Study On The Proliferation And Senescence Of Stem Cells From Apical Papilla By MiR-141-3p Targeting Regulation Of YAP

MicroRNAs(miRNAs)are a group of short,non-coding endogenous RNAs that function by binding to the 3 untranslated region of target mRNA at the post-transcriptional level.More and more evidences have shown that miRNAs play an important role in many biological processes such as proliferation,senescence and differentiation of mesenchymal stem cells,but the mechanism is still unclear.In this paper,we will discuss the effects of miR-141-3p on the proliferation and senescence of stem cells from apical papilla(SCAPs)and their mechanisms from the following three parts.Part ?.Effects of miR-141-3p on proliferation and senescence of SCAPsObjective To observe the changes in cell proliferation and senescence,after changing the expression of miR-141-3p in SCAPs.Methods Primary SCAPs were isolated and cultured by enzyme digestion,phenotypic markers were detected by flow cytometry and immunofluorescence staining,and the cells were identified.The transfection effect was verified by real-time quantitative PCR,and cell proliferation was detected by CCK-8,flow cytometry and EDU.Western Blot,RT-PCR,galactosidase staining and immunofluorescence staining were used to detect the expression of cell senescence related indexes.Results We found that the increase of miR-141-3p inhibited the proliferation of SCAPs.At the same time,SCAPs are aging ahead of their time.On the contrary,interference with miR-141-3p inhibitor can promote SCAPs proliferation and delay their senescence.Conclusions miR-141-3p inhibited the proliferation of SCAPs,and the senescence of them was ahead of time.Part ?.Prediction and Verification of miR-141-3p target genesObjective To find possible target genes downstream of miR-141-3p and explore possible pathway mechanisms involved.Methods The downstream target genes of miR-141-3p were verified by miRDB,miRTARbase,miRWalk,TargetScan software prediction and dual-luciferase assay detection.Results Through database screening and dual-luciferase assay,we confirmed the sequence specific binding relationship between YAP gene and miR-141-3p.Conclusions yes-associated protein(YAP)was identified as a potential target gene of miR-141-3p.Part ?.Effects of YAP on proliferation and senescence of SCAPsObjective To inhibit the downstream target gene YAP of miR-141-3p and observe the changes of SCAPs proliferation and senescence.Methods The transfection effect was verified by real-time quantitative PCR,and cell proliferation was detected by CCK-8,flow cytometry and EDU.Western Blot,RT-PCR,galactosidase staining and immunofluorescence staining were used to detect the expression of cell senescence related indexes.Results After interfering with the expression of YAP,the proliferation of SCAPs was inhibited,and SCAPs entered the senescence state prematurely.Conclusions After a loss of YAP,the function of YAP on SCAPs proliferation and senescence was opposite to that of miR-141-3p inhibitor.Collectively,these findings proposed a novel avenue whereby miR-141-3p impeded SCAPs proliferation and promoted senescence through the post-transcriptional downregulation of YAP.