Mechanisms Of Resisting The Expression Of FAK Signaling Pathway To Promote The Proliferation Of Human Pulmonary Artery Smooth Muscle Cells

Objective:1） To observe the variations of FAK and survivin expressionsin HPASMCs stimulated by the different concentrations of recombinanthuman RELM-β.2) To investigate whether RELM-βcould lead to abnormalproliferation of HPASMCs though upregulating FAK signal pathway, and itscorrelations among them.Methods: Take the normal tissues around the lung cancer and culture themin vitro to5-6generations, then randomly divide them into4groups: Cgroup (control group),rhRELM-β25ng/ml group, rhRELM-β50ng/ml group，rhRELM-β100ng/ml group. To detect the mRNA expression level of FAK andsurvivin by the method of Real time RT-PCR and Western blotting methodto detect the protein expression quantity of FAK and survivin. With theImmunohistochemistry method to detect positioning expression of FAK inHPASMCs and PI flow cytometry for cell cycle detection. To investigatethe investigating correlations between FAK and survivin, We construct thesiRNAs (small interfering RNA) of FAK and survivin, then transfect themrespectively into the HPASMCs cultured in vitro. Then detect the mRNAexpression level of FAK and survivin respectively by the method of RT-PCR.Results: Comparing with the control group, the mRNA and proteinexpression level of FAK and survivin are significantly up-regulated inother groups, especially in rhRELM-β50ng/ml group. The rhRELM-β hassignificantly increased the proportion of cells at S period and decreasedit at the G0/G1period. The siRNA of FAK does down-regulate the mRNA expression level of survivin in HPASMCs, while the siRNA of suvivin cannotdo the same.Conclusion: The results suggest that the RELM-β has up-regulated theexpression level of FAK and survivin when promoting the proliferation ofHPASMCs. It shows that the FAK is locating on the upstream of survivinin the signal-transmitting pathway when improving the HPASMCsproliferation. Both FAK and survivin are playing a vital role in theabnormal proliferation of HPASMCs induced by the RELM-β. Objective: We are aimed to investigate whether (RELM-β) could lead toabnormal proliferation of human pulmonary arterial smooth muscle cells(HPASMCs) through upregulating FAK mediated by activated FAK-survivinsignal pathway.Materials and methods: Take the normal tissues around the lung cancerand culture them in vitro to5-6generations,then cell was randomlydivided into2groups: C group (control group), rhRELM-β50ng/ml group.Cells was collected at1、2、4、8h,Western blotting were used to detectprotein variations of FAK,surviving,ROCK in HPASMCs. And flow cytometrywas analyzed to cell apoptosis rate.At the sametime, A smallinterferingRNA (siRNA) to knock down the expression of FAK to inhibit theactivation of FAK in primary HPASMCs which were challenged with rhRELM-β50ng/ml was constructed.5-bromo-2-deoxyuridine(BrdU) was used toassess cell proliferation by incorporation. Western blotting andimmunofluorescence technique were used to detect expression and locationof FAK、pFAK、survivin.Results: The research suggest that Expression of FAK andp-FAK,survivin,ROCK were upregulated by rhRELM-β50ng/ml at differenttime, accompanying with proliferation of HPASMCs. FAKsiRNA not onlyprevented survivin expression significantly but also suppressedproliferation of HPASMCs exposed to RELM-β. In addition,, Theproliferation of HPASMCs was partly inhibited by FAKsiRNAConclusion:Activated FAK played a vital role in abnormal proliferation of HPASMCs induced by RELM-β, which might bepartly as a result ofupregulating expression of survivn、ROCK.