LIN28A inhibits DUSP family phosphatases and activates MAPK signaling pathway to maintain pluripotency in porcine induced pluripotent stem cells
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。Abstract: LIN28A, an RNA-binding protein, plays an important role in porcine induced pluripotent stem cells (piPSCs). However, the molecular mechanism underlying the function of LIN28A in the maintenance of pluripotency in piPSCs remains unclear. Here, we explored the function of LIN28A in piPSCs based on its overexpression and knockdown. We performed total RNA sequencing (RNA-seq) of piPSCs and detected the expression levels of relevant genes by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and immunofluorescence staining. Results indicated that piPSC proliferation ability decreased following LIN28A knockdown. Furthermore, when LIN28A expression in the shLIN28A2 group was lower (by 20%) than that in the negative control knockdown group (shNC), the pluripotency of piPSCs disappeared and they differentiated into neuroectoderm cells. Results also showed that LIN28A overexpression inhibited the expression of DUSP (dual-specificity phosphatases) family phosphatases and activated the mitogen-activated protein kinase (MAPK) signaling pathway. Thus, LIN28A appears to activate the MAPK signaling pathway to maintain the pluripotency and proliferation ability of piPSCs. Our study provides a new resource for exploring the functions of LIN28A in piPSCs.
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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