Abstract
BackgroundSmall nucleolar RNA host gene 3 (Snhg3) is a long non-coding RNA (lncRNA) that was shown to participate in the tumorigenesis of certain cancers. However, little is known about its role in embryonic stem cells (ESCs).MethodsHere, we investigated the role of Snhg3 in mouse ESCs (mESCs) through both loss-of-function (knockdown) and gain-of-function (overexpression) approaches. Alkaline phosphatase staining, secondary colony formation, propidium iodide staining, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to access self-renewal capacity, whereas immunofluorescence, qRT-PCR, and embryoid body formation were performed to examine pluripotency. In addition, the effect of Snhg3 on mouse embryonic development was determined based on the morphological changes, blastocyst rate, and altered pluripotency marker (Nanog, Oct4) expression. Moreover, the relationship between Snhg3 and key pluripotency factors was evaluated by chromatin immunoprecipitation qPCR, qRT-PCR, subcellular fractionation, and RNA immunoprecipitation. Finally, RNA pull-down and mass spectrometry were applied to explore the potential interacting proteins of Snhg3 in mESCs.ResultsWe demonstrated that Snhg3 is essential for self-renewal and pluripotency maintenance in mESCs. In addition, Snhg3 knockdown disrupted mouse early embryo development. Mechanistically, Snhg3 formed a positive feedback network with Nanog and Oct4, and 126 Snhg3-interacting proteins were identified in mESCs.ConclusionsSnhg3 is essential for mESC self-renewal and pluripotency, as well as mouse early embryo development.
Highlights
Small nucleolar RNA host gene 3 (Snhg3) is a long non-coding RNA that was shown to participate in the tumorigenesis of certain cancers
Snhg3 is highly expressed in mouse embryonic stem cells (ESCs) (mESCs) and decreased after differentiation To explore the potential functions of Snhg3 in mESCs, we first compared the expression levels between mESCs and Mouse embryonic fibroblast (MEF). quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that the expression of Snhg3 was much higher in mESCs than in MEFs (~ 9fold; Fig. 1a)
Knockdown of Snhg3 impairs mESC self-renewal To understand the role of Snhg3 in mESCs, we first transfected two efficient and specific targeting Snhg3 siRNA oligos into mESCs to knock down its expression (Fig. 2a)
Summary
Small nucleolar RNA host gene 3 (Snhg3) is a long non-coding RNA (lncRNA) that was shown to participate in the tumorigenesis of certain cancers. Small nucleolar RNA host gene 3 (Snhg3) is a newly discovered lncRNA that was identified as a biomarker of malignant status or poor prognosis in several types of cancers including lung cancer, hepatocellular carcinoma, glioma, ovarian cancer, and colorectal cancer [11,12,13,14,15]. The detailed functions and associated mechanisms of Snhg in mESCs are not clear. We characterized its regulatory functions and associated molecular mechanisms with respect to mESC self-renewal and pluripotency and early mouse embryonic development. Our study identify Snhg as an additional important player in ESC regulation, and indicate its potential value for optimizing ESC generation, aimed at accelerating stem cell translational medicine
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