The Mechanism And Functional Network Study Of Small Nuclealous RNA In Self-Renewal Of Human Pluripotency Stem Cells

Background and objectiveEmbryonic stem cells(ESCs)are the strongest Pluripotention of all pluripotency cells,owing great clinical application prospection,especially in regenerative tissue engineering.However,To fully explore their potential requires a deeper understanding of the molecular basis of stemness.Human umbilical cord blood derived mesenchymal stem cells(uMSC),which have been successfully used in regenerative treatment of various diseases,and some of the usage is FDA approved.Although they showed great promise in cell-based therapies,substantial challenges still need to be overcome before they can be used in clinical practice.It is known that MSCs can only be passaged in a limited period in vitro.And after this certain period,the cells began to lose their capacity in attenuating diseases,either undergone differentiation or became senescence.How to promote their self-renewing span and maintain their proliferation and differentiation capacity is vital for their usage in the future.Emerging evidence suggests that small nucleolar RNAs(snoRNAs)are actively involved in cell proliferation especially in tumor cells,but their roles in stem cells are seldom studied.And recent discoveries indicated that some snoRNAs can generate lineage conserved small and functional RNAs after enzyme digestion,namely small nucleolus derived RNA(sdRNAs).These sdRNAs have the characteristics of miRNAs.These discoveries about the relation between sdRNAs and miRNAs have widened the functional roles of snoRNAs in biological context.Since many studies showed that specific snoRNAs can generate sdRNA,but the molecular mechanism and the function of sdRNA in stem cells have not been fully investigated.In this study,we tried to provide an available model to investigate the cellular function and the underlying mechanism of some specific snoRNAs in stem cells.PartⅠ C/D Box Small Nucleolar RNA 3A(U3)promotes the Self-Renewal of Human Embryonic Stem CellsMethods:(1)Investigated RNA-sequence to identify snoRNAs which have difference expression between differentiation and ground state.(2)we tried to overexpress and inhibit U3 and U3-sd RNA by transfecting to investigat the effect on uMSC pluripotency by Real-time PCR,Northern Blot,AP et al.(3)we identified the cellular localization of U3-sd RNA in hESCs using RNA in situ hybridization and nuclear-plasma separation.(4)To identify the generational localization of U3-sd RNA In hESCs using Incubation in vitro and nuclear-plasma separation.(5)Designed six kinds of mutant transcripts that either mutated the characteristic binding sites to investigate the potential mechanism of U3-sd RNA generation.Results:(1)U3 has similar expression level in different cell types.However,U3-sd RNA,which derived from U3 was significantly different in the expression level around different cell types.It present as hESCs>uMSCs>293T.(2)The expression levels of U3 and U3-sd RNA were significantly higher than that of hESCs,and the clones were significantly increased,whereas effect was on the opposite after U3-sd RNA was inhibited.(3)U3-sd RNA dominantly expressed in cytoplasm.(4)Northern Blot results after in vitro cleavage showed that the hESCs cytoplasm and U3 co incubation could generate more U3-sd RNA.(5)Overexpressed the six mutant transcripts,hESCs clone formation rate of Mut-2,Mut-1 group showed no significant difference between the control group and the control group.while it was improved in the other four mutant transcripts.Conclusions:U3 can maintain even improve hESCs pluripotency.This function is mainly obtained by derived U3-sd RNA.In addition,the production of sdRNA is mainly related to the characteristic structure of snoRNA.Part ⅡH/ACA Box Small Nucleolar RNA 7A promotes the Self-Renewal of Human Umbilical Cord Mesenchymal Stem CellsMethords:(1)we first established the cell models by knocking down DKC1 and NOP10,two core proteins that form H/ACA box snoRNA related snoRNP,in uMSCs.After conformation of the knockdown efficiency of the DKC1 and NOP10 specific siRNA by Real-time PCR and Western Blot Western Blot BlotWestern Blot.(2)we analyzed the global snoRNAs expression in both differentiated and undifferentiated state of the MSCs using high through-put small RNA sequencing data.(3)we tried to overexpress and inhibit SNORA7 A by transfecting to investigat the effect of ectopic SNORA7 A on uMSC self-renenwal by Real-time PCR/Western Blot、EDU、cloning、Doubling time.(4)we designed two kinds of mutant transcripts that either mutated the core protein binding sites(Mut-S)or the 28 s RNA complimentary binding sites(Mut-B)based on known structural characteristics,then investigated the effect of ectopic SNORA7 A on uMSC self-renenwa.We then validated the expression of SNORA7 A among several cell lines with different pluripotent capacity by Real-time PCR/Western Blot/EDU/cloning/Doubling time(5)We evaluated the effect of SNORA7 A on uMSC osteogenic and adipogenic differentiation regulation.Results:(1)H/ACA box SnoRNA is vital to the maintaining and proliferation of uMSC;(2)SNORA7A is correlated with the proliferation state of uMSC;(3)Ectopic SNORA7 A promotes u MSC self-renewal;(4)Inhibition of SNORA7 A greatly impaired the self-renewal capacity of uMSC;(5)The functions of SNORA7 A are snoRNP and rRNA dependent;(6)DKC1 is required for SNORA7 A to exert its function;(7)SNORA7A inhibits osteogenic and adipogenic differentiation of uMSC Conclusions: The endogenous SNORA7 A is critical for the proliferation of uMSC through recruiting snoRNPs.Moreover,SNORA7 A as one of the critical snoRNAs that involved in the uMSC self-renewal in vitroConclusions Different snoRNA play different roles in the self-renewal of stem cells,and the mechanisms are different.The endogenous U3 can maintain even improve hESCs pluripotency.This function is mainly obtained by derived U3-sd RNA.The endogenous SNORA7 A is critical for the proliferation of uMSC through recruiting snoRNPs.