The Expression Change Of MET Associated Genes When BMSCs Are Chemically Induced To Neuron-like Cells Differentiation

BackgroundIn1987, Friedenstein and others discovered bone marrow mesenchymal stem cells (BMSCs). Subsequent studies found that such mesenchymal stem cells are widely present in umbilical cord blood, bone marrow, and even the peripheral circulating blood., According to the embryonic tissue source, mesenchymal cells originate from the mesoderm in the embryo development phase of gestrulation. Since these stem cells remain relatively unlimited proliferative capacity in vivo, they are also easy to be cultured and purified in vitro. Moreover, BMSCs can be induced to undergo the directed differentiation under the stimulation of cytokines or chemical reagents, forming a variety of mesenchymal cell types, or even trans-differentiating into neuron-like cells. So BMSCs are recognized aswell-used stem cell source for both basic research and clinical treatment.Mesenchymal-epithelial transition (MET) and the reverse process of epithelial-mesenchymal transition (EMT) refer to the process that epidermal-derived cells transite to obtain the features of mesodermal-derived cell types under some physiological and pathological conditions, vice versa.. At present, MET and EMT are roughly grouped into three categories:(1) used in embryonic development gastrulation.(2) happening in wound healing and fibrosis ((3) for cancer pathogenesis.Not only changes in cell morphology and many other features, but cell proteins levels for membrane protein and transcription factors are also changed. Protein molecule changes include:membrane protein (E-cadherin, N-cadherin), cytoplasmic protein (Vimentin), growth factors (TGF-P, EGF), and transcription factors (Snail, Slug, ZEB1and ZEB2), etc..Therecent reports indicate that Wnt/p-catenin signaling pathway i involved in the regulation of physiological and pathological processes of EMT/MET. For instance, study in the development in the renal tubules showed that knocking out Wnt4a gene could effectively block the formation of mesenchymal stem cell migration and tissue formation of renal tubular epithelial cells. Another study using gene knock-down method to interference Wnt/β-catenin pathway proved to effectively reverse Wnt-mediated cancer pathogenesi.BMSCs with mesenchymal characteristics when induced by the chemical inducer, differentiate into neuron-like cells which are of the characteristics of epithelial cells., It is not yet documented whether this MET process is similar to the other types known and well-accepted EMT/MET changes in gene expression, cell proliferation and molecular mechanisms.Our laboratory had successfully induced by using chemical inducers differentiation of rat BMSCs into neuron-like cells. We also proved that differentiated cells expressed neural stem cell-specific marker proteins NSE, nestin, etc., and took on the electrophysiological characteristics of neurons. To illustrate the changes of MET-related molecules in the process from BMSCs to neuron-like cells under chemical inducer,, it will help further exploring the molecular mechanism of MET/EMT, providing support for the study of MET/EMT-related disease pathology (cancer pathogenesis, etc.). Meanwhile, based on its convenience of in vitro culture, BMSCs could be used as MET/EMT model for cell research in vitro. ObjectiveIn this study, rat BMSCs (P3-P5) induced into neuron-like cells by chemical reagents DMSO/BHA and detected before and after induction by RT-PCR to check cell surface proteins (E-cadherin, N-cadherin, etc.) expression variation and MET related molecules Slug, Snail, ZEB1, ZEB2, vimintin, Twist, etc. gene expression changes. Meanwhile, the use of flow cytometry detect the cell cycle, to compare cell proliferation characteristics before and after induction. To explore whether Wnt/β-catenin signals are involved in the regulation of MET, we will check pathway related genes(C-myc, Axin2, β-catenin) expression changes. Method1. SD rat femur bone marrow extraction. Ages4-6weeks, weight between100-120g, male SD rats were sacrificed by neck dislocation, sterilized in75%alcohol, under sterile conditions femur peeled, cut on both sides, with DMEM/F12medium, respectively, flushing the marrow cavity from the ends of the incision, to collect all the cells in the culture dish, and after centrifugation, washed with PBS, inoculated into sterile flasks (PO), the at37℃,5%CO2and saturated humidity culture.2. BMSC in pure culture. First, in cell culture after PO24h, replaced with fresh medium and discard non-adherent cells. When PO cultured cells were grown to cover the bottom of the bottle about60%-70%, according to the ratio of1:2for cell passaging, the P3-generation cellular (cell morphology showed a high degree of uniformity and high proliferative properties)can be used to the next experiment.3. BMSCs induced by chemically induced cells into neuron-like cells. Choose4th generation (P3-P5) of BMSCs cells induced group inducible medium (10%FBS in DMEM-F12medium containing bFGF10ng/ml,), in the control group (non-induced group) use complete medium, for24hours. After pre-induction group induced culture was discarded, use DMEM-F12medium with2%DMSO and200μmol/L BHA for chemical induction. Replace the control group the same amount of complete medium. Continue to observe changes in cell morphology. Stop induce about4-6hours by the visual cell differentiation.4. MET related molecules expression detection. Total RNA of experimental and control groups was extracted by using RNA extraction kits. Measure the concentration by Nanodrop accurately, and the same amount of RNA was reverse transcribed into cDNA. Using relative quantitative PCR to detect MET related molecules (E-cadherin, N-cadherin, changes Slug, Snail, ZEB1, ZEB2, virnintin, Twist) and other protein expression levels in BMSCs and neural-like cells before and after induction, and the calculation according to the experimental group and the control group get-σTT compared to the relative ratio of gene expression level. GAPDH as reference gene, ROX reference for the fluorescence of sample.5. MET pathway may regulate Wnt/β-catenin pathway target genes should be detected. Using the same method as above to obtain RNA and reverse as cDNA, relative quantitative PCR assay in the experimental group and the control group C-myc, Axin2, β-catenin expression variation in BMSCs and neural-like cells before and after induction.-σTT calculate the experimental group compared with the control group to obtain the relative ratio of gene expression levels GAPDH as reference gene, ROX reference for the fluorescence of sample.6. BMSCs and neuron-like cell proliferation characteristic detected by flow cytometry. Take the experimental group and a control group of cells, fixed by ethanol and using PI staining. Detected cell cycle distribution by using flow cytometry.7. Statistical analysis:The results of this experiment were analyzed using statistical software SPSS17.0process. Measurement data for comparison with the same time point between the experimental group and control group two-sample t test, count data were examined using statistical data as mean±standard deviation (±s) said. In order to test the level of a=0.05, P<0.05was considered statistically significant.Results1. To obtain bone marrow cells in vitro after purification (> P3), BMSCs were high purity of cells of uniform morphology, most flat spindle cells. And maintaining proliferative properties, dense cells together side by side showing spiral arrangement.2. DMSO/BHA induced BMSCs cells form in the4-6hours after the presentation part of the cytoplasm to the nucleus shrink centralized direction, rounded cell bodies narrow, elongated protrusion formed into a mesh contact with the surrounding cells, which appear typical of neurons like cells.3. Relative quantitative PCR shows MET related molecules expression changes. Chemically induced BMSCs to neuron-like cells,"M (interstitial)" feature molecule (Slug, Snail, ZEB1, ZEB2, Vimintin, Twist, etc.) was significantly reduced.4. Relative quantitative PCR shows MET related signal Wnt/β-catenin pathway target genes changes. Chemically induced BMSCs to neuron-like cells, the target gene (C-myc, β-catenin) was significantly reduced.5. Flow cytometry showed, G2/S phase was significantly decreased in chemically induced cells.Conclusion:DMSO/BHA inducing BMSCs to differentiate into neuron-like cells outside the commonwealth, promoting changes of the MET related genes,"M (interstitial)" gene expression was significantly reduced. Wnt/β-catenin pathway activity reduced in the MET, indicate that the regulation may be involved in the process.