Bone Marrow Mesenchymal Stem Cells Induce Ht - 29 Occurred Between The Pre - Mrna Alternative Splicing Regulation Of Emt

Objectives:1. To study the effects of bone marrow derived mesenchymal stem cells (BMSCs) on the growth and differentiation of the tumors formed by HT-29cells and HCT-8cells.2. To explore the influence of BMSCs microenvironment on the Epithelial to mesenchymal transition of HT-29cells and to analyse the mechanisms of the differertial splicing outcome of FGFR2pre-mRNA.3. To study the regulation of PTB on the biological behaviors of HT-29cell.Methods:1.In vivo, nude mice were divided into5groups—BMSCs group, HT-29group, HT-29+BMSCs group, HCT-8group, and HCT-8+BMSCs group, then cell suspensions were subcutaneously injected into nude mice. Tumors were removed after7weeks.The volumes and weights of the tumors were measured and HE staining was performed. The expression of an epithelial marker—E-CADHERIN was detected using RT-PCR and immunohistochemistry staining.2. HT-29cells were co-cultured with BMSCs, the morphology of HT-29cells was observed, and the expression of EMT related genes and FGFR2splice factors, splicing of FGFR2,CD44and ENAH were detected using RT-PCR.3. Knock down of PTB was performed on HT-29cells, expression of EMT related genes and splicing of FGFR2, CD44and ENAHwere detected.Results:1. In vivo, no tumor was observed in the BMSCs group, and no distinct differences were found in the volume and weight of tumors between the HT-29+BMSCs group and the HT-29group. But the tumors of the HT-29+BMSCs group were poorly differentiated and expressed lower E-CADHERIN compared with that of HT-29group. No marked differences in volume,weight and differentiation level of the tumors was found in the HCT-8group and the HCT-8+BMSCs group. 2. In vitro, after coculturing with BMSCs, morphological changes of HT-29cells were observed and were consistent with the expression of EMT related genes. Switch to the mesenchymal cell specific splicing of FGFR2,CD44and ENAH were detected. Down regulations of TIA1, TIAL1, ESRP1, ESRP2, hnRNPM, hnRNPH, hnRNPF, ASF/SF2, RBM38, PTB and C-MYC were detected, no obvious changes in the expression of F0X2, MRG15and hnRNPA1were seen.3. Knocking down of PTB deprived some of the epithelia propertities of HT-29cells, which was accompanied with splicing switch of FGFR2, CD44and ENAH, meanwhile there is down regulation of epithelial specific gene E-CADHERJN and ESRP1/2, but upregulation of the mesenchymal specific gene VIMENTIN and upregulation of three EMT reguLators SNAIL, SLUG and TWIST were not seen, no differences in the expression of MRG15was detected.Conclusions:1. Triple amount of BMSCs failed to influence the weights and volumes of the tumors formed by HT-29cells and HCT-8cells. But the existence of BMSCs lowered the differentiation level of the HT-29tumors.2. BMSCs microenvironment induced EMT and splicing switch of FGFR2,CD44and ENAH of HT-29cell.3. PTB may be involved in the maintenance of the epithelial properities of HT-29cell through regulating the expression of ESRPland ESRP2.