| Background:Pulmonary arterial hypertension(PAH) is a severe disease closely related to increased pulmonary vascular resistance chronic pulmonary, and its main clinical features are the abnormal progressive increasing of pulmonary vasospasm, pulmonary vascular remodeling,and pulmonary thrombosis. Severe PAH could lead to in situ thrombosis, right heart failure and death,if not treated timely. Its pathogenesis include the abnormal cellular, molecular and genetic factors of medium and other ways, the damage of vascular endothelial cells, the increasing of pulmonary vasoconstriction and vascular remodeling, Inflammation and immune disorders are also involved. The imbalance of the vascular active substances also contribute to its occurrence. the hypoxic pulmonary artery vasoconstriction,(HPV)and the hypoxic pulmonary vascular remodeling(HPVR) are the two main courses of physiology and pathology leading to PAH.,especially the significant role of proliferation of PASMCs. Although the pathogenesis of PAH is not completely elucidated and is still explored, the more and more attention are paid to the imbalance of proliferation and apoptosis(especially PASMCs).microRNAs(miRNAs) in eukaryotic cells are approximately 22 nucleotide noncoding small RNAs that regulate gene expression at the post-transcriptional level through translational repression or mRNA degradation. miRNAs can bind to complimentary sequences in the 3'UTRs of mRNAs of target gene and negatively regulate the translation and/or post-transcriptional process of target gene,which further affect the biological behavior of cells including development, differentiation,proliferation and apoptosis. There are more than 300 miRNA gene clusters in the human genome, miR- 17 family called miR- 106 – b family, is composed of six similar in sequence, structure and seed sequence(AAAGUGCU)), highly conserved among species of the mature miRNA body composition. Many studies have shown that the miR- 17 families involved in mammalian organs like hearts, lungs, immune system development, and is closely related to the occurrence of tumors. The fact has been widely concerned.The mitofusin2 is the member of the Mitochondria fusion protein family. It Is not only an important element in maintaining the mitochondria normal l morphology and molecular characteristics of the necessary important elements, but also has a regulating cell proliferation, differentiation, apoptosis and signal transduction, and other functions at the same time. The study have found that Mfn2 in vascular smooth muscle cells(VSMCs) could inhibit the PI3K/Akt signal pathway, increase the mitochondrial outer membrane permeability, and then activate the mitochondrial apoptotic pathways, finally promote VSMCs) apoptosis. Mfn2 plays an important role in the VSCMs proliferation of cardiovascular disease, with the in-depth study, whether the PASMCs has the same effect in HPH is not clear.In recent years, many studies have found that miR- 17 can be deregulatedd through the PDLIM5 or BMPR2 miR- 17 in the expression regulation of cell proliferation or apoptosis, participating in the regulation of PASMCs, thus leads the occurrence of PAH. However, miR- 17 in pulmonary hypertension disease is involved in research and animal models, miR- 17 and Mfn2 play an important role in hypoxia-induced animal model of PAH. Whether the miR- 17 can be regulated through targeted Mfn2 in vitro human clinical study of PASMCs was not reported. Therefore, we hypothesize that miR- 17 may be targeted by regulating the expression of the Mfn2 thus inhibit PASMCs proliferation and delay theprogress of pulmonary hypertension. Aims:The subject intend to normal lung tissue and pulmonary hypoxia-induced pulmonary arterial smooth muscle cells as materials,process simulate the process of chronic hypoxia. qRT-PCR and Western blot(WB) to detect miR-17 Mfn2 and protein expression in hypoxia-induced PAH and normoxic models of human lung tissue-17 target genes.; through dual luciferase report to verify the site of action of miR;This subject explored the miR- 17 and its target genes of anoxic PASMCs proliferation, apoptosis and related proteins. Methods:1. To simulate the process of PAH, cells were transferred in a special hypoxia incubator with 3% O2, 5% CO2 and balance nitrogen. Then the expression levels of miR-17 and Mfn2 in PASMCs exposed to hypoxia and normoxic control were determined by qRT-PCR and western blot, respectively.2. To investigate the effect of miR-17 in PASMC proliferation and apoptosis, PASMCs were transfected with anti-miR-17 or control inhibitor(anti-miR-NC). Proliferation of PASMCs exposed to normoxia orhypoxia was determined by using CellTiter-Glo Luminescent cell viability assay kit. Western blot assay was used to determine the protein expression level of the proliferation marker PCNA.The apoptosis rate was measured by a flow cytometry. And the Caspase-3 activity was also measured.3. The PASMCs were transfected with miR-NC, miR-17 mimics, anti-miR-17 or anti-miR-NC. A dual-luciferase reporter assay was conductedat 48 h after transfection. To determine whether miR-17 suppress the expression of Mfn2 protein, we conducted western blot to detect the expression in cells transfected with miR-NC, miR-17 mimics, anti-miR-17 or anti-miR-NC. To investigatethe underlying mechanism that miR-17 regulates proliferation and apoptosis of PASMCs under hypoxic condition, PASMCs were transfected with siMfn2 to inhibite Mfn2 expression. Then, the cell viability and increased apoptosis were observed. The pro-apoptotic protein cleaved Caspas-3 and the proliferation marker PCNA were observed by western blot. Results:1. miR-17 expression was upregulated and MFN2 protein was downregulated in PAH tissues compared with matched normal lung tissues. In addition, expression of miR-17 was also increased in hPASMCs exposed to hypoxiacompared with that in normoxic control. A reduction of MFN2 protein level was observed in hPASMCs treated with hypoxia compared to controls.2. The number of viable cells was greater in the anti-miR-17 group as compared with the anti-miR-NC control group, indicating that inhibition of miR-17 suppressed hypoxia-induced hPASMC proliferation. Western blot assay showed that the protein expression level of the proliferation marker PCNA was decreased in hPASMCs transfected with anti-miR-17 compared with that in hPASMCs transfected with anti-miR-NC. The number of apoptotic hPASMCs was remarkably increased in the anti-miR-17 group compared with the control group. The Caspase-3 activity in hPASMCs transfected with miR-17 inhibitor was increased when compared with that in hPASMCs transfected with negative controls.3. A decrease in relative luciferase activity was observed when cells were co-transfected with miR-17 mimics and WT MFN 3'UTR, whereas no change was found in cells co-transfected with miR-17 and mutant Mfn2 3'UTR. The protein expression level of Mfn2 was higher in the anti-miR-17 group than that in the anti-miR-NC group. Decreased cell viability and increased apoptosis and Caspase-3 activity were observed in the anti-miR-17 + siMfn2 group as compared with the anti-miR-NC + siNC group. Moreover, an upregulation of the pro-apoptotic protein cleaved Caspas-3 and a downregulation of the proliferation marker PCNA were observed. Conclusion1. miR-17 is upregulated and Mfn2 is downregulated in PASMCs treated with hypoxia and lung tissues of PAH patients.2. Inhibition of miR-17 suppresses hypoxia-induced PASMC proliferation and promotes hypoxia-induced PASMC apoptosis in vitro.3. miR-17 regulates hypoxia-induced PASMC proliferation and apoptosis through directly targeting Mfn2 modulation.In summary, our data demonstrated that miR-17/Mfn2 regulatory pathway is induced by hypoxia-mediated PASMC proliferation and reduced apoptosis important regulatory mechanism. miR-17 may be the prevention and treatment of pulmonary hypertension potential therapeutic targets. |
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