| BackgroundsPeriodontal ligament (PDL) is a self-renewal system and contains a group of mesenchymal stem cells (MSCs) which can present different phenotypes. These cells were then defined as Periodontal ligament stem cells (PDLSCs). Since isolated from PDL, PDLSCs were given great attention. Recent studies have confirmed that PDLSCs play a key role in the periodontal homeostasis and defect repair. It not only has the two characteristics of self-renewal and multipotent differentiation potential, but it also owns the unique characteristic of formation of cementum-like and PDL-like structures different from other MSCs. Therefore, PDLSCs were considered to be the most promising seed cells in periodontal engineering. However, the developments of PDLSCs were largely restrained by limited resources, easy aging, the age and disease status of donors, and so on.There is a large, integrated and coordinated network composed of kinds of signaling pathways that accounts for cell fates and behaviors. At appropriate time or space, these signaling systems results in markedly different cell fates:Senescence, Apoptosis, Differentiation or Proliferation. The Hippo pathway was a newly discovered protein kinase cascade pathway associated with organ size control or tumor formation. Yes-associated protein (YAP), a main effector of this pathway, was often deactivated by upstream molecules. YAP was often detected overexpression in tumor, but has been also observed in the stem or progenitor cells of multiple tissues. It plays an important part in stem/progenitor cell maintenance and differentiation. There were evidences confirm that the Hippo pathway was involved in tooth development of mouse and altering the expression of YAP of dental epithelial cells can lead to abnormal morphogenesis and structure. Nevertheless, there are no relevant reports on the role of YAP in control of PDLSCs so far.Besides, there are many signaling pathways regulating cell fates or behaviors, such as well-known the TGF-? pathway or the Wnt pathway. A number of examples demonstrate crosstalk between Hippo and other pathways, with implications for stem cell biology. Nowadays the mechanism of proliferation and differentiation of PDLSCs is not very clear and it's essential to study the role of YAP in the maintenance and self-renewal of PDLSCs.ObjectiveIn this study, we generated YAP knockdown Human-periodontal ligament stem cells (h-PDLSCs) by means of RNAi to identify the effects of YAP on the biological behavior of h-PDLSCs, in the terms of proliferation, apoptosis, cell cycle and osteogenic differentiation.Materials and Methods1.The h-PDLSCs were isolated by method of enzymatic digestion of tissue, and then purified by method of limited dilution. The cell surface markers were identified by flow cytometry (FC).2.Non-specific and YAP-targeted siRNAs were synthesized. There were there sequences of the later. They were respectively transfected into h-PDLSCs by LipofectamineTM 2000. These four groups of cells were set as Control Negative (NC) group, siRNA-1/2/3groups. In addition, another group of cells with only LipofectamineTM 2000 intervention was set as the blank group. The expression of YAP were identified by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot.3.On the 1st,2nd,3rd,4th and 5th day after transfection, proliferation activity of h-PDLSCs was detected by CCK-8.72 hours after transfection, Changes in cell cycle were detected by Cell Cycle Analysis Kit through flow cytometry. Besides, the downstream target gene of YAP, CTGF (Connective tissue growth factor),associated with cell growth, were also detected by Western blot.4.72 hours after transfection, the apoptosis rate of h-PDLSCs were detected by Cell Apoptosis Analysis Kit through (FC).5.Osteogenic-Related Genes (RUNX2, OCN, ALP, Collagen I) of transfected h-PDLSCs were also detected by RT-PCR.6.Results were analyzed by SPSS 19.0, and P<0.05 was considered statistically significant.ResultsFlow cytometry results proved that primary derived cells were h-PDLSCs, as expressing markers of MSCs such as CD73 and CD44 (98%?99.4%), and lacking of hematopoietic cell markers such as CD45, HLA-DR (0.28±0.012%). Expression of YAP mRNA and protein of siRNA-1 group and siRNA-2 group were both significantly down-regulated after 48h for transfection (P<0.01). No obvious difference was found in the expression of YAP protein between 48 and 72 h. Proliferation activity of siRNA-1 group and siRNA-2 group detected by CCK-8 was inhibited. FC results also showed that apoptosis rate of siRNA-1 group and siRNA-2 group was increased and their cell cycle was changed as the proportion of G0/G1 and S phases elevated (P<0.01) and G2 phase decreased (P<0.05). Besides, the expression of CTGF were also down-regulated. Interestingly, expression of osteogenic-related genes (Collagen ?, ALP, OCN) mRNA, especially for OCN, were elevated and no significant changes were found in term of RUNX2.ConclusionKnocking down YAP gene of h-PDLSCs by siRNA could inhibit proliferation activity, induce apoptosis, and inhibit the cell cycle. Thus, YAP could regulate the proliferation and apoptosis of h-PDLSCs. Changes in proliferation may not only be associated with cell cycle or apoptosis, but also CTGF. What's more, some early osteogenic-related genes (Collagen ?, ALP, OCN) were elevated which may indicate that YAP was associated with osteogenic differentiation. Our study suggests that YAP might play a key role in regulating biological behavior of h-PDLSCs. However, further research is needed. |
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