The Molecular Mechanism Of The Long Non-coding RNA Gtl2 Regulating Transcription Factor Oct4 In Early Mouse Embryos

The Gtl2 gene is located in the Dlk1-Dio3 imprinting interval at the end of mouse chromosome 12,and its long non-coding RNA Gtl2 plays an important role in embryonic development and tumorigenesis.Previous studies in our lab about mouse early embryos found that Gtl2 expressed in the morula stage and ultimately localized in the blastocyst inner cell mass(ICM).Functional studies showed that interference with Gtl2 expression did not affect the blastocyst development rate,but it affected hatching,outgrowth and ICM formation.Further studies revealed that down-regulation of Gtl2 expression resulted in the decreased expression of Oct4 that is the blastocyst ICM-specific transcription factor,suggesting that Gtl2 may play a role by affecting Oct4.Previous experiments have confirmed that Gtl2 may act as a ceRNA(competing endogenous RNA)to regulate the expression of Oct4 through the miRNA pathway,thereby playing a role in early embryos.Therefore,the aim of this study is to screen for candidate miRNAs that may bind to Gtl2 and regulate Oct4,and to reveal the possible molecular mechanism of Gtl2 regulates Oct4 in mouse early embryos.Firstly,using bioinformatics website tools in conjunction with the published papers,we screened the candidate miRNAs with potential binding ability to Gtl2 and confirmed by experiments that they could down regulate the expression of Oct4.They are let-7c(2 binding sites),miR-24,miR-29 a,miR-31 and miR-135 b.In addition,the dual luciferase reporter system confirmed that the both binding sites of let-7c have significant binding ability to Gtl2,and miR-24,miR-29 a and miR-135 b can also bind to Gtl2.Only miR-31 did not detect binding to Gtl2.Therefore,we selected let-7c,miR-24,miR-29 a,and miR-135 b as candidate miRNAs for further study.At the cellular level,we verified the interaction of candidate miRNAs with endogenous Gtl2 using MLTC-1 cells(mouse testicular stromal cell tumor cells)with high expression of Gtl2.The results showed that only let-7c had significant binding to endogenous Gtl2.We further demonstrated the interaction of let-7c with endogenous Gtl2 by using N2 a cell(mouse neuroblastoma cell)expressing Gtl2.Therefore,we used let-7c as the final candidate miRNA for further validation at the embryo level.At the embryonic level,we confirmed by rescue experiments that Gtl2 can rescue the loss of ICM in the growth of blastocysts in vitro caused by let-7c overexpression and increase the expression level of Oct4,thereby restoring embryonic developmental capacity.It is indicated that Gtl2 can interact with let-7c in early embryos,affect the expression of Oct4 by let-7c,and then regulate the formation of ICM in early embryos.In summary,this study screened candidate miRNAs that bind to Gtl2 and regulate Oct4 expression,and confirmed that let-7c has the binding ability to endogenous Gtl2 at the cellular level,and then elucidated the molecular mechanism that let-7c mediated Gtl2 to regulate Oct4.The research results of this thesis provide the basis for understanding Gtl2 on early embryonic development and ICM formation and differentiation,and provide theoretical basis and reference for related researches on embryonic stem cells(ESC)and induced pluripotent stem cells(iPSC).