The Role Of Hsa-miR-335-3p In The Differentiation Of Human Periodontal Ligament Stem Cells (PDLSCs) Into Cementoblast-like Cells

Cementum is a sort of mineralized tissue which attaches to the very outer surface of the root of tooth. When periodontitis and abnormal mechanical forces lead to the degeneration of periodontal tissues, the healthy cementum is essential for new epithelial attachment and the regeneration of periodontal tissues. As a sort of adult stem cells,PDLSCs possess the ability to self-renew and the potential of multi-directional differentiation. Under suitable extra-cellular microenvironment, PDLSCs can be used as the ideal seed cells for regeneration of the periodontal tissues which include cementum.Plenty of investigations have proved that micro RNAs play crucial roles during biological processes of stem cells, such as growth and differentiation. Our studies want to show that whether micro RNA plays a role in the differentiation of PDLSCs toward cementoblast-like cells.Objective: The purpose of this study is to demonstrate whether hsa-mi R-335-3p plays a role in the differentiation of PDLSCs into cementoblast-like cells.Materials and methods: We acquired homogeneous populations of PDLSCs by the application of screening immune magnetic beads. Cell morphological appearance and immunocytochemical staining were evaluated. PDLSCs were induced by the apical tooth germ cell-conditioned medium(APTG-CM) and RT-PCR was carried out to verify the relative gene expression level of hsa-mi R-335-3p compared to the non-induced control. Adenoviruses that are modified with specific micro RNA sequences respectively were used to transfect the PDLSCs, and the transfected PDLSCs were cultured in APTG-CM. The relative expression level of cementoblast-like cells related genes(bone sialoprotein, cementum-derived attachment protein, cementum protein 1) were tested through RT-PCR and Western Blot at the time-point of 7days, 14 days and 21 days after the transfected PDLSCs were cultured in APTG-CM.Results: The positive immunomagetic sorted cells exhibited several characteristics of PDLSCs, as indicated by morphological characters and positive immunocytochemical stainings of STRO-1 and CD146. The gene expression level of hsa-mi R-335-3p is lower in the induced group relative to the non-induced control group. After the transfected PDLSCs were cultured in APTG-CM for 7 days, 14 days and 21 days, the respective results of RT-PCR for the related gene expression levels show: the relative gene expression level is lower when compared with the up-regulated group and is higher relative to the down-regulated group. Meanwhile, the higher expression levels in the 21 days samples are of statistic significance.Conclusion: Our findings suggest that hsa-mi R-335-3p has an impact on the differentiation of PDLSCs into cementoblast-like cells as a negative regulator.