Institute of Clinical Pharmacology, Key Laboratory of Anti-inflammatory and Immune Medicine, Ministry of Education, Anhui Collaborative Innovation Center of Anti-inflammatory and Immune Medicine, Anhui Medical University, 81# Meishan Road, Hefei, Anhui, China.
Faculty of Medicine, School of Biomedical Sciences, The Chinese University of Hong Kong, Lo Kwee-Seong Integrated Biomedical Sciences Building, Shatin, N.T 622A, Hong Kong, Special Administrative Region of China.
Stem Cell Res Ther. 2018 Mar 21;9(1):71. doi: 10.1186/s13287-018-0813-5.
The regulatory role of long noncoding RNAs (lncRNAs) have been partially proved in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).
In the current study, we investigated mouse ESC (mESC) self-renewal, differentiation, and proliferation in vitro by knocking down a lncRNA, growth arrest specific 5 (Gas5). A series of related indicators were examined by cell counting kit-8 (CCK-8) assay, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), Western blot, alkaline phosphatase staining, propidium iodide (PI) staining, Annexin V staining, competition growth assay, immunofluorescence, and chromatin immunoprecipitation (ChIP)-qPCR. An in vivo teratoma formation assay was also performed to validate the in vitro results. qRT-PCR, fluorescence-activated cell sorting (FACS), alkaline phosphatase staining, and immunofluorescence were used to evaluate the role of Gas5 during mouse iPSC reprogramming. The regulatory axis of Dicer-miR291a-cMyc-Gas5 and the relationship between Gas5 and Tet/5hmC in mESCs was examined by qRT-PCR, Dot blot, and Western blot.
We identified that Gas5 was required for self-renewal and pluripotency of mESCs and iPSCs. Gas5 formed a positive feedback network with a group of key pluripotent modulators (Sox2, Oct4, Nanog, Tcl1, Esrrb, and Tet1) in mESCs. Knockdown of Gas5 promoted endodermal differentiation of mESCs and impaired the efficiency of iPSC reprogramming. In addition, Gas5 was regulated by the Dicer-miR291a-cMyc axis and was involved in the DNA demethylation process in mESCs.
Taken together, our results suggest that the lncRNA Gas5 plays an important role in modulating self-renewal and pluripotency of mESCs as well as iPSC reprogramming.
长链非编码 RNA（lncRNAs）的调控作用已在胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs）中得到部分证实。
在本研究中，我们通过敲低 lncRNA 生长停滞特异性 5（Gas5）来研究体外小鼠胚胎干细胞（mESC）的自我更新、分化和增殖。通过细胞计数试剂盒-8（CCK-8）检测、定量逆转录聚合酶链反应（qRT-PCR）、Western blot、碱性磷酸酶染色、碘化丙啶（PI）染色、Annexin V 染色、竞争生长试验、免疫荧光和染色质免疫沉淀（ChIP）-qPCR 检测一系列相关指标。还进行了体内畸胎瘤形成试验来验证体外结果。通过 qRT-PCR、荧光激活细胞分选（FACS）、碱性磷酸酶染色和免疫荧光来评估 Gas5 在小鼠 iPSC 重编程过程中的作用。通过 qRT-PCR、Dot blot 和 Western blot 检测 Dicer-miR291a-cMyc-Gas5 调控轴和 Gas5 与 mESCs 中 Tet/5hmC 之间的关系。
我们发现 Gas5 是 mESCs 和 iPSCs 自我更新和多能性所必需的。Gas5 与一组关键的多能调节剂（Sox2、Oct4、Nanog、Tcl1、Esrrb 和 Tet1）在 mESCs 中形成正反馈网络。Gas5 的敲低促进了 mESCs 的内胚层分化，并降低了 iPSC 重编程的效率。此外，Gas5 受 Dicer-miR291a-cMyc 轴调控，并参与 mESCs 中的 DNA 去甲基化过程。
综上所述，我们的研究结果表明，lncRNA Gas5 在调节 mESCs 的自我更新和多能性以及 iPSC 重编程中发挥着重要作用。
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