shRNA and Thoc5 overexpression lentivirus were used to modulate the expression of Thoc5. RNA-Seq combined with RIP-Seq was used to explore the target mRNAs that directly bind to THOC5, and FISH was used to confirm its subcellular localization. Results: Immunohistochemical staining showed significantly increased THOC5 expression in the calcified artery of CKD patients. Besides, calcification-induced increase of THOC5 expression was found in both in vivo and in vitro calcification models. The overexpression of Thoc5 relieves the calcification and osteogenic differentiation of VSMCs significantly in vitro, which is mainly manifested by the reduction of calcium ion deposition and the decreased expression of osteogenic markers. Furthermore, RNA-Seq revealed that THOC5 overexpression in osteogenic-induced VSMCs closely resembled the gene expression changes induced on TGF-ß treatments in cultured VSMCs. In addition, overexpression of THOC5 alleviates the exacerbation of calcification in vivo. Our previous studies found that THOC5 displayed limited binding to VSMCs genomic DNA, so RIP-Seq was selected to detect target genes of THOC5. It was found that THOC5 directly interacts with Guanylate exchange factors (GEFs) mRNAs, and is required for their export. Thereby THOC5 maintaining RhoA GTPase activation contributes to increasing the expression of VSMCs contraction marker, which maintains the contraction phenotype of VSMCs. ROCK (Rho-kinase) inhibitor Y-27632 reversed the protective role of THOC5 on osteoblastic transdifferentiation and calcification, as well as the maintenance of the spindle morphology of VSMCs. Conclusions: Our data introduce the binding of THOC5 to GEFs as a novel mechanism contributing to maintaining VSMCs homeostasis and imply THOC5 as a potential intervention node for vascular calcification diseases. Overall design: To ascertain how THOC5 influences VSMCs global gene expression of VSMCs during the osteogenic transition. We performed RNA sequencing (RNA-Seq) on THOC5 overexpression VSMCs treated with Pi for 6 days . Then, We performed RNA immunoprecipitation sequencing (RIP-Seq) against THOC5 in VSMCs, both with or without Pi treatment, to assess the transcriptomic binding of THOC5.