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LIN28A inhibits DUSP family phosphatases and activates MAPK signaling pathway to maintain pluripotency in porcine induced pluripotent stem cells

Xiao-Long Wu Zhen-Shuo Zhu Xia Xiao Zhe Zhou Shuai Yu Qiao-Yan Shen Ju-Qing Zhang Wei Yue Rui Zhang Xin He Sha Peng Shi-Qiang Zhang Na Li Ming-Zhi Liao Jin-Lian Hua

Xiao-Long Wu, Zhen-Shuo Zhu, Xia Xiao, Zhe Zhou, Shuai Yu, Qiao-Yan Shen, Ju-Qing Zhang, Wei Yue, Rui Zhang, Xin He, Sha Peng, Shi-Qiang Zhang, Na Li, Ming-Zhi Liao, Jin-Lian Hua. LIN28A inhibits DUSP family phosphatases and activates MAPK signaling pathway to maintain pluripotency in porcine induced pluripotent stem cells. Zoological Research, 2021, 42(3): 377-388. doi: 10.24272/j.issn.2095-8137.2020.375
Citation: Xiao-Long Wu, Zhen-Shuo Zhu, Xia Xiao, Zhe Zhou, Shuai Yu, Qiao-Yan Shen, Ju-Qing Zhang, Wei Yue, Rui Zhang, Xin He, Sha Peng, Shi-Qiang Zhang, Na Li, Ming-Zhi Liao, Jin-Lian Hua. LIN28A inhibits DUSP family phosphatases and activates MAPK signaling pathway to maintain pluripotency in porcine induced pluripotent stem cells. Zoological Research, 2021, 42(3): 377-388. doi: 10.24272/j.issn.2095-8137.2020.375

LIN28A抑制DUSP家族磷酸酶并激活MAPK信号通路来维持猪诱导多能干细胞的多能性

doi: 10.24272/j.issn.2095-8137.2020.375

LIN28A inhibits DUSP family phosphatases and activates MAPK signaling pathway to maintain pluripotency in porcine induced pluripotent stem cells

Funds: This work was supported by the National Key Research, Development Program of China-Stem Cell and Translational Research (2016YFA0100200), National Natural Science Foundation of China (32072806, 31572399, 61772431, 62072377), Program of Shaanxi Province Science and Technology Innovation Team (2019TD-036), Fundamental Research Funds for the Central Universities, Northwest A & F University (Z1090219146, Z102022004)
More Information
  • 摘要: 作为RNA结合蛋白,LIN28A在猪诱导多能干细胞(piPSCs)中发挥着重要的作用。然而,LIN28A维持piPSCs多能性的分子机制尚不清楚。该文通过过表达和干扰LIN28A来探究其在piPSCs中的作用。我们对piPSCs进行了全转录组测序(RNA-seq),并通过实时荧光定量聚合酶链式反应(qRT-PCR)、蛋白质印迹法(Western Blot)和免疫荧光染色检测相关基因的表达水平。我们的结果表明,干扰LIN28A后,piPSCs的增殖能力明显下降。此外,当干扰组中的LIN28A的表达量低于阴性对照组(shNC)表达量的20%时,piPSCs会丧失多能性,并向神经外胚层细胞分化。结果还表明LIN28A能抑制DUSP家族磷酸酶的活性进而激活丝裂原活化蛋白激酶(MAPK)信号通路。因此,LIN28A可激活MAPK信号通路进而维持piPSCs的多能性和增殖能力。该文为探索LIN28A在piPSCs中的作用提供了新思路。
    #Authors contributed equally to this work
  • Figure  1.  Effects of LIN28A on piPSC proliferation ability

    A: LIN28A mRNA expression levels in shNC, shLIN28A1, and shLIN28A2 groups with DOX were detected by qRT-PCR. Data are mean±SEM. **: P<0.01. n=3. B: Left: PCNA and LIN28A protein expression levels in shNC, shLIN28A1, and shLIN28A2 groups with DOX were detected by western blotting. Data are mean±SEM. n=2. Right: Quantitative analysis of PCNA and LIN28A protein expression levels in shNC, shLIN28A1, and shLIN28A2 groups with DOX is shown in histogram. Data are mean±SEM. **: P<0.01. n=3. C: Left: Morphology and AP staining of shNC, shLIN28A1, and shLIN28A2 groups with DOX. Scale bar: 100 μm for phase and 100 μm for AP. Right: Area ratio of colonies in shNC, shLIN28A1, and shLIN28A2 groups with DOX. **: P<0.01. D: Growth curve in shNC, shLIN28A1, shLIN28A2, OENC, and OELIN28A groups with DOX. Data are mean±SEM, *: P<0.05, **: P<0.01. n=3. E: Morphology and AP staining of the negative control overexpression group without DOX (OENC+DOX−), negative control overexpression group with DOX (OENC+DOX+), OELIN28A group without DOX (OELIN28A+DOX−), and OELIN28A group with DOX (OELIN28A+DOX+). Scale bar: 100 μm. F: Number of colonies in OENC+DOX−, OENC+DOX+, OELIN28A+DOX−, and OELIN28A+DOX+ groups. Data are mean±SEM. *: P<0.05, **: P<0.01. G: mRNA expression levels of pluripotent genes in OENC+DOX−, OENC+DOX+, OELIN28A+DOX−, and OELIN28A+DOX+ groups were detected by qRT-PCR. Data are mean±SEM. *: P<0.05, **: P<0.01. n=3. ns: No significance.

    Figure  2.  Effects of LIN28A on piPSC pluripotency

    A: Left: Morphology of OEOCT4-shNC and OEOCT4-shLIN28A2 groups without DOX. Scale bar: 100 μm. Right: Area ratio of colonies in OEOCT4-shNC and OEOCT4-shLIN28A2 groups without DOX. **: P<0.01. B: Morphology of OEOCT4-shNC and OEOCT4-shLIN28A2 groups without DOX using fluorescence microscopy. Scale bar: 400 μm. C: LIN28A mRNA expression levels in OEOCT4-shNC and OEOCT4-shLIN28A2 groups without DOX were detected by qRT-PCR. Data are mean±SEM. *: P<0.05, **: P<0.01. n=3. D: EdU staining of OEOCT4-shNC and OEOCT4-shLIN28A2 groups cultured on third day without DOX. Scale bar: 400 μm. E: Percentage of EdU-positive cells in OEOCT4-shNC and OEOCT4-shLIN28A2 groups cultured on third day without DOX. Scale bar: 400 μm. Data are mean±SEM. *: P<0.05. n=3. F: AP staining of OEOCT4-shNC and OEOCT4-shLIN28A2 groups without DOX. Scale bar: 100 μm for AP. G: Quantification of AP-positive colonies in OEOCT4-shNC and OEOCT4-shLIN28A2 groups without DOX. Data are mean±SEM. *: P<0.05, **: P<0.01. n=3.

    Figure  3.  LIN28A inhibited expression of differentiation-related genes

    A: Number of up- and down-regulated DEGs in OEOCT4-shNC and OEOCT4-shLIN28A2 groups without DOX. B: Heat map shows expression of pluripotent genes in OEOCT4-shNC and OEOCT4-shLIN28A2 groups without DOX based on RNA-seq. C: Gene Ontology enrichment analysis of up-regulated DEGs, highlighting positive regulation of cell differentiation, neuronal differentiation, and negative regulation of cell proliferation. D: Heat map showing expression of genes involved in negative regulation of cell proliferation based on RNA-seq. E: Heat map showing expression of genes involved in positive regulation of cell differentiation and neuronal differentiation based on RNA-seq. F: mRNA expression levels of genes involved in positive regulation of cell differentiation, neuronal differentiation, and negative regulation of cell proliferation were detected by qRT-PCR. Data are mean±SEM. *: P<0.05, **: P<0.01. n=3.

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  • 收稿日期:  2021-02-16
  • 录用日期:  2021-05-14
  • 网络出版日期:  2021-05-17
  • 刊出日期:  2021-05-18

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