The Role And Mechanism Of MiR-320a Inhibits Angiotensinâ…¡-induced Proliferation In Human Aortic Vascular Smooth Muscle Cells By Targeting MMP-9

PartⅠThe express validation of differential miRNAs in human aortic vascular smooth muscle cellsObjective To detect expression levels of chip differential miRNAs in HAVSMC.Methods ① 38 differential expressed miRNAs were selected from the known gene microarray of TAD/NA.② Design the corresponding primers of miRNAs. ③ cell passage cultivation of HAVSMC. ④ total RNA extraction and RT-PCR.Results 38miRNAs were all expressed in HAVSMC,12 of them were in high level, 8 were in moderate expression and 18 were in low.Conclusion The expression of selected miRNAs exist differences in HAVSMC. KeyPart ⅡHigh Content Screening (HCS) for functional miRNAsObjective To screen functional miRNAs for HAVSMC.Methods ① preliminary experiment cell transfection by slow virus/adenovirus to determine virus vector; ②select 20 miRNAs from upon ones;③divide the 20 miRNAs into overexpression and down-expression, build the viral vectors; ④to observe the cell proliferation by cell counting based on the Cellomics HCS systemResults ①adenovirus was more sensitive to the cell;②Cells of each group growth well for 5 consecutive days, but the proliferation of each group exist difference.Conclusion 14 miRNAs are functional to HAVSMC proliferation. HCS system is accurate, stable and high credibility for miRNAs screening.Part ⅢmiR-320a inhibits angiotensin Ⅱ-induced proliferation and migration in human aortic vascular smooth muscle cells by targeting MMP-9Objective To investigate the role and mechanism of miR-320a on Ang Ⅱ induced HAVSMC.Methods To establish a cell culture system in vitro, ①transfection rate of miR-320a mimics was measured by RT-PCR.②using CCK-8 to verify the function of angiotensin Ⅱ to Human Aortic Vascular Smooth Muscle Cells with the change of time and concentration,also the effect of miR-320a on the cell proliferation which induced by angiotensinⅡ; ③detect the effect of miR-320a on cell migration by scratch and transwell; ④verify the expression level of MMP9-mRNA after mimics transfection by RT-PCR;⑤ test the expression level of MMP-9 and MAPK/ERK protein in HAVSMC by Western Blot.Results Ang Ⅱ can promote human aortic vascular smooth muscle cells proliferation remarkably, which is positively correlated with action time and concentration; miR-320a overexpression inhibits angiotensin Ⅱ-induced proliferation and migration in HAVSMC; miR-320a overexpression inhibits express of MMP9, both in mRNA and protein level; miR-320a overexpression inhibits express of ERK1/2 protein.Conclusion miR-320a inhibits angiotensin Ⅱ-induced proliferation and migration in human aortic vascular smooth muscle cells by targeting MMP-9; MAPK/ERK pathway maybe related to the regulation.