MiR-146a-5p/Notch Signaling Is Involved In The Proliferation And Differentiation Of Human Dental Pulp Stem Cells

Objective: The main purpose of this study is to explore whether mi R-146a-5p is related to the status of human dental pulp stem cells(DPSCs)and stem cells from the apical papilla(SCAPs)as well as its role in the differentiation and proliferation of DPSCs.Methods:(1)DPSCs and SCAPs were purified by magnetic-activated cell sorting with specific STRO-1 antibody.Real-time q PCR(q RT-PCR)was used to determine seven differential mi RNAs(mi R-224-5p ? mi R-1247-5p ? mi R-3065-3p ? mi R-452-5p ?mi R-767-5p?mi R-4284?mi R-146a-5p)expression of DPSCs and SCAPs which were screened by the Next generation sequencing in the previous study.(2)The expression of mi R-146a-5p was measured by qRT-PCR during mineralization induction in DPSCs.(3)mi R-146a-5p expression in DPSCs was detected after stably transfected by mi R-146a-5p overexpression and inhibition lentivirus by q RT-PCR.(4)Alizarin red staining,alkaline phosphatase(ALP)staining,ALP activity and the expression levels of osteogenic and odontogenic marker gene(RUNX2,OSX,OCN,ALP and DSPP)were analyzed after transfection and mineralization induction.The impact of mi R-146a-5p on proliferation of DPSCs was analyzed by using CCK8 assays.(5)The expression of NOTCH1 and HES1 were measured by q RT-PCR during mineralization induction in DPSCs.(6)Interference of NOTCH1 was performed to investigate its role in the differentiation and proliferation of DPSCs.(7)Bioinformatic analyses,luciferase assays combined with qRT-PCR and Western Blot were utilized to identify the targets interacting with mi R-146a-5p.Results:(1)The expression of mi R-224-5p?mi R-1247-5p?mi R-452-5p?mi R-767-5p?mi R-4284 and mi R-146a-5p were downregulated in SCAPs compared with DPSCs,and mi R-146a-5p expression was the most different,but no significant changes in mi R-3065-3p expression.(2)qRT-PCR detection results showed that mi R-146a-5p expression increased during mineralization induction in DPSCs.(3)mi R-146a-5p expression was markedly upregulated/downregulated in DPSCs transfected with lentivirus for mi R-146a-5p overexpression/inhibition.(4)mi R-146a-5p overexpression in DPSCs resulted in formation of more calcified nodules and resulted in elevated alkaline phosphatase(ALP)staining,alkaline phosphatase activity and osteogenic and odontogenic marker gene expression levels,whereas it inhibited the proliferation of DPSCs.These results were the opposite after mi R-146a-5p inhibition.(5)q RT-PCR detection results showed that NOTCH1 and HES1 expression decreased during mineralization induction in DPSCs.(6)Interference with Notch signaling was verified to enhance DPSC differentiation and suppress proliferation.(7)Bioinformatic analyses,luciferase assays,qRT-PCR and Western Blot identified that mi R-146a-5p directly targeted the 3'UTR of NOTCH1.Conclusion:(1)It was confirmed that mi R-146a-5p inhibited DPSC proliferation and promoted differentiation partially by suppressing the Notch signaling.(2)Low levels of mi R-146a-5p may contribute to maintaining DPSCs in an undifferentiated state and could be used to aid in expanding cells in vitro for tooth tissue engineering and dental pulp tissue regeneration.