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Effects of RNAi on hypoxia inducible factor--1#alpha# activity and proliferation of hypoxic pulmonary artery smooth muscle cells in rat

Zhang Wei, Cao Yue, Zhang Yu, Ma Qisheng, Ma Lan, Ge Rili

Research Center for High Altitude Medicine, Qinghai University.Xining 810001,Qinghai

Abstract

Pulmonary vascular remodeling is one of the major characteristics of hypoxia-induced pulmonary hypertension, mainly represented by over-proliferation of pulmonary artery smooth muscle cells (PASMCs). Hypoxia inducible factor-1#alpha# (HIF-1#alpha#) is a transcription factor which is produced by the cells exposed to hypoxia. HIF-1#alpha# up-regulates the expression of many hypoxia response genes (HRGs) for the body to adapt to hypoxia and maintain homeostasis. The expression of HIF-1#alpha# in the PASMCs is remarkably elevated under hypoxic condition and it stimulates the proliferation of PASMCs. In this experiment, we used gene clone technology to design and synthesize two siRNAs based on the sequence of HIF-1#alpha# mRNA. They were separately subcloned into the plasmid of pGenesil- 1 containing U6 promoter. The pGenesil- 1 vector of the RNA interference eukaryotic expression vector specific to HIF-1#alpha# gene was constructed. DNA sequencing of the plasmid verified the successful construction of the HIF-1#alpha# RNAi. We isolated and cultured the PASMCs of rat. The pGenesil-1 vector was transferred into the PASMCs with METAFECTENE in vitro. The positive cell clones transfected with pGenesil-1 were obtained after being screened with 400#mu#g/ml G418. These PASMCs were cultured in normoxia and hypoxia. After 48 h, the effects of RNAi on the expression of HIF-1#alpha# mRNA were detected by RT-PCR. The cellular growth activities were assayed by MTT colorimetry and flow cytometry in vitro. The results showed that for the PASMCs cultured in hypoxia for 48 h, the cell proliferation of blank group and control group were remarkably increased and the HIF-1#alpha#mRNA expressions were up-regulated, while the cell proliferation of the treatment groups did not increase and the HIF-1#alpha# mRNA expressions were not up-regulated. In conclusion, we successfully constructed the recombinant plasmid of RNAi and transfected them into the PASMCs in vitro. The RNAi inhibited the expression of HIF-1#alpha# mRNA in the PASMCs, and subsequently it remarkably suppressedthe proliferation of PASMCs in hypoxia. These results indicate that HIF-1#alpha# plays a pivotal role in PASMC proliferation.

Key words: Pulmonary artery;Smooth muscle cell;hypoxia inducible factor-1#alpha#;RNA interference;cell proliferation

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Citing This Article：

Zhang Wei, Cao Yue, Zhang Yu, Ma Qisheng, Ma Lan, Ge Rili. Effects of RNAi on hypoxia inducible factor--1#alpha# activity and proliferation of hypoxic pulmonary artery smooth muscle cells in rat. Acta Physiol Sin 2006; 58 (1): (in Chinese with English abstract).