The Effect Of Tumor Suppressor PTEN Overexpression On The Apoptosis And Cell Cycle Of Human Airway Smooth Muscle Cells

Background:Bronchial asthma is chronic allergic airway inflammatory disease, eosinophils, mast cells, T lymphocytes and other inflammatory cells, airway epithelial cells and cellular components are involved in this disease and has a complex immune mechanism. The main performance of asthma are the infiltration of eosinophils in the airway, increased mucus secretion and airway responsiveness because of inhaling external allergens and nonspecific stimuli. The airflow obstruction in asthma can be reversible, partly irreversible or even irreversible completely, leading to the complex clinical manifestations and to the more difficulty of treatment, and moreover which will influence disease prognosis. Currently treatment of anti-inflammatory can not cure asthma, and also can not eliminate airway hyperresponsiveness completely. There is a significant "miss-link" between airway inflammation and airway hyperresponsiveness. As a key characteristics of asthma-airway remodeling, it linked airway inflammation and airway hyperresponsiveness together. Airway remodeling refers to structural changes include loss of epithelial integrity thickening of basement membrane, subepithelial fibrosis, goblet cell and submucosal gland enlargement, increased smooth muscle mass, decreased cartilage integrity, and increased airway vascularity. As one of the major structural components of airway remodeling, the status of airway smooth muscle cells(ASMC) get more and more attention. ASMCs are not only the damaged target cells during airway inflammation, but also the effector cells actively participate in the pathological process of asthma (airway inflammation, airway remodeling and airway hyperresponsiveness), and they are associated with the aggravating illness. The excessive proliferation and reduced apoptosis of ASMC play an important role in asthma. Unfortunately, the diagnosis and treatment of asthma now in domestic and foreign are emphasizing the control of airway inflammation, but not put enough attention on airway remodeling, and the current treatment is not effective to prevent or even reverse the occurrence and development of airway remodeling. Because of this, in recent years, ASMC has become a new focus of asthma, more and more scholars suggest that we should target ASMC in the research and treatment of bronchial asthma. Therefore, it is important to further reveal relevant mechanism of ASMC in asthma, which can open new ideas in asthma treatment.PTEN gene(phosphase and tensin homology deleted on chromosome ten), also known as MMAC1 gene or TEP-1 gene, was located at chromosome 10q 23.3, and has a full-length of 200 Kb. It was discovered by three research groups in 1997. The PTEN gene was the first dual-specificity phosphates activity of tumor suppressor gene, playing an important role in regulating embryonic development, cell cycle, cell growth, apoptosis, migration, cytoskeleton and so on.The previous research shows that the expression changes of PTEN gene were found during human multiple tumors, such as prostate cancer, breast cancer, gliomas, endometrium and ovarian cancer. With the development of research, people also found the PTEN gene play an important role in non-neoplastic disease, such as myocardial hypertrophy, hypertension atherosclerosis, bronchus asthma, renal fibrosis and ischemic stroke. PTEN gene encodes a protein consisting of 403 amino acids, and plays an important role in maintaining the stability of normal cells. It belongs to the members of protein tyrosine phosphatase family, has a dual specific protein phosphatase activity, which can adjust signal transduction mediated by tyrosine phosphatase and serine/threonine phosphatase. PTEN protein may regulate complex signal network system directly or indirectly through its lipid phosphatase/protein phosphatase activities. PTEN protein can regulate phosphatidyl inositol-3-kinase signaling pathways (PKB/Akt signal PI3K/cascade), mitogen activated protein kinase (MAPK) signaling pathways (Ras-Raf-MEK1/2-ERK1/2 signal cascade), and can up-regulate the expression of CDKI such as p21 and p27, down-regulate the expression of CyclinD1, while those signal molecule influence the cell growth and survival. Since previous study shows that PTEN can influence the growth and survival of tumor cells, and vascular smooth muscle cells and fibroblast through adjusting the PI3K/Akt, MAPK/ERK and cyclin, so we conclud that PTEN has the similar effect to ASMCs. Therefore, further study of PTEN on PI3K/Akt, MAPK, the CDKI and the cyclin will clarify the regulation mechanism of PTEN on ASMC growth and survival, and we hope for providing theoretical and experimental basis about in PTEN in asthma airway remodeling.Objectives:ASMCs were transfected with adenovirus vector which carry the wild-type PTEN cDNA, to investgate the effect of over-expression of PTEN gene on ASMC apoptosis and cell cycle pathway, using Ad-GFP transfection and a blank as control groups.Methods:1. Human airway smooth muscle cells (ASMC) isolation, culture and identification:Samples of normal lung small bronchi were obtained from resected lobes of patients, and then airway smooth muscle cells were cultured in vitro with mature methods in our laboratory group and the related literature reference, the third to eighth generations of which were used for experiments. We observed cell growth patterns under the inverted microscope, and anti-smooth muscleα-SM actin monoclonal antibody immunocytochemistry dye were used for the identification of airway smooth muscle cells.2. groups of Experiment:cells were divided into the following three groups:①over-expression group (Ad-PTEN):ASMCs were transfected with adenovirus vector carring human PTEN gene in vitro at the multiplicity of infection 100 (MOI of 100 PFU per cell);②ngative control group (Ad-GFP):the multiplicity of infection 100 of adenovirus-mediated green fluorescent protein(GFP) were transfected into human ASMCs in vitro;③blank control group (DMEM):complete medium DMEM were added to culture ASMCs in vitro.3. Adenovirus transfected ASMC:ASMCs in logarithmic growth phase were selected to add with adenovirus serum-free DMEM medium diluents and then cells were placed at the 37℃, saturated humidity,5% CO2 incubator.6-8 hours after incubation the complete medium was supplied. After 24h incubation, changed the new complete medium. Transfection results/rat were observed under inverted fluorescence microscope and flow cytometry.4. CCK-8 assay was used to detect the cytotoxicity of adenovirus transfection and the proliferation of ASMC after PTEN gene over-expression.5. Observed the cell apoptosis using Hoechst-33342 staining assay.6. Cell apoptosis was detected by FCM after PTEN gene overexpression through PE-7AAD double-staining.7. Cell cycle progress was detected by FCM after PTEN gene over-expression using PI-staining.8. The expression levels of protein including PTEN, p-Akt, p-ERK1/2, Total-Akt, Total-ERK1/2, cleaved-Caspases-3, Caspases-9,p21, CyclinD1andβ-actin in three groups were detected with Western blots.9. Data are expressed as mean±SD. Two groups of comparisons was were performed using two-sample t test. Multiple comparisons were performed using one-way ANOVA (LSD/Bonferroni). Statistical significance was set at P<0.05. All statistical analysis was operated with SPSS13.0 software.Results:1. Under the inverted phase contrast microscope at 100-fold, the ASMCs show a fusiform or polygon before confluence; after confluence some regional of cells possessed fascicular form and typical "peak-and-valley" appearance. And the cultured cells were positive immunostaining forα-SM actin. Under 400-fold microscopeα-SM actin distributed uniformly in the cytoplasm, and showed brown parallel filamentous structure. Passed to the 3-4 generation, the ASMCs purity is about 95%.2. After 24 hours transfection, ASMCs were successfully transfected with adenovirus vector and displayed fluorescent green light under the inverted fluorescence microscope. Afer 48 hours transfection, ASMCs displayed fluorescent green light at low magnification under the inverted fluorescence microscope. Afer 48 hours transfection with MOI=100, the efficiency was (95.19±0.39)% and (96.20±0.33)% using the inverted fluorescence microscope and the FCM test.3. CCK-8 assay results revealed that the OD values among Ad-PTEN,Ad-GFP, mock groups were statistically significant difference(F=480.63,106.644,89.592, P =0.000), the OD values of Ad-PTEN were lower than those of Ad-GFP and mock groups(P<0.05), this indicated that overexpression of PTEN can inhibit the growth of ASMCs. While there was no significant difference between Ad-GFP group and mock group, this demonstrate that there was no influence on cell growth and no cell toxicity after adenovirus transfection(t=-0.327,1.149,-1.732, P=0.748,0.270, 0.105).4. Cell apoptosis detected by Hoechst-33342 staining assay shows that after 1,2,3.5,7 days transfection, the apoptosis rate among Ad-PTEN, Ad-GFP and mock groups was not statistically difference((F=1.650,0.412,0.085, P=0.245,0.674, 0.919). This indicated PTEN overexpression can not induce ASMCs apoptosis.5. Cell apoptosis detected by FCM through PE-7AAD double-staining shows that after 2,3.5,7 days transfection, the apoptosis rate among Ad-PTEN, Ad-GFP and mock groups was not statistically difference(F=1.988,2.569,0.127,0.064, P=0.191, 0.131,0.882,0.939). This also indicated PTEN overexpression can not induce ASMCs apoptosis.6. Detection of the expression of PTEN protein with Western blots shows that after transfected with Ad-PTEN, the expression of PTEN of Ad-PTEN group increased; while Ad-GFP and mock group had no significant changed.7. Detection the expression of Akt protein with Western blots shows that compared to the Ad-GFP and mock group, the expression of p-Akt markedly decreased in the Ad-PTEN group, While there was no significant changed about the expression of total-Akt during the three groups.8. Detection the expression of ERK protein with Western blots shows that the expression of p-ERK and total-ERK during the three groups was no significant difference.9. Detection the expression of cleaved-Caspases-3 protein with Western blots shows that the expression of cleaved-Caspases-3 can not be detected among the three groups.10. Detect of the cell cycle progress using PI staining ASMCs via flow cytometry shows that after 48 hours transfection, The G0-G1 phase cells of Ad-PTEN group was significantly increased, while S and G2/M was the opposite, that is, the cell cycle was arrested at G0/G1 phase.There were statistically significant difference among Ad-PTEN,Ad-GFP, mock groups (F=8.333, P=0.005). Cell at G0/G1 phase of Ad-PTEN group were lower than those of Ad-GFP and mock groups(P<0.05).11. Detection the expression of p21 protein with Western blots shows that compared to the Ad-GFP and mock group, the expression of p21 markedly increased in the Ad-PTEN group, while Ad-GFP and mock group had no significant changed.12. Detection the expression of CyclinD1 protein with Western blots shows that compared to the Ad-GFP and mock group, the expression of CyclinD1 markedly decreased in the Ad-PTEN group, while Ad-GFP and mock group had no significant changed.Conclusions:1. Ad-GFP can infect HASMCs efficiently, the infection efficiency was about 95% at 100 MOI, and it is a safe gene vector with no cell cytotoxicity in vitro.2. Overexpression of PTEN can not induce ASMCs apoptosis after tested by Hoechst-33342 staining assay, FCM and western blots. The result was not similar with other conclusion in which PTEN can induce some kind of cell apoptosis.3. Over-expression of PTEN gene can inhibit the proliferation and the cell cycle of ASMC, namely, proportion of cells in G0-G1 phase increased, while S phase and G2/M phase cells decreased. This is in conformity with the previous study.4. Over-expression of PTEN gene can inhibit cell proliferation through down-regulating the level of p-Akt,but has no influence on the level of p-ERK1/2,total-Akt,total-ERK1/2.5. Over-expression of PTEN gene can regulate the cell cycle progress through down-regulating the level of CyclinD1 and up-regulating the level of p21.