Journal of Molecular Medicine

, Volume 94, Issue 3, pp 291–300 | Cite as

TGF-β induces miR-30d down-regulation and podocyte injury through Smad2/3 and HDAC3-associated transcriptional repression

  • Lin Liu
  • Wenjun Lin
  • Qin Zhang
  • Wangsen CaoEmail author
  • Zhihong LiuEmail author
Original Article


The microRNA-30 family plays important roles in maintaining kidney homeostasis. Patients with focal segmental glomerulosclerosis (FSGS) have reduced miR-30 levels in glomerulus. TGF-β represses miR-30s in kidney podocytes, which leads to cytoskeleton damage and podocyte apoptosis. In this study, we investigated the mechanism by which TGF-β represses miR-30d in vitro. The human miR-30d promoter contains multiple copies of Smad binding element-like sequences. A fragment of 150 base pairs close to the transcription start site was negatively regulated by TGF-β to a similar extent as the 1.8 kb promoter, which was blocked by histone-deacetylase inhibition. TGF-β specifically enhanced HDAC3 expression. Knockdown of HDAC3 by shRNA or a selective inhibitor RGFP966 significantly relieved the repression of miR-30d mRNA and the promoter transcription. TGF-β promoted HDAC3 association with Smad2/3 and NCoR and caused their accumulation at the putative Smad binding site on the miR-30d promoter, which was prohibited by TSA or RGFP966. Furthermore, TSA or RGFP966 treatment reversed TGF-β-induced up-regulation of miR-30d targets Notch1 and p53 and alleviated the podocyte cytoskeleton damage and apoptosis. Taken together, these findings pinpoint that TGF-β represses miR-30d through a Smad2/3-HDAC3-NCoR repression complex and provide novel insights into a potential target for the treatment of podocyte injury-associated glomerulopathies.

Key message

  • MiR-30d promoter is negatively regulated by TGF-β.

  • TGF-β down-regulates miR-30 through Smad signaling pathway.

  • HDAC3 and NCoR are recruited by Smad2/3 to mediate miR-30d repression by TGF-β.

  • HDAC3 acts as a critical player in TGF-β-induced miR-30d repression and podocyte injuries.


TGF-β miR-30d HDAC3 Smad Podocyte Transcriptional repression 



This work was supported by research grants from National Basic Research Program of China 973 Program No.2012CB517606, National Nature Science Foundation of China (81271301 and 81470940), and the Major International (Regional) Joint Research Project (81320108007).

Conflict of interest

The authors declare that they have no competing interests.


  1. 1.
    John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS (2004) Human MicroRNA targets. PLoS Biol 2:e363CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Zhao JJ, Lin J, Zhu D, Wang X, Brooks D, Chen M, Chu ZB, Takada K, Ciccarelli B, Admin S et al (2014) miR-30-5p functions as a tumor suppressor and novel therapeutic tool by targeting the oncogenic Wnt/beta-catenin/BCL9 pathway. Cancer Res 74:1801–1813CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Kao CJ, Martiniez A, Shi XB, Yang J, Evans CP, Dobi A, deVere WRW, Kung HJ (2014) miR-30 as a tumor suppressor connects EGF/Src signal to ERG and EMT. Oncogene 33:2495–2503CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Zaragosi LE, Wdziekonski B, Brigand KL, Villageois P, Mari B, Waldmann R, Dani C, Barbry P (2011) Small RNA sequencing reveals miR-642a-3p as a novel adipocyte-specific microRNA and miR-30 as a key regulator of human adipogenesis. Genome Biol 12:R64CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Wu J, Zheng C, Fan Y, Zeng C, Chen Z, Qin W, Zhang C, Zhang W, Wang X, Zhu X et al (2014) Downregulation of microRNA-30 facilitates podocyte injury and is prevented by glucocorticoids. JASN 25:92–104CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Ketley A, Warren A, Holmes E, Gering M, Aboobaker AA, Brook JD (2013) The miR-30 microRNA family targets smoothened to regulate hedgehog signalling in zebrafish early muscle development. PLoS One 8:e65170CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Frederick JP, Liberati NT, Waddell DS, Shi Y, Wang XF (2004) Transforming growth factor beta-mediated transcriptional repression of c-myc is dependent on direct binding of Smad3 to a novel repressive Smad binding element. Mol Cell Biol 24:2546–2559CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Xi Q, Wang Z, Zaromytidou AI, Zhang XH, Chow-Tsang LF, Liu JX, Kim H, Barlas A, Manova-Todorova K, Kaartinen V et al (2011) A poised chromatin platform for TGF-beta access to master regulators. Cell 147:1511–1524CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Blahna MT, Hata A (2012) Smad-mediated regulation of microRNA biosynthesis. FEBS Lett 586:1906–1912CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Kato M, Putta S, Wang M, Yuan H, Lanting L, Nair I, Gunn A, Nakagawa Y, Shimano H, Todorov I et al (2009) TGF-beta activates Akt kinase through a microRNA-dependent amplifying circuit targeting PTEN. Nat Cell Biol 11:881–889CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Khan SN, Khan AU (2010) Role of histone acetylation in cell physiology and diseases: an update. Clin Chim Acta; Intl J Clin Chem 411:1401–1411CrossRefGoogle Scholar
  12. 12.
    Shahbazian MD, Grunstein M (2007) Functions of site-specific histone acetylation and deacetylation. Annu Rev Biochem 76:75–100CrossRefPubMedGoogle Scholar
  13. 13.
    Oberoi J, Fairall L, Watson PJ, Yang JC, Czimmerer Z, Kampmann T, Goult BT, Greenwood JA, Gooch JT, Kallenberger BC et al (2011) Structural basis for the assembly of the SMRT/NCoR core transcriptional repression machinery. Nat Struct Mol Biol 18:177–184CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Zhang S, Cai X, Huang F, Zhong W, Yu Z (2008) Effect of trichostatin a on viability and microRNA expression in human pancreatic cancer cell line BxPC-3. Exp Oncol 30:265–268PubMedGoogle Scholar
  15. 15.
    Scott GK, Mattie MD, Berger CE, Benz SC, Benz CC (2006) Rapid alteration of microRNA levels by histone deacetylase inhibition. Cancer Res 66:1277–1281CrossRefPubMedGoogle Scholar
  16. 16.
    Chen DQ, Pan BZ, Huang JY, Zhang K, Cui SY, De W, Wang R, Chen LB (2014) HDAC 1/4-mediated silencing of microRNA-200b promotes chemoresistance in human lung adenocarcinoma cells. Oncotarget 5:3333–3349CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Lodrini M, Oehme I, Schroeder C, Milde T, Schier MC, Kopp-Schneider A, Schulte JH, Fischer M, De Preter K, Pattyn F et al (2013) MYCN and HDAC2 cooperate to repress miR-183 signaling in neuroblastoma. Nucleic Acids Res 41:6018–6033CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Zhang X, Chen X, Lin J, Lwin T, Wright G, Moscinski LC, Dalton WS, Seto E, Wright K, Sotomayor E et al (2012) Myc represses miR-15a/miR-16-1 expression through recruitment of HDAC3 in mantle cell and other non-Hodgkin B-cell lymphomas. Oncogene 31:3002–3008CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Wu C, Jin B, Chen L, Zhuo D, Zhang Z, Gong K, Mao Z (2013) MiR-30d induces apoptosis and is regulated by the Akt/FOXO pathway in renal cell carcinoma. Cell Signal 25:1212–1221CrossRefPubMedGoogle Scholar
  20. 20.
    Saleem MA, O’Hare MJ, Reiser J, Coward RJ, Inward CD, Farren T, Xing CY, Ni L, Mathieson PW, Mundel P (2002) A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression. JASN 13:630–638PubMedGoogle Scholar
  21. 21.
    Yin SS, Cao WS (2015) Toll-like receptor signaling induces Nrf2 pathway activation through p62-Triggered Keap1 degradation. Mol Cell Biol 35:2673–2683CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Qi W, Chen X, Holian J, Mreich E, Twigg S, Gilbert RE, Pollock CA (2006) Transforming growth factor-beta1 differentially mediates fibronectin and inflammatory cytokine expression in kidney tubular cells. Am J Physiol Renal Physiol 291:F1070–1077CrossRefPubMedGoogle Scholar
  23. 23.
    Wotton D, Lo RS, Swaby LA, Massague J (1999) Multiple modes of repression by the Smad transcriptional corepressor TGIF. J Biol Chem 274:37105–37110CrossRefPubMedGoogle Scholar
  24. 24.
    Long J, Matsuura I, He D, Wang G, Shuai K, Liu F (2003) Repression of Smad transcriptional activity by PIASy, an inhibitor of activated STAT. Proc Natl Acad Sci U S A 100:9791–9796CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Yuan H, Reddy MA, Sun G, Lanting L, Wang M, Kato M, Natarajan R (2013) Involvement of p300/CBP and epigenetic histone acetylation in TGF-beta1-mediated gene transcription in mesangial cells. Am J Physiol Renal Physiol 304:F601–613CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Aarenstrup L, Flindt EN, Otkjaer K, Kirkegaard M, Andersen JS, Kristiansen K (2008) HDAC activity is required for p65/RelA-dependent repression of PPARdelta-mediated transactivation in human keratinocytes. J Invest Dermatol 128:1095–1106CrossRefPubMedGoogle Scholar
  27. 27.
    Sun Z, Feng D, Fang B, Mullican SE, You SH, Lim HW, Everett LJ, Nabel CS, Li Y, Selvakumaran V et al (2013) Deacetylase-independent function of HDAC3 in transcription and metabolism requires nuclear receptor corepressor. Mol Cell 52:769–782CrossRefPubMedGoogle Scholar
  28. 28.
    Bantscheff M, Hopf C, Savitski MM, Dittmann A, Grandi P, Michon AM, Schlegl J, Abraham Y, Becher I, Bergamini G et al (2011) Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes. Nat Biotechnol 29:255–265CrossRefPubMedGoogle Scholar
  29. 29.
    Ozsolak F, Poling LL, Wang Z, Liu H, Liu XS, Roeder RG, Zhang X, Song JS, Fisher DE (2008) Chromatin structure analyses identify miRNA promoters. Genes Dev 22:3172–3183CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Chung AC, Huang XR, Meng X, Lan HY (2010) miR-192 mediates TGF-beta/Smad3-driven renal fibrosis. J Am Soc Nephrol: JASN 21:1317–1325CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Long X, Miano JM (2011) Transforming growth factor-beta1 (TGF-beta1) utilizes distinct pathways for the transcriptional activation of microRNA 143/145 in human coronary artery smooth muscle cells. J Biol Chem 286:30119–30129CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Davis BN, Hilyard AC, Nguyen PH, Lagna G, Hata A (2010) Smad proteins bind a conserved RNA sequence to promote microRNA maturation by Drosha. Mol Cell 39:373–384CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Yu Y, Wang Y, Ren X, Tsuyada A, Li A, Liu LJ, Wang SE (2010) Context-dependent bidirectional regulation of the MutS homolog 2 by transforming growth factor beta contributes to chemoresistance in breast cancer cells. MCR 8:1633–1642CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Simonsson M, Kanduri M, Gronroos E, Heldin CH, Ericsson J (2006) The DNA binding activities of Smad2 and Smad3 are regulated by coactivator-mediated acetylation. J Biol Chem 281:39870–39880CrossRefPubMedGoogle Scholar
  35. 35.
    Chen CR, Kang Y, Siegel PM, Massague J (2002) E2F4/5 and p107 as Smad cofactors linking the TGFbeta receptor to c-myc repression. Cell 110:19–32CrossRefPubMedGoogle Scholar
  36. 36.
    Nomura T, Khan MM, Kaul SC, Dong HD, Wadhwa R, Colmenares C, Kohno I, Ishii S (1999) Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor. Genes Dev 13:412–423CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Chen S, El-Dahr SS (2013) Histone deacetylases in kidney development: implications for disease and therapy. Pediatr Nephrol 28:689–698CrossRefPubMedGoogle Scholar
  38. 38.
    Brilli LL, Swanhart LM, de Caestecker MP, Hukriede NA (2013) HDAC inhibitors in kidney development and disease. Pediatr Nephrol 28:1909–1921CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Noh H, Oh EY, Seo JY, Yu MR, Kim YO, Ha H, Lee HB (2009) Histone deacetylase-2 is a key regulator of diabetes- and transforming growth factor-beta1-induced renal injury. Am J Physiol Renal Physiol 297:F729–F739CrossRefPubMedGoogle Scholar
  40. 40.
    Wang X, Liu J, Zhen J, Zhang C, Wan Q, Liu G, Wei X, Zhang Y, Wang Z, Han H et al (2014) Histone deacetylase 4 selectively contributes to podocyte injury in diabetic nephropathy. Kidney Int 86:712–725CrossRefPubMedGoogle Scholar
  41. 41.
    Tabata T, Kokura K, Ten Dijke P, Ishii S (2009) Ski co-repressor complexes maintain the basal repressed state of the TGF-beta target gene, SMAD7, via HDAC3 and PRMT5. Genes Cells: Devoted Mol Cell Mech 14:17–28CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.National Clinical Research Center of Kidney Diseases, Jinling HospitalNanjing University School of MedicineNanjingChina
  2. 2.The Key lab of Jiangsu molecular MedicineNanjing University School of MedicineNanjingChina

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