RNA sequencing analysis identifies stiffness and survivin-associates genes regulating cellular functions

Introduction: Cell proliferation and migration is critical for normal development and wound healing. However, in the context of neointima and plaque formation in atherosclerosis, it can lead to adverse outcomes. Several mechanobiological processes, such as an increase in vascular stiffness, become active but remain understudied during or after neointima and plaque formation, particularly regarding uncontrolled cell proliferation and migration. Previous studies by others have demonstrated that survivin is highly expressed during neointima formation after vascular injury in rabbits and inhibition of survivin decreases neointima formation, suggesting its regulatory role in vascular repair and injury. We have recently identified survivin (also known as BIRC5) as a novel mechanoprotein that is upregulated in stiff environments, such as those found in neointima and atherosclerotic plaque. Thus, we hypothesize that survivin is important in regulating stiffness-mediated cell proliferation and migration, as well as possibly other biological processes. However, the detailed molecular mechanisms behind this stiffness- and survivin-dependent proliferation are still unknown, which prompted us to undertake RNA sequencing (RNA-seq) analysis to investigate how survivin modulates stiffness-mediated cell proliferation and migration. RNA-seq analysis enables us to predict potential molecular players regulated by stiffness and/or survivin, crucial in proliferation and migration-related pathway, while also uncovering new cellular functions.

Materials and

Methods: To investigate the role of survivin in stiffness-dependent cellular functions, primary human VSMCs transfected with control or survivin siRNA were plated on low stiffness (2-8 kPa; “soft”) and high stiffness (20-24 kPa; “stiff”), which approximate the physiological stiffness of a healthy mouse femoral artery and those observed after vascular injury or during atherosclerosis, respectively. Cell cycle progression and proliferation were assessed through immunoblotting or RT-qPCR for cell cycle markers (cyclin D1 and cyclin A), as well as EdU incorporation or cell counting assays. For RNA sequencing analysis, RNA samples isolated from: 1) control siRNA-treated VSMCs cultured on soft and stiff hydrogels, and 2) survivin-siRNA transfected VSMCs cultured on stiff hydrogels, were sent to the University at Buffalo Genomics and Bioinformatics Core. For RNA-seq data analysis, we used DSeq2 analysis along with Sample Correlation metrics, Gene Ontology, Cytoscape, and Ingenuity Pathway Analysis (IPA).

Results, Conclusions, and Discussions: Immunoblotting and RT-qPCR showed that survivin knockdown in VSMCs led to a decrease in stiffness-mediated protein (Fig. 1A) and mRNA (Fig. 1B-D) expression of survivin, cyclin D1 and cyclin A. Additionally, survivin inhibition reduced cell proliferation, as indicated by EdU incorporation (Fig. 1E) and counting (Fig. 1F). These functional assays collectively demonstrated the essential role of survivin in the observed responses of VSMCs to increased stiffness. We then analyzed RNA-seq data to further investigate how survivin mediates the effects of ECM stiffness on cell cycling and proliferation in VSMCs. A total of 15,615 genes were initially identified. These genes were filtered based on a fold-change threshold of ±1.5 and a significance threshold of p-adjusted value < 0.05. 336 genes total were in the control siRNA-stiff vs control siRNA-soft dataset (“stiffness-regulated dataset”), 1,060 genes in the control siRNA-stiff vs survivin siRNA-stiff (“survivin-regulated dataset”)t, and the overlapping genes (“stiffness- and survivin-regulated dataset”) were 72 genes (Fig. 2A). In the stiffness-regulated dataset (Fig. 2), gene ontology (GO) analysis for upregulated DEGs (Fig. 2B) revealed highly enriched processes including cell migration, morphogenesis, and development, while downregulated DEGs (Fig. 2C) exhibit highly enriched metabolic-related functions. Figures 2D/2E also revealed top HUB genes linked to growth, differentiation, and metabolic-related genes. Additionally, top Canonical Pathways were related to Cell Cycle regulation and Smooth Muscle Contraction (Fig. 2E). In the survivin-regulated dataset (Fig. 3), the GO analysis highlighted survivin’s role in immune response, cell cycle regulation, and development. HUB Genes related to the immune system, development, and cell cycle regulation were also revealed in figures 3C/3D. Additionally, top Canonical Pathways were associated with Interferon Signaling, Hepatic Fibrosis Signaling Pathway, and more. In figure 4 (stiffness- and survivin-regulated dataset), GO analysis revealed processes related to development, differentiation, and actin filament, along with top HUB genes from Cytoscape. Here, we identified distinct cell proliferation-related processes in both survivin and stiffness-dependent datasets, indicating survivin’s predicted roles in these processes and multifunctionality, along with inflammation, development, cell signaling and more predicted networks and pathways also highlight potential target genes in survivin and stiffness dependent pathway.

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