Upregulation of SRF Is Associated With Hypoxic Pulmonary Hypertension by Promoting Viability of Smooth Muscle Cells via Increasing Expression of Bcl-2

Abstract

The aim of study was to investigate the involvement of hypoxia-induced upregulation of serum response factor (SRF) and its downstream effector, B cell leukemia-2 (Bcl-2), in hypoxia-induced pulmonary hypertension (PH). Immunohistochemistry analysis and western blot analysis were used to detect the levels of SRF and Bcl-2 in rats exposed to hypoxia. Furthermore, the regulatory relationship between SRF and Bcl-2 was investigated in PASMCs using real-time PCR and western-blot analysis. We found that mPAP (mean pulmonary arterial pressure) and WA (the ratio of vascular wall area to external diameter) were increased after exposure to hypoxia, while LA (the ratio of vascular lumen area to total area) decreased after exposure to hypoxia. The immunohistochemistry analysis displayed a substantial increase in SRF and Bcl-2 in pulmonary arterial walls after 14 days of hypoxia. And the western blotting showed that SRF and Bcl-2 protein levels were much higher after 7 days of hypoxia and then remained at a high level. And then the levels of SRF and Bcl-2 in pulmonary artery smooth muscle cells (PASMCs) exposed to hypoxia were substantially suppressed following introduction of SRF siRNA, and the level of Bcl-2 was remarkably inhibited by Bcl-2 siRNA, while Bcl-2 siRNA had no effect on SRF level. Finally, SRF siRNA, and Bcl-2 siRNA significantly reduced viability of PASMCs exposed to hypoxia, and enhanced apoptosis of PASMCs exposed to hypoxia. These data validated that SRF responded to hypoxia, which subsequently was involved in pulmonary hypertension by abnormally promoting viability of PASMCs via modulating expression of Bcl-2. J. Cell. Biochem. 118: 2731-2738, 2017. © 2017 Wiley Periodicals, Inc.

Microbiota Imbalance Contributes to COPD Deterioration by Enhancing IL-17a Production via miR-122 and miR-30a

“…To confirm the regulatory relationship between IL-17a and miR-122, miR-125b, and miR-30a, we cloned the 3′ UTR of IL-17a containing the binding sites for miR-122, miR-125b, and miR-30a into pcDNA vectors (Promega, Madison, WI, USA) to generate a wild-type luciferase vector of IL-17a 3′ UTR. 40 , 41 At the same time, we used a Quick Change mutagenesis assay kit (Stratagene, San Diego, CA, USA) following the standard protocol provided by the assay kit manufacturer to induce site-directed mutations in the miR-122, miR-125b, and miR-30a binding sites of IL-17a 3′ UTR, respectively, to generate various mutant vectors of IL-17a 3′ UTR. In the next step, PASMCS cells were co-transfected with wild-type or mutant luciferase vectors of IL-17a 3′ UTR in conjunction with miR-122, miR-125b, or miR-30a mimics using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the standard protocol provided by the assay kit manufacturer.…”

Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

“…The experiments were performed as previously described. 56 , 57 TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from PASMCs or tissue samples. An All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia, Rockville, MD, USA) was used to determine the relative expression of H19, miR-200a, and miR-675-3p that has been normalized to the expression of small RNA U6.…”

Combined Effects of PVT1 and MiR-146a Single Nucleotide Polymorphism on the Lung Function of Smokers with Chronic Obstructive Pulmonary Disease

“…The experiments were performed as previously described 29 , 30 . An RNeasy Mini kit (Qiagen, Germany) was utilized to extract the total RNA from the fibroblasts, PASMCs, and tissue samples.…”