RNA 结合蛋白 Lin28A 差异表达可调控牙周膜干细胞的成骨分化.

BACKGROUND: The cellular activity of Lin28A can be influenced in several ways, including the self-renewal of embryonic stem cells, somatic cell reprogramming, individual growth and development, tissue metabolism, and carcinogenic consequences. However, neither domestically nor internationally have its effects on periodontal ligament stem cell ability to differentiate into osteoblasts been well explored; the underlying mechanisms are not known. OBJECTIVE: To investigate the impact of Lin28A on periodontal ligament stem cell capacity for osteogenic differentiation. METHODS: By using conventional enzyme digestion, periodontal ligament stem cells were extracted and cultured. The distribution of Lin28A in these cells and the differential expression of Lin28A at different time points of osteogenic induction were detected by qRT-PCR. Lentiviral vectors with Lin28A overexpression and interference expression were constructed and transfected into periodontal ligament stem cells. Fluorescence microscopy was used to observe the transfection effect. qRT-PCR was used to verify the expression difference of Lin28A after lentivirus transfection. The expression levels of osteoblastic genes alkaline phosphatase, Runt-related transcription factor 2 and osteopontin, alkaline phosphatase staining and alizarin red staining results were further detected from positive and negative aspects. The protein expression level of Runt-related transcription factor 2 was detected by western blot assay. Bioinformatics techniques and dual luciferase were used to predict and verify the relationship between Lin28A-related let-7 family members. The periodontal ligament stem cells differentially expressing Lin28A were cultured through osteoblastic induction, and the miRNA with the most obvious expression change during osteoblastic differentiation was determined. RESULTS AND CONCLUSION: (1) Lin28A was enriched in the nucleus of periodontal ligament stem cells, and the expression of Lin28A increased at different time points after osteogenic induction. (2) In the periodontal ligament stem cell models with Lin28A overexpression, alkaline phosphatase staining and alizarin red staining were significantly deeper than those in the blank control group; the expression levels of alkaline phosphatase, Runt-related transcription factor 2 and osteopontin were increased 3.3, 5.1 and 2.2 times, respectively, compared with the blank ones (P < 0.01). In cell models that interfered with Lin28A expression, alkaline phosphatase staining and alizarin red staining were also decreased compared with the control group. The levels of alkaline phosphatase, Runt-related transcription factor 2 and osteopontin decreased 0.19-fold, 0.61-fold and 0.1-fold, respectively (P < 0.01). (3) The protein level of Runt-related transcription factor 2 was consistent with the mRNA difference tendency. The expression of Runt-related transcription factor 2 was increased in the overexpression group and decreased in the interference group. (4) Bioinformatics predicted that Lin28A was associated with eight let-7 family members, which were let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g and let-7i, respectively. After screening, the expression levels of let-7a and let-7c showed significant changes with Lin28A expression, and let-7a showed the most significant trend. Double luciferase binding assay confirmed that let-7a was directly bound to the 3'-UTR of Lin28A and was involved in the osteogenic differentiation of periodontal ligament stem cells. (5) The results suggested that Lin28A was involved in the regulation of osteogenic differentiation of periodontal ligament stem cells, with a positive correlation. That was, overexpression of Lin28A promoted the osteogenic ability of periodontal ligament stem cells, and the osteogenic ability of periodontal ligament stem cells was weakened after interference of Lin28A expression. The osteogenic differentiation of periodontal ligament stem cells was regulated by Lin28A . [ABSTRACT FROM AUTHOR]