Hypoxia, Survivin As A Downstream Signaling Molecule Of FAK On Proliferation Of Human Pulmonary Artery Smooth Muscle Cells

ObjectiveTo understand whether survivin be involved in the proliferation of humanpulmonary artery smooth muscle cells(HPASMCs)in hypoxia in order to explore thepricing mechanism of pulmonary hypertension.MethodsPrimary HPASMCs was cultured.The cells are preconditioned by hypoxia for0h、0.5h、1h、2h、4h、8h、12h. The protein expression of survivin was detectedBy western blotting and immunofluorescence staining, the expression of survivinmRNA was surveyed by realtime–PCR. In addition, the ability of cell proliferationwas watched by Cell Counting Kit-8(CCK8)in hypoxia,At the same time the abilityof migration of HPASMCs in hypoxia was observed by cell migration test.ResultsThe experiment by western blotting and immunofluorescence staining show thatthe protein expression of survivin is increased significantly by hypoxicpreconditioning. It reached to peak in hypoxia at2h. Likewise, the expression ofsurvivi mRNA is increased by realtime-PCR.which peaked in hypoxia at2h. theability of proliferation by CCK8test is significantly enhanced in hypoxa at12h、24h、48h. Meanwhile the ability of migration of HPASMCs is significantly elevated inhypoxia.ConclusionThe protein expression of survivin in HPASMCs is increased in hypoxia, whichsuggest survivin participate in the proliferation of HPSMCs in hypoxia. ObjectiveTo explore whether focal adhesion kinase (FAK) via survivin signal pathwaypromote the proliferation of HPASMCs in hypoxia.MethodsPrimary HPASMCs was cultured. Three FAKsiRNA sequences synthetic designwas in vitro. And then the target sequence to inhibit expression of FAKmRNA ofHPASMCs for the highest inhibition rate(at least70%) was screening. Afterwardswhich was transtected into cell by lipofectin2000.The cells are grouped into nomoxicblank group,nomoxic FAKsiRNA transfecting group,nomoxic noncontrol siRNAtransfecting group,hypoxic blank group,hypoxic FAKsiRNA transfectinggroup,hypoxic noncontrol siRNA transfecting group. What is more,the change ofFAK and survivin protein expression in these groups was detected by westernblotting,so do FAKmRNA and survivin mRNA by realtime–PCR. simultaneouslythe ability of cell proliferation was observed.The ability of cell proliferation waswatched by CCK8and the change of protein expression by EDUimmunofluorescence.ResultsComparing with the nomoxic noncontrol siRNA transfecting group, the nomoxicblank group has no significant difference,but the nomoxic FAKsiRNA transfectinggroup has remarkly difference,which the protein and mRNA expression of FAKdecreased significantly. Besides, the expression of survivin protein and survivinmRNA have no significant difference；Comparing with the hypoxic noncontrol siRNA transfecting group， the hypoxic blank group has no significant difference,but thehypoxic FAKsiRNA transfecting group has a significant difference,which the proteinand mRNA expression of FAK along with survivin were decreased significantly. Inaddition, the survivin protein and survivin mRNA expression of the nomoxic survivinsiRNA transfecting group and the hypoxic survivin siRNA transfecting group werecut down significantly,while the FAK protein and FAKmRNA expression have aunchanged.Furthermore, Cell proliferation test by EDU immunofluorescence andCCK8showed that there are a significantly less in the nomoxic and hypoxicFAKsiRNA transfecting group than noncontrol siRNA transfecting group.ConclusionsThe result displays that knocking out FAK gene can reduce the expression ofsurvivin of HPSMCs in hypoxia, and inhibit the proliferation of HPASMCs.but afterknocking out survivin gene,the expression of FAK protein and FAKmRNA have nosignificant change.Experimental results reveals that survivin as the signaling molecules downstreamof FAK,It participate the proliferation of HPSMCs in hypoxia.