Effects Of α5-nAChR/HIF-1α/VEGF Axis On Proliferation And Apoptosis Of Human Lung Cancer Cells

ObjectiveLung cancer is a common form of lung malignant tumor, the mortality rate is the highest among the cancer deaths.The year of2008, Genome-wide association studies (GWAS) showed that,the mutation of nicotinic acetylcholine repeptor genomic cluster CHRNA5/A3/B4locates in chromosomal region15q which has closed relation with smoking-related lung cancer.The CHRNA5gene encoding a5-nAChR is particularly relevant with lung cancer.nAChRs are five polymers composed of alpha and beta subunits homology or heteromeric, nAChR are universally expressed in central and peripheral nervous system.Currently, more function research of nAChRs are in the normal tissues than in the tumor.If we can clear the function of nAChRs subnits in the lung cancer cells, the therapy on the basis of the nAChRs may provide new research ideas and therapeutic targets for the treatment of lung cancer.To explore the influence of small interfering RNA (siRNA) down-regulating a5-nAChR expression on HIF-1αand VEGF expression of human lung cancer in vitro, and further discussion the influence of α5-nAChR/HIF-1α/VEGF axis on cell proliferation and apoptosis in the lung cancer.Methods1. α5-nAChR、 HIF-1α and VEGF mRNA expressions were detected by Real Tmie-PCR,protien expression were detected by western blot and the protien expression of VEGF were detected by ELISA, after various concentration nicotine treatment on A549cells.2. siRNA fragment of CHRNA5encoding a5-nAChR was constructed and transfected into human lung cancer A549cell line with lipofectamine2000. The inhibition effect was detected by Real Tmie-PCR and Western blot. After CHRNA5-siRNA transfection, the expression of HIF-1α and α5-nAChR were detected by Real Tmie-PCR and Western blot respectively and the expression of VEGF was detected by ELISA.3. Nicotine-induced A549cell proliferation was detected by MTT assay.4. The influence on apoptosis was detected by Western blot.Results1. Real Time-PCR showed that expressions of α5-nAChR and VEGF were significantly increased at lμmol/L nicotine treatment compared with other concentration groups(P<0.05),but the expression of HIF-1αhad not change obviously (P>0.05)2. Western blot showed that expression ofa5-nAChR and HIF-1αwere significantly increased at1μmol/L nicotine treatment compared with other concentration groups,but the expression of a5-nAChR and HIF-1α were decreased at5μmol/L.protein expression of a5-nAChR and HIF-lashowed concentration-dependence of nicotine (P<0.05)3. ELISA showed that expression of VEGF was significantly increased at1μmol/L nicotine treatment compared with other concentration groups.but decreased at5μmol/L,protein of VEGF showed concentration-dependence of nicotine (P<0.05)4. Real Time-PCR showed that siRNA group expression of a5-nAChR、 HIF-1αand VEGF were decreased compared with the0u mol/L Nicotine group and si-NC group (P<0.05).the (1μ mol/L Nicotine+siRNA) group expression of a5-nAChR、HIF-laand VEGF were decreased compared with the1μmol/L Nicotine group and1μ mol/LNicotine+si-NC group (P<0.05), after CHRNA5-siRNA transfection.5. Western Blot showed that siRNA group expression of a5-nAChR and HIF-1α were decreased compared with the0μ mol/L Nicotine group and si-NC group (P <0.05). the (1μ mol/L Nicotine+siRNA) group expression of a5-nAChR and HIF-1αwere decreased compared with the1μ mol/LNicotine group and1μ mol/LNicotine+si-NC group (P<0.05), after CHRNA5-siRNA transfection. 6. ELISA showed that, siRNA group expression of VEGF was decreased compared with the0μ mol/L Nicotine group and si-NC group (P<0.05).the (1μ mol/L Nicotine+siRNA) group expression of VEGF was decreased compared with the1u mol/L Nicotine group and1μ mol/LNicotine+si-NC group (P<0.05), after CHRNA5-siRNA transfection.7. MTT assay showed that,after CHRNA5-siRNA transfection, nicotine-induced A549cell proliferation was significantly inhibited (P<0.05).8. Western blot showed that siRNA group the expression of bcl-2and survivin were significantly decreased compared with the0μ mol/LNicotine group and si-NC group (P<0.05). the (1μ mol/L Nicotine+siRNA) group expression of bcl-2and survivin compared with the1μ mol/L Nicotine group and1μ mol/LNicotine+si-NC group (P<0.05), after CHRNA5-siRNA transfection.Conclusion1. Nicotine up-regulating the expression of a5-nAChR、 HIF-1α and VEGF, which presented a dose-dependent manner.2. After CHRNA5-siRNA transfection, nicotine-induced A549cell proliferation was significantly inhibited,and the expression of HIF-laand VEGF mRNA and protein were significantly decreased,,showed that a5-nAchR played an important role in nicotine-induced A549cell proliferation via HIF-la/VEGF axis.3. After CHRNA5-siRNA transfection, the expression of HIF-1α VEGF、 bcl-2and survivin protein were significantly decreased, showed thata5-nAchR can regulate the infuluence of HIF-1α/VEGF on cell apoptosis, which suggested that a5-nAChR/HIF-1α/VEGF axis may be one of the important molecular mechanmism of smoking related lung cancer.