Analysis of crucial genes, pathways and construction of the molecular regulatory networks in vascular smooth muscle cell calcification

Vascular calcification (VC) accompanies the trans-differentiation of vascular smooth muscle cells (VSMCs) into osteo/chondrocyte-like cells and resembles physiological bone mineralization. However, the molecular mechanisms underlying VC initiation and progression have remained largely elusive. The aim of the present study was to identify the genes and pathways common to VSMC and osteoblast calcification and construct a regulatory network of non-coding RNAs and transcription factors (TFs). To this end, the Gene Expression Omnibus dataset GSE37558 including mRNA microarray data of calcifying VSMCs (CVSMCs) and calcifying osteoblasts (COs) was analyzed. The differentially expressed genes (DEGs) were screened and functionally annotated and the microRNA (miRNA/mRNA)-mRNA, TF-miRNA and long non-coding RNA (lncRNA)-TF regulatory networks were constructed. A total of 318 DEGs were identified in the CVSMCs relative to the non-calcified VSMCs, of which 43 were shared with the COs. The CVSMC-related DEGs were mainly enriched in the functional terms cell cycle, extracellular matrix (ECM), inflammation and chemotaxis-mediated signaling pathways, of which ECM was enriched by the DEGs for the COs as well. The protein-protein interaction network of CVSMCs consisted of 281 genes and 3,650 edges. There were 30 hub genes in this network, including maternal embryonic leucine zipper kinase (MELK), which potentially regulates the differentially expressed TF (DETF) forkhead box (FOX)M1 and is a potential target gene of Homo sapiens miR-485-3p and miR-181d. The TF-miRNA network included 251 TFs and 60 miRNAs, including 10 DETFs such as FOXO1 and snail family transcriptional repressor 2 (SNAI2). Furthermore, the lncRNAs H19 imprinted maternally expressed transcript (H19) and differentiation antagonizing non-protein coding RNA (DANCR) were predicted as the upstream regulators of FOXO1 and SNAI2 in the lncRNA-TF regulatory network. DANCR, MELK and FOXM1 were downregulated, and H19, FOXO1 and SNAI2 were upregulated in the CVSMCs. Taken together, the CVSMCs and COs exhibited similar molecular changes in the ECM. In addition, the MELK-FOXM1, H19/DANCR-FOXO1 and SNAI2 regulatory pathways likely mediate VSMC calcification.