Studies On MiRNAs Which Regulate PLAP-1Expression In Esteogenic Differentiation Of Periodontal Ligament Cells

Periodontal tissues are highly specific organs supporting the teeth, including the gums, periodontal membrane and the mineralized tissues of cementum and alveolar bone. Due to its constituent diversity and the interaction between soft and hard connective tissue, reconstruction and healing of periodontal tissue is more complex than that of the average soft tissue. Normal periodontal tissue has regeneration capacity. During orthodontic tooth movement, several events happen, like remodeling of periodontal ligament, cementum, new bone formation, especially the phenomenon of osteoblast. They are physiological responses, not pathological damage. Injury inflammation of periodontal disease spread to the periodontal ligament and alveolar bone. This will cause periodontal attachment loss and even lead to tooth loss. Its ability to regenerate is very limited, affecting the function and esthetics. The most effective modern concept of periodontal treatment required to achieve the regeneration of periodontal tissues (periodontal tissue regeneration, PDTR), and new alveolar bone, cementum and collagen fibers. This is an important issue. The regulation of periodontal tissue regeneration cells to develop periodontal tissue is the direction of future research.Periodontal ligament cells (PDLCs) is a mesenchymal cell populations with a heterogeneous composition of the diversity of characteristics, including fibroblasts, osteoblasts, cementum cells and their precursor cells, mesenchymal stem cells. They have ability to differentiate into osteoblasts and cementoblasts. So they have high osteogenic differentiation potential. Interestingly, under normal circumstances, the PDL is tough and elastic connective tissue and maintaining homeostasis of periodontium. Periodontal ligament cells play a vital role in maintaining this balance and stability of the process. Studies have shown that variety of factors of periodontal ligament cells, such as genes, proteins, and cytokines, involved in the regulation of tissue regeneration and homeostasis of periodontium. However, the molecular mechanisms and signaling pathways are not clear in terms of how periodontal ligament cells regulate the regeneration and homeostasis of periodontium.Periodontal ligament associated protein-1(PLAP-1), a specific marker of the periodontal ligament, is an important negative regulator of osteogenic differentiation in maintaining homeostasis of periodontium. It plays an important role in the metabolism of the periodontal ligament, periodontal tissue remodeling, regeneration and maintenance of periodontal tissue.2001, Yamada, S et al. named PLAP-1for the first time and reported its high expression in periodontal tissues, and confirmed that PLAP-1expression levels increased with the mineralization of human periodontal ligament cells. Subsequently, a number of data showed that FGF-2inhibits PLAP-1expression while BMP-2upregulates PLAP-1expression. PLAP-1played a negative role in periodontal ligament cell differentiation and mineralization of the periodontal ligament. Periodontal ligament cells in mice as a model, overexpression of PLAP-1inhibited the alkaline phosphatase activity and the formation ability of ore nodules by regulating the activity of BMP-2. PLAP-1hinders the binding of BMP-2and its receptor BMPR-IB, thereby inhibiting the BMP-2signaling pathway and mineralization differentiation.MicroRNAs (miRNAs) provide an additional level of gene regulation beyond that of transcription factors. miRNAs are a class of small noncoding RNA molecules that regulate gene expression post-transcriptionally in eukaryotic cells. miRNAs are-22nucleotide single-stranded RNAs that silence gene expression by binding to target mRNAs. Mature miRNAs target the3’untranslated regions (3’UTR) of genes by complementary base-pair binding.Previous studies have shown that osteogenesis of PDLCs, a complex process in which multiple signaling pathways are involved, is controlled by many growth factors and transcription factors. However, the mechanism of PLAP-1expression regulation is not clear, particularly in the post-transcriptional level. So far, there is no report on PLAP-1gene regulation by miRNAs during osteogenic differentiation. Also, there is no report on differential expression of miRNAs during osteogenic differentiation of PDL. It is a new field to test whether certain miRNAs could be able to regulate PLAP-1gene expression and lead to differentiation of PDLCs. Therefore, the aim of this study was to determine which miRNAs are responsible for regulating PLAP-1expression and osteogenic differentiation of PDLCs.Objective:Observe and verify in vitro culture of periodontal ligament cells and gene expression of PLAP-1in the process of osteogenic differentiation of PDLCs.Methods:In this study, MTT assay, Alizarin red staining, immunohistochemistry, ALP, RT-PCR and Western blot were performed.Results:The results showed that PLAP-1genes highly expressed in human periodontal ligament cells. IHC results showed that PLAP-1was mainly in the perinuclear cytoplasm. Expression level of PLAP-1increased gradually with the mineralization of periodontal ligament cells. The status of cell proliferation and mineralization of periodontal ligament cells was good.Conclusion:These results suggest that periodontal ligament cells used in this subject maintained its original features, such as the ability of osteogenic differentiation, cell proliferation and normal expression level of PLAP-1. So, these cells are suitable for further research. Objective:Screen and verify microRNAs which regulate expression of PLAP-1gene. Detect the expression of PLAP-1gene and related miRNAs in human periodontal ligament cells in the process of osteogenic differentiation.Methods:We used Targetscan, mirBase, microRNA.org and mirGen-miRanda four different bioinformatics software to predict miRNAs targeting PLAP-1gene, respectively.307potential microRNAs were found and5miRNAs were chosen based on the prediction by more than one program. In order to further confirm the function of these miRNAs in regulating PLAP-1gene, a dual luciferase reporter system and quantitative PCR methods were employed in this study.Results:MicroRNAs were predicted by Targetscan, mirBase, microRNA.org and mirGen-miRanda. Results showed that five microRNAs might regulate PLAP-1expression. They are miR-144, miR-26a, miR-26b, miR-21and miR-101. miR-21and miR-101can inhibit luciferase activity significantly (p-<0.05, p-<0.001) in dual luciferase reporter assay. Results from LSD group comparison showed that psiPLAP-1+mir21and psiPLAP-1+mir101have significant difference (p less than0.001), while there was no significant difference (p=0.873, p=0.792, p=0.854) when comparing psiPLAP-1+mir26a, psiPLAP-1+mir26b,and psiPLAP-1+mir144respectively with psiPLAP-1+Blank group. Expression level of PLAP-1gene increased with time in the process of osteogenic differentiation(F=291.982, P0.001). Expression level of miR-21and miR-101declined with time in the process of osteogenic differentiation (F=181.266, P<0.001; F=40.233, P<0.001).There was significant difference on expression level of PLAP-1when miR-21or miR-101was overexpressed transitly (F=120.691, P=0.001). Expression level of PLAP-1gene was significantly reduced when miR-21or miR-101was overexpressed, comparing with that of normal cells (P=0.012, P=0.002).Conclusion:miR-21and miR-101can bind to3’UTR of PLAP-1and regulate its expression. miR26a, miR26b and miR144are not able to do so. Expression level of miR-21and miR-101was inversely related with PLAP-1during osteogenic differentiation of PDLCs. Overexpression of miR-21or miR-101can reduce PLAP-1mRNA and protein level. miR-21and miR-101were chosen for further study.Objective:Analyze binding sites of miR-21and miR-101on3’UTR of PLAP-1.Methods:Targetscan and MicroRNA.org were used to predict the binding sites on PLAP-1gene. To further confirm the binding site, mutations were created and tested.Results:The combination of Targetscan, and MicroRNA.org software analysis predicted binding sites of miR-21or miR-101targeting to PLAP-1genes. Results from dual luciferase reporter assay showed that there was significant difference between psiPLAP-13’UTR group and negative control groups (p<0.001). Results from LSD group comparison showed that psiPLAP-1+mir21and psiPLAP-1+mir101have significant difference (p less than0.001) while there is no significant difference (p=0.901) between psiPLAP-13’UTR of+Blank and psiPLAP-13’UTR+NC. Results of site-directed mutagenesis showed no differences (p=0.114, p>0.05) between mir21,mirl01, Blank and NC groups.Conclusion:Determined the binding sites of miR-21and miR-101on PLAP-1gene. has-miR-101was chosen as a further study to explore its function on expression regulation of PLAP-1.Objective:Illustrate the function of miR-101in regulating the PLAP-1gene and osteogenic differentiation of PDLCs.Methods:Firstly, lentivirus of miR-101overexpression and downexpression were obtained. Pre-experiment determined MOI values of the periodontal ligament cells. In the process of proliferation and osteogenic differentiation induced by miR-101overexpression or downexpression, MTT, Alizarin red staining, ALP activity assay, quantitative PCR and Western blot were employed to test cell proliferation, osteogenic differentiation, ALP activity, and expression level of PLAP-1.Results:MTT assay results showed that the main effect of grouping and time, time and group interaction effects were statistically significant. Control group and mir101group have a similar growth trend at four time points while anti-mir101grows slower than the first two. The time factor, the OD value of MTT in mir101group and anti-mir101group were decreased compare with the control group, and the difference was statistically significant (P<0.001and P<0.001).ALP activity data showed that the main effect and interaction effects of grouping and time are statistically significant. anti-mir101group had lowest ALP activity on day0the highest ALP activity on day14. Regardless time factor, the difference of ALP activity was statistically significant (P<0.001and P<0.001) when the control group and anti-miR-101group compared with miR-101group.ALP activity in the groups of day7and day14have increased compared with day0, and the difference was statistically significant (P=0.003and P<0.001).Variance analysis of QPCR data showed that grouping and time as main effects was significantly different. Expression level of PLAP-1gene was lowest on day21of miR-101over-expression group. Expression level of PLAP-1increased with time in control group while it was opposite in miR-101overexpression group. Expression level of PLAP-1gene in anti-miR-101group was basically the same on day7and day14but increased on day21compared with miR-101group. Expression level of PLAP-1gene was increased on day14and day21in control and anti-miR-101groups compared with that of day7, and the difference was statistically significant (P=0.025and P=0.003). Control and miR-101overexpression groups were significantly different (p<0.01) on day7, day14and day21.These results suggested that miR-101overexpression inhibited PLAP-1gene expression while anti-miR-101increased PLAP-1expression. Results of mineralized nodule formation and Western blot showed that mineralization nodule formation number and PLAP-1protein expression were increased in miR-101overexpression group compared with the control group. While mineralization nodule formation number and PLAP-1protein expression were decreased in anti-miR-101group compared with the control group.Conclusion:These results indicate that the miR-101overexpression significantly inhibited PLAP-1gene expression, weakened the negative regulatory role of PLAP-1in osteogenic differentiation. It increased cell proliferation and osteogenic differentiation. Meanwhile, anti-miR-101significantly increased PLAP-1gene expression, enhanced the negative regulatory role of PLAP-1in osteogenic differentiation, inhibited cell proliferation and osteogenic differentiation.