The Effect Of Tumor Suppressor PTEN Overexpression On The Proliferation Of Human Airway Smooth Muscle Cells Cultured In Vitro

Background:Airway remodeling is one of the basic features of bronchial asthma,which correlates airway inflammation and airway hyperresponsiveness.Airway remodeling in asthma refers to the structural changes occurring in the airway wall,including epithelial cell hyperplasia and metaplasia,subepithelial fibrosis,muscle cell proliferation and angiogenesis,of which airway smooth muscle cells(airway smooth muscle cell,ASMC) is the main structural component.The biological characteristics of asthmatic ASMC changes greatly,including hyperplasia,hypertrophy,and migration,which can not only enhance the contractility of bronchi responsing to stimuli,thus increases airway hyperresponsiveness,but also promote the airway wall thickening,leading to an increase in the basic airway resistance and the occurrence of irreversible airflow obstruction.Unfortunately,no current therapy can prevent or reverse airway remodeling and its impact on lung function.So there is an urgent need for a better understanding of the mechanism of airway remodeling,ASMC proliferation and migration,thus provides theoretical and experimental basis for new drugs which can inhibit proliferation and migration of ASMC,therefore stop or even reverse airway remodeling.Homologous of tensin and auxiliary protein,phophatase and tensin homolog deleted on chromosone 10(PTEN) is a new tumor suppressor gene discovered in recent years,it is also the first tumor suppressor gene holding phosphatase activity found so far,having an important impact on cell growth,proliferation and differentiation,apoptosis,adhesion,migration and blood vessel growth,and many other biological behavior of cells.With study in-depth,researchers also found it also plays an important role in the non-neoplastic diseases,such as cardiac hypertrophy, hypertension,atherosclerosis,bronchial asthma etc..Previous research has confirmed that PTEN can inhibit the proliferation of tumor cells,cardiac and vascular smooth muscle cells through pathways of PI3K,FAK and ERK,which are also the main signal pathways mediating ASMC proliferation and migration.Whether it has a similar impact on the proliferation of ASMC,there are no relevant reports at present.We inferred that the overexpression of PTEN may have a similar inhibitory effect on HASMC.Objectives:Plasmids which carry the full-length wild-type PTEN cDNA was transfected into cultured HASMC,to investgate the effect of over-expression of PTEN on HASMC proliferation and cell cycle progress,HASMC transfected with empty vector and normal HASMC as control.Methods:1.Samples of relatively normal small bronchi were obtained from resected lobes of patients with lung cancer,and then airway smooth muscle cells were cultured in vitro with mature methods in our laboratory.The forth to eighth generations of airway smooth muscle cells were used for experiments.Anti-smooth muscle a actin monoclonal antibody is used for the identification of airway smooth muscle cells.2.Plasmids carrying the full-length of wild-type PTEN cDNA(p-WT-PTEN) and the empty plasmid(P-EGFP) were transformed into Escherichia coli DH5a to amplify, Screen with Ampicillin,select positive clones,culture bacterium,and then extract plasmids with omega endotoxin-free plasmid extraction kit.Double restrictive digestion,and gene sequencing were used to identified the rightness of plasmid. Large amount of plasmid was extracted and reserved under -20℃circumstance.3.Experiment grouping:cells were divided into three groups:p-WT-PTEN transfected group(Group A),p-EGFP transfected group(Group B),and non-transfected group(Group C).Cells were transfected with Lipofectamin 2000 according to its' procedure.Transfection results were observed under inverted fluorescence microscope.4.MTS/PMS method was used to detect the proliferation of HASMCs.5.Cell cycle progress of PI-staining cells was detected by flow cytometry.6.Total RNA of each group was axtracted with Trizol,the expression levels of PTENmRNA was detected by RT-PCR method.7.HASMCs cultured in 6-culture plates were transfected as the above-mentioned method.After 48 hours of transfection,PTEN protein expression was detected with immunohistochemistry SP method,and was observed and photographed under inverted microscope.8.Data are expressed as mean±SEM.Statistical comparisons were performed using one-way ANOVA(LSD).Statistical significance was set at P＜0.05.All statistical analysis was operated with SPSS 11.5 software. Results:1.The ASMCs,with 1-2 nucleus,show a fusiform or polygon before confluence, Under microscope,the cultured cells possessed typical "hill-and-valley" appearance and were positive immunostaining for a--actin.Passed to the 4th generation,the ASMCs purity is about 95%.2.After digested by Xbaâ… and Hindâ…¢,agarose gel electrophoresis of p-WT-PTEN show two bands,the molecular weight of each is 5.4 kb and 1.3 kb respectively,the same size as the pGZdXZ and the inserted PTEN cDNA.Extracted plasmids were delivered to Beijing Nuosai Company for sequencing,and the sequence result was fully consistent with Genebank by BLAST.3.Under inverted fluorescence microscope,ASMCs which were successfully transfected with plasmids display fluorescent green light.4.MTS/PMS were used to detect the proliferation of HASMCs.The absorbance (A490 value) of the p-WT-PTEN transfected cells were lower than those in non-transfected group and the empty vector transfected group(P＜0.05),while there was no significant difference between empty vector transfected group and on-transfected group(P＞0.05).5.Flow cytometry was used to detect the effect of plasmid transfection on the cell cycle progress of HASMCs.The proportion of p-WT-PTEN transfected cells in Go / G₁ phase was significantly greater than that of empty vector transfected and no-transfected group,while S and G₂ / M was the opposite(P＜0.05),there was no significant difference between empty vector transfected group and non-transfected group(P＞0.05).6.RT-PCR was used to amplificate PTEN mRNA of p-WT-PTEN transfected group,empty plasmid transfected group and the control group,its results show that the level of PTEN mRNA expression of p-WT-PTEN transfected group was higher than that of empty plasmid transfected group and the control group.7.48 hours after the transfection of the plasmid,the immunohistochemical results of PTEN protein show that PTEN protein was expressed in the nucleus and cytoplasm, and its expression is more significantly in the nucleus than in the cytoplasm,while empty plasmid transfected Group and the control group had only weak expression of PTEN protein.Conclusions:1.Same as in the other cells,the overexpression of PTEN can inhibit the proliferation of ASMCs in vitro,proportion of cells in G₀/G₁ phase was increase, while S and G₂ / M phase cells was reduced.2.p-WT-PTEN successfully transfected the HASMCs cultured in vitro,and PTEN was overexpressed in gene and protein level.