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.
-
Key words:
- LIN28A /
- MAPK /
- Pluripotency /
- piPSCs /
- DUSP
-
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.
-
[1] Balzer E, Heine C, Jiang Q, Lee VM, Moss EG. 2010. LIN28 alters cell fate succession and acts independently of the let-7 microRNA during neurogliogenesis in vitro. Development, 137(6): 891−900. doi: 10.1242/dev.042895 [2] Buganim Y, Faddah DA, Cheng AW, Itskovich E, Markoulaki S, Ganz K, et al. 2012. Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase. Cell, 150(6): 1209−1222. doi: 10.1016/j.cell.2012.08.023 [3] Burdon T, Stracey C, Chambers I, Nichols J, Smith A. 1999. Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Developmental Biology, 210(1): 30−43. doi: 10.1006/dbio.1999.9265 [4] Caunt CJ, Keyse SM. 2013. Dual-specificity MAP kinase phosphatases (MKPs): shaping the outcome of MAP kinase signalling. FEBS Journal, 280(2): 489−504. doi: 10.1111/j.1742-4658.2012.08716.x [5] Chandrasekaran S, Zhang J, Sun Z, Zhang L, Ross CA, Huang YC, et al. 2017. Comprehensive mapping of pluripotent stem cell metabolism using dynamic genome-scale network modeling. Cell Reports, 21(10): 2965−2977. doi: 10.1016/j.celrep.2017.07.048 [6] Chen HF, Chuang HC, Tan TH. 2019. Regulation of dual-specificity phosphatase (DUSP) ubiquitination and protein stability. International Journal of Molecular Sciences, 20(11): 2668. doi: 10.3390/ijms20112668 [7] Chen HX, Guo RP, Zhang Q, Guo HC, Yang M, Wu ZF, et al. 2015. Erk signaling is indispensable for genomic stability and self-renewal of mouse embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America, 112(44): E5936−E5943. doi: 10.1073/pnas.1516319112 [8] Cheng D, Guo YJ, Li ZZ, Liu YJ, Gao X, Gao Y, et al. 2012. Porcine induced pluripotent stem cells require LIF and maintain their developmental potential in early stage of embryos. PLoS One, 7(12): e51778. doi: 10.1371/journal.pone.0051778 [9] Chomczynski P, Sacchi N. 2006. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nature Protocols, 1(2): 581−585. doi: 10.1038/nprot.2006.83 [10] Cornacchia D, Zhang C, Zimmer B, Chung SY, Fan YJ, Soliman MA, et al. 2019. Lipid deprivation induces a stable, naive-to-primed intermediate state of pluripotency in human PSCs. Cell Stem Cell, 25(1): 120−136. doi: 10.1016/j.stem.2019.05.001 [11] Deathridge J, Antolović V, Parsons M, Chubb JR. 2019. Live imaging of ERK signalling dynamics in differentiating mouse embryonic stem cells. Development, 146(12): dev172940. doi: 10.1242/dev.172940 [12] Esteban MA, Xu JY, Yang JY, Peng MX, Qin DJ, Li W, et al. 2009. Generation of induced pluripotent stem cell lines from tibetan miniature pig. Journal of Biological Chemistry, 284(26): 17634−17640. doi: 10.1074/jbc.M109.008938 [13] Ezashi T, Telugu BPVL, Alexenko AP, Sachdev S, Sinha S, Roberts RM. 2009. Derivation of induced pluripotent stem cells from pig somatic cells. Proceedings of the National Academy of Sciences of the United States of America, 106(27): 10993−10998. doi: 10.1073/pnas.0905284106 [14] Faunes F. 2020. Role and Regulation of Lin28 in Progenitor Cells During Central Nervous System Development. New York: Springer. [15] Gao XF, Nowak-Imialek M, Chen X, Chen DS, Herrmann D, Ruan DG, et al. 2019. Establishment of porcine and human expanded potential stem cells. Nature Cell Biology, 21(6): 687−699. doi: 10.1038/s41556-019-0333-2 [16] Gao Y, Li H, Han Q, Li Y, Wang TX, Huang CY, et al. 2020. Overexpression of DUSP6 enhances chemotherapy-resistance of ovarian epithelial cancer by regulating the ERK signaling pathway. Journal of Cancer, 11(11): 3151−3164. doi: 10.7150/jca.37267 [17] Hackett JA, Surani MA. 2014. Regulatory principles of pluripotency: from the ground state up. Cell Stem Cell, 15(4): 416−430. doi: 10.1016/j.stem.2014.09.015 [18] Hafner M, Max KEA, Bandaru P, Morozov P, Gerstberger S, Brown M, et al. 2013. Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition. RNA, 19(5): 613−626. doi: 10.1261/rna.036491.112 [19] Haraguchi S, Kikuchi K, Nakai M, Tokunaga T. 2012. Establishment of self-renewing porcine embryonic stem cell-like cells by signal inhibition. Journal of Reproduction and Development, 58(6): 707−716. doi: 10.1262/jrd.2012-008 [20] Hiratsuka T, Bordeu I, Pruessner G, Watt FM. 2020. Regulation of ERK basal and pulsatile activity control proliferation and exit from the stem cell compartment in mammalian epidermis. Proceedings of the National Academy of Sciences of the United States of America, 117(30): 17796−17807. doi: 10.1073/pnas.2006965117 [21] Hou DR, Jin Y, Nie XW, Zhang ML, Ta N, Zhao LH, et al. 2016. Derivation of porcine embryonic stem-like cells from in vitro-produced blastocyst-stage embryos. Scientific Reports, 6: 25838. doi: 10.1038/srep25838 [22] Hu X, Wang M, Cao L, Cong L, Gao YJ, Lu JW, et al. 2020. MiR-4319 suppresses the growth of esophageal squamous cell carcinoma via targeting NLRC5. Current Molecular Pharmacology, 13(2): 144−149. doi: 10.2174/1874467212666191119094636 [23] Kalkan T, Bornelov S, Mulas C, Diamanti E, Lohoff T, Ralser M, et al. 2019. Complementary activity of ETV5, RBPJ, and TCF3 drives formative transition from naive pluripotency. Cell Stem Cell, 24(5): 785−801. doi: 10.1016/j.stem.2019.03.017 [24] Kobayashi T, Kozlova A. 2018. Lin28a overexpression reveals the role of Erk signaling in articular cartilage development. Development, 145(15): dev162594. doi: 10.1242/dev.162594 [25] Kumar RM, Cahan P, Shalek AK, Satija R, Daleykeyser AJ, Li H, et al. 2014. Deconstructing transcriptional heterogeneity in pluripotent stem cells. Nature, 516(7529): 56−61. doi: 10.1038/nature13920 [26] Liao J, Wu Z, Wang Y, Cheng L, Cui C, Gao Y, et al. 2008. Enhanced efficiency of generating induced pluripotent stem (iPS) cells from human somatic cells by a combination of six transcription factors. Cell Research, 18(5): 600−603. doi: 10.1038/cr.2008.51 [27] Liu MQ, Qin Y, Hu QS, Liu WS, Ji SR, Xu WY, et al. 2021. SETD8 potentiates constitutive ERK1/2 activation via epigenetically silencing DUSP10 expression in pancreatic cancer. Cancer Letters, 499: 265−278. doi: 10.1016/j.canlet.2020.11.023 [28] Ma FL, Du XM, Wei YD, Zhou Z, Clotaire DZJ, Li N, et al. 2019. LIN28A activates the transcription of NANOG in dairy goat male germline stem cells. Journal of Cellular Physiology, 234(6): 8113−8121. doi: 10.1002/jcp.27593 [29] Ma YY, Yu T, Cai YX, Wang HY. 2018. Preserving self-renewal of porcine pluripotent stem cells in serum-free 3i culture condition and independent of LIF and b-FGF cytokines. Cell Death Discovery, 4: 21. [30] Marks H, Kalkan T, Menafra R, Denissov S, Jones K, Hofemeister H, et al. 2012. The transcriptional and epigenomic foundations of ground state pluripotency. Cell, 149(3): 590−604. doi: 10.1016/j.cell.2012.03.026 [31] Moss EG, Lee RC, Ambros V. 1997. The cold shock domain protein LIN-28 controls developmental timing in C. elegans and is regulated by the lin-4 RNA. Cell, 88(5): 637−646. doi: 10.1016/S0092-8674(00)81906-6 [32] Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, et al. 2001. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocrine Reviews, 22(2): 153−183. [33] Polesskaya A, Cuvellier S, Naguibneva I, Duquet A, Moss EG, Harel-Bellan A. 2007. Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis by increasing translation efficiency. Genes & Development, 21(9): 1125−1138. [34] Richards M, Tan SP, Tan JH, Chan WK, Bongso A. 2004. The transcriptome profile of human embryonic stem cells as defined by SAGE. Stem Cells, 22(1): 51−64. doi: 10.1634/stemcells.22-1-51 [35] Romer-Seibert JS, Hartman NW, Moss EG. 2019. The RNA-binding protein LIN28 controls progenitor and neuronal cell fate during postnatal neurogenesis. FASEB Journal, 33(3): 3291−3303. doi: 10.1096/fj.201801118R [36] Sang H, Wang D, Zhao S, Zhang JX, Zhang Y, Xu J, et al. 2019. Dppa3 is critical for Lin28a-regulated ES cells naïve-primed state conversion. Journal of Molecular Cell Biology, 11(6): 474−488. doi: 10.1093/jmcb/mjy069 [37] Shaul YD, Seger R. 2007. The MEK/ERK cascade: from signaling specificity to diverse functions. Biochimica Et Biophysica Acta (BBA)-Molecular Cell Research, 1773(8): 1213−1226. doi: 10.1016/j.bbamcr.2006.10.005 [38] Shyh-Chang N, Daley GQ. 2013. Lin28: primal regulator of growth and metabolism in stem cells. Cell Stem Cell, 12(4): 395−406. doi: 10.1016/j.stem.2013.03.005 [39] Tsanov KM, Daley GQ. 2017. Signaling through RNA-binding proteins as a cell fate regulatory mechanism. Cell Cycle, 16(8): 723−724. doi: 10.1080/15384101.2017.1302205 [40] Tsanov KM, Pearson DS, Wu ZT, Han A, Triboulet R, Seligson MT, et al. 2017. LIN28 phosphorylation by MAPK/ERK couples signalling to the post-transcriptional control of pluripotency. Nature Cell Biology, 19(1): 60−67. doi: 10.1038/ncb3453 [41] Wei YD, Du XM, Yang DH, Ma FL, Yu XW, Zhang MF, et al. 2021. Dmrt1 regulates the immune response by repressing the TLR4 signaling pathway in goat male germline stem cells. Zoological Research, 42(1): 14−27. [42] Xu BS, Zhang KX, Huang YQ. 2009. Lin28 modulates cell growth and associates with a subset of cell cycle regulator mRNAs in mouse embryonic stem cells. RNA, 15(3): 357−361. doi: 10.1261/rna.1368009 [43] Xu JJ, Zheng Z, Du XG, Shi BB, Wang JC, Gao DF, et al. 2020. A cytokine screen using CRISPR-Cas9 knock-in reporter pig iPS cells reveals that Activin A regulates NANOG. Stem Cell Research & Therapy, 11(1): 67. [44] Xue BH, Li Y, He YL, Wei RY, Sun RZ, Yin Z, et al. 2016. Porcine pluripotent stem cells derived from IVF embryos contribute to chimeric development in vivo. PLoS One, 11(3): e0151737. doi: 10.1371/journal.pone.0151737 [45] Yang DH, Moss EG. 2003. Temporally regulated expression of Lin-28 in diverse tissues of the developing mouse. Gene Expression Patterns, 3(6): 719−726. doi: 10.1016/S1567-133X(03)00140-6 [46] Yermalovich AV, Osborne JK, Sousa P, Han A, Kinney MA, Chen MJ, et al. 2020. Author correction: Lin28 and let-7 regulate the timing of cessation of murine nephrogenesis. Nature Communications, 11(1): 1327. doi: 10.1038/s41467-020-14944-3 [47] Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, et al. 2008. The ground state of embryonic stem cell self-renewal. Nature, 453(7194): 519−523. doi: 10.1038/nature06968 [48] Yu JY, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian SL, et al. 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science, 318(5858): 1917−1920. doi: 10.1126/science.1151526 [49] Zhang J, Ratanasirintrawoot S, Chandrasekaran S, Wu ZT, Ficarro SB, Yu CX, et al. 2016. LIN28 regulates stem cell metabolism and conversion to primed pluripotency. Cell Stem Cell, 19(1): 66−80. doi: 10.1016/j.stem.2016.05.009 [50] Zhang SQ, Guo YJ, Cui Y, Liu YJ, Yu T, Wang HY. 2015. Generation of intermediate porcine iPS cells under culture condition favorable for mesenchymal-to-epithelial transition. Stem Cell Reviews and Reports, 11(1): 24−38. doi: 10.1007/s12015-014-9552-x [51] Zhang SQ, Xie YL, Cao HX, Wang HY. 2017. Common microRNA-mRNA interactions exist among distinct porcine iPSC lines independent of their metastable pluripotent states. Cell Death & Disease, 8(8): e3027. [52] Zhu H, Shyh-Chang N, Segrè AV, Shinoda G, Shah SP, Einhorn WS, et al. 2011. The Lin28/let-7 axis regulates glucose metabolism. Cell, 147(1): 81−94. doi: 10.1016/j.cell.2011.08.033 [53] Zhu ZS, Pan Q, Zhao WX, Wu XL, Yu S, Shen QY, et al. 2021. BCL2 enhances survival of porcine pluripotent stem cells through promoting FGFR2. Cell Proliferation, 54(1): e12932. [54] Zhu ZS, Wu XL, Li Q, Zhang JQ, Yu S, Shen QY, et al. 2021. Histone demethylase complexes KDM3A and KDM3B cooperate with OCT4/SOX2 to define a pluripotency gene regulatory network. FASEB Journal. doi: 10.1096/fj.202100230R. -
下载:
点击查看大图
计量
- 文章访问数: 1507
- HTML全文浏览量: 642
- PDF下载量: 178
- 被引次数: 0
