Alteration Of PTEN Gene Expression Affects The Proliferation Of Airway Smooth Muscle Cells And Signaling Pathway

Background:Bronchial asthma is a chronic airway inflammatory disease which involved with various cells, inflammatory mediators, cytokines and the complex immune mechanisms. The main performances are airway eosinophilia, mucus hypersecretion, and hyperresponsiveness to inhaled allergens and to non-specific 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. Now generally agreed that the main causes are the continuous airway injury and structural changes, that is, airway remodeling. The histologic changes consist of airway wall thickening, epithelial injury and hyperplasia, subepithelial fibrosis, goblet cell differentiation and mucus metaplasia, myofibroblastic proliferation and muscle cell hyperplasia and hypertrophy, vascular anomalies, matrix protein deposition, gland hyperplasia and hypertrophy. Now widely recognized that airway smooth muscle cell proliferation is closely related to airway hyperresponsiveness and drug effects in addition to contributes to airway remodeling. Unfortunately, asthma diagnosis and treatment program now in domestic and foreign are emphasizing the control of airway inflammation, but not put enough emphasis on airway remodeling, and the current treatment is not effective to prevent or even reverse the occurrence and development of airway remodeling. In recent years, airway smooth muscle cell has become a new hot spot in asthma research and, in increasing researchers'opinion, should be turned into the main target of asthma research and treatment.PTEN gene (phosphase and tensin homology deleted on chromosome ten), also known as MMAC1 gene or TEP-1 gene and located at chromosome 10q 23.3, is so far the first dual-specificity phosphates activity of tumor suppressor gene, playing an important role in regulating in cell growth, apoptosis, migration, signal transduction and so on. Researches have found that a variety of tumors in humans, such as prostate cancer, glioma, breast cancer, endometrial cancer and ovarian cancer, all have changes in PTEN gene. 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, renal fibrosis and ischemic stroke etc. PTEN gene encodes a protein consisting of 403 amino acids, and plays an important role in maintaining the stability of normal cells. PTEN protein can code dual substrate specificity phosphatase and so can block phosphatidylinosito1-3-kinase signaling pathway (PI3K/PKB/AKT) and mitogen-activated protein kinase (MAPK) signaling pathway (Ras-Raf-MEK1/2-ERK1/2) which are also the main signal pathways mediating ASMC proliferation and migration. Therefore, it is worth studying whether PTEN can regulate airway smooth muscle cell hyperplasia, hypertrophy, and promote apoptosis via inhibiting PI3K/PKB/AKT and MAPK signal pathway. We inferred that the over-expression of PTEN gene may have a inhibitory effect on ASMC; In contrast silencing the PTEN gene expression have a reverse impact. Objectives:ASMCs were respectively transfected with adenovirus vector which carry the wild-type PTEN cDNA and adenovirus-mediated shRNA interference vector of human PTEN cDNA, to investgate the effect of alteration of PTEN gene expression on ASMC proliferation and signaling pathway, and Ad-GFP transfection and a blank as controls.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 and a relative literature reference, the fourth to eighth generations of which were used for experiments. Under the inverted microscope to observe cell growth patterns and anti-smooth muscleα-SM actin monoclonal antibody immunocytochemistry dye is used for the identification of airway smooth muscle cells.2. Experiment grouping:cells were divided into the following four groups:①interference group (Ad-GFP-shRNA-PTEN):ASMCs were transfected with adenovirus-mediated shRNA interference vector of human PTEN gene in vitro at high multiplicity of infection (MOI of 100 PFU per cell);②negative control group (Ad-GFP):the 100 multiplicity of infection 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;④over-expression group (Ad-GFP-PTEN):ASMCs were transfected with adenovirus vector of human PTEN gene in vitro at high multiplicity of infection (MOI of 100 PFU per cell). 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. Four hours after incubation the complete medium was supplied and one day later the medium was replaced. Transfection results were observed under inverted fluorescence microscope.4. MTS/PMS colorimetric assay was used to detect the proliferation of ASMC after PTEN gene expression have changed.5. Cell cycle progress of PI-staining cells was detected by flow cytometry after PTEN gene expression have changed.6. The expression levels of protein in each group including PTEN, p-AKTser473, p-ERK1/2, Total-Akt, Total-ERK1/2 together with the endogenous reference GAPDH was detected with Western blot method.7. 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 SPSS13.0 software.Results:1. Under 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 for a-SM actin. Under 400-fold microscope a-SM actin distributed uniformly in the cytoplasm, and showed brown parallel filamentous structure. Passed to the 4th generation, the ASMCs purity is about 95%.2.48 hours after transfection, ASMCs which were successfully transfected with adenovirus vector displayed fluorescent green light under inverted fluorescence microscope.3. Detection of PTEN protein expression with Western blot method:after transfected with Ad-GFP-shRNA-PTEN, PTEN protein expression of the interference group decreased, and after transfected with Ad-GFP-PTEN, PTEN protein expression of over-expression group increased; while negative control group and blank control group had no significant changed in PTEN protein expression.4. Detection of AKT protein expression with Western blot method:compared to the two control group, p-AKTser473 protein expression markedly increased in the interference group, whereas significantly reduced in over-expression group. While four groups had no significant changed in total-Akt protein expression.5. Detection of ERK1/2 protein expression with Western blot method:compared to the two control group, p-ERK1/2 protein expression markedly decreased in the interference group, whereas showed no significant difference in over-expression group. While four groups had no significant changed in total-ERK protein expression.6. MTS/PMS colorimetric assay results revealed that, compared to the two control group, The cells of the interference group grew more quickly (P<0.01), whereas that of over-expression group grew more slowly. While there was no significant difference between negative control group and blank control group (P= 1.000).7. Detect of the cell cycle progress of single-PI staining ASMCs via flow cytometry.48 hours after transfection, the majority of cells in the interference group escape G1 phase and leap into the S phase significantly; The G0-G1 phase cells of over-expression group was significantly increased, while S and G2/M was the opposite, that is, the cell cycle was arrested at G0/G1 phase. The interference group cells mainly concentrate in S phase, but over-expression cells mainly in G0/G1 phase. Compared with the two control groups, the differences were statistically significant (P<0.05). Conclusions:1. Same as in the other cells, alteration of PTEN gene expression can regulate the proliferation of ASMCs in vitro. Over-expression of PTEN gene can inhibit the proliferation of ASMC and the progress of the cell cycle, namely, proportion of cells in G0-G1 phase increased, while S phase and G2/M phase cells decreased; On the contrary, silencing the expression of PTEN can promote ASMC proliferation and cell cycle progress, namely, proportion of S phase and G2/M phase cells increased and that of the G0-G1 phase reduced.2. With the intervention of PTEN gene, the PI3K-Akt and Ras/Raf/MEK/ERK pathway contact each other, that is, the so-called Cross-talk, as follows:silencing the expression of PTEN gene induces phosphorylation and activation of Akt, which could inhibit the ERK1/2's phosphorylation and activation. It also showed that PI3K-Akt signaling pathway is the predominant adjusting means in the process of PTEN gene regulation in airway smooth muscle cell proliferation.