Tumor Biology

, Volume 37, Issue 10, pp 13155–13166 | Cite as

Downregulation of HMGB1 by miR-34a is sufficient to suppress proliferation, migration and invasion of human cervical and colorectal cancer cells

  • Karthik Subramanian Chandrasekaran
  • Anusha Sathyanarayanan
  • Devarajan KarunagaranEmail author
Original Article


High mobility group box 1 (HMGB1) is a ubiquitous nuclear protein known to be highly expressed in human cervical (CaCx) and colorectal (CRC) cancers, and sustained high levels of HMGB1 contribute to tumourigenesis and metastasis. HMGB1-targeted cancer therapy is of recent interest, and there are not many studies on miRNA-mediated HMGB1 regulation in these cancers. Since miRNA-based therapeutics for cancer is gaining importance in recent years, it was of interest to predict miRNAs targeting HMGB1. Based on the identification of a potential miR-34a response element in HMGB1–3′ untranslated region (3′UTR) and an inverse correlation between HMGB1 and miR-34a expression levels in CaCx and CRC tissues, from a subset of the local population as well as a large sampling from TCGA database, experiments were performed to validate HMGB1 as a direct target of miR-34a in CaCx and CRC cells. Ectopic expression of miR-34a decreased the wild-type HMGB1–3′UTR luciferase activity but not that of its mutant in 3′UTR luciferase assays. While forced expression of miR-34a in CaCx and CRC cells inhibited HMGB1 mRNA and protein levels, proliferation, migration and invasion, inhibition of endogenous miR-34a enhanced these tumourigenic properties. siRNA-mediated HMGB1 suppression imitated miR-34a expression in reducing proliferation and metastasis-related events. Combined with the disparity in expression of miR-34a and HMGB1 in clinical specimens, the current findings would help in not only understanding the complexity of miRNA-target regulatory mechanisms but also in designing novel therapeutic interventions in CaCx and CRC.


microRNA-34a HMGB1 Human cervical cancer Human colorectal cancer Tumourigenicity 



This work was supported by an exploratory research grant (to D.K.) and a project associateship (to K.S.C.) from the Centre for Industrial Consultancy and Sponsored Research, Indian Institute of Technology Madras and a senior research fellowship (to A.S.) from the Council of Scientific and Industrial Research, Government of India. The authors would like to thank Dr. Prabhavathy Devan, Indian Institute of Technology Madras, Dr. Radha Bai Prabhu, Institute of Obstetrics and Gynaecology and Government Hospital for Women and Children, Government of India and Dr. Shankar Srinivasan, Consultant Medical Oncologist, Apollo Specialty Hospitals, Chennai, India, for their help in procurement of clinical specimens and Dr. Rao Srinivasa Rao, Nuffield Department of Surgical Sciences, University of Oxford, Oxford, for his help in analysing data from TCGA. TCGA Research Network ( is acknowledged for providing access to their data. The following were kind gifts: C33A, SiHa, SW480 and SW620 cells from Dr. Ygal Haupt, Peter MacCallum Cancer Centre, Victoria, Australia; HCT116WT cells from Dr. Bert Vogelstein, Sidney Kimmel Comprehensive Cancer Center, Baltimore, USA and CaSki cells from Dr. Sudhir Krishna, National Centre for Biological Sciences, Bangalore, India.

Compliance with ethical standards

Conflicts of interest


Ethical approval

Informed consent was obtained from all individuals included in this study.


  1. 1.
    American Cancer Society. Cancer Facts & Figures. Cancer Facts Fig. 2015. 2015;Google Scholar
  2. 2.
    Moghimi-Dehkordi B. An overview of colorectal cancer survival rates and prognosis in Asia. World J. Gastrointest Oncol. 2012;4:71.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Hawes SE, Kiviat NB. Are genital infections and inflammation cofactors in the pathogenesis of invasive cervical cancer? J Natl Cancer Inst. 2002;94:1592–3.CrossRefPubMedGoogle Scholar
  4. 4.
    Haggar FA, Boushey RP. Colorectal cancer epidemiology: incidence, mortality, survival, and risk factors. Clin Colon Rectal Surg. 2009;22:191–7.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Zur Hausen H. Papillomavirus infections — a major cause of human cancers. Biochim Biophys Acta - Rev Cancer. 1996;1288:F55–78.CrossRefGoogle Scholar
  6. 6.
    Mohandas KM. Colorectal cancer in India: controversies, enigmas and primary prevention. Indian J Gastroenterol. 2011;30:3–6.CrossRefPubMedGoogle Scholar
  7. 7.
    Davalos AR, Kawahara M, Malhotra GK, Schaum N, Huang J, Ved U, et al. p53-dependent release of alarmin HMGB1 is a central mediator of senescent phenotypes. J Cell Biol. 2013;201:613–29.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Grosschedl R, Giese K, Pagel J. HMG domain proteins: architectural elements in the assembly of nucleoprotein structures. Trends Genet. 1994;10:94–100.CrossRefPubMedGoogle Scholar
  9. 9.
    Goodwin GH, Sanders C, Johns EWA. New Group of Chromatin-Associated Proteins with a high content of acidic and basic amino acids. Eur J Biochem. 1973;38:14–9.CrossRefPubMedGoogle Scholar
  10. 10.
    McKinney K, Prives C. Efficient specific DNA binding by p53 requires both its central and C-terminal domains as revealed by studies with high-mobility group 1 protein. Mol Cell Biol. 2002;22:6797–808.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Brickman JM, Adam M, Ptashne M. Interactions between an HMG-1 protein and members of the Rel family. Proc Natl Acad Sci U S A. 1999;96:10679–83.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Ranzato E, Martinotti S, Patrone M. Emerging roles for HMGB1 protein in immunity, inflammation, and cancer. ImmunoTargets Ther. 2015;4:101.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Harris HE, Andersson U, Pisetsky DS. HMGB1: a multifunctional alarmin driving autoimmune and inflammatory disease. Nat Rev Rheumatol. 2012;8:195–202.CrossRefPubMedGoogle Scholar
  14. 14.
    Andersson U, Tracey KJ. HMGB1 is a therapeutic target for sterile inflammation and infection. Annu Rev Immunol. 2011;29:139–62.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Xu Y, Chen Z, Zhang G, Xi Y, Sun R, Chai F, et al. HMGB1 overexpression correlates with poor prognosis in early-stage squamous cervical cancer. Tumor Biol. 2015;36:9039–47.CrossRefGoogle Scholar
  16. 16.
    Pang X, Zhang Y, Wei H, Zhang J, Luo Q, Huang C, et al. Expression and effects of high-mobility group box 1 in cervical cancer. Int J Mol Sci. 2014;15:8699–712.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Choi YR, Kim H, Kang HJ, Kim N-G, Kim JJ, Park K-S, et al. Overexpression of high mobility group box 1 in gastrointestinal stromal tumors with KIT mutation. Cancer Res. 2003;63:2188–93.PubMedGoogle Scholar
  18. 18.
    Sezer C. The role of high mobility group box 1 (HMGB1) in colorectal cancer. Med Sci Monit. 2014;20:530–7.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Volp K, Brezniceanu ML, Bosser S, Brabletz T, Kirchner T, Gottel D, et al. Increased expression of high mobility group box 1 (HMGB1) is associated with an elevated level of the antiapoptotic c-IAP2 protein in human colon carcinomas. Gut. 2006;55:234–42.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Guo ZS, Liu Z, Bartlett DL, Tang D, Lotze MT. Life after death: targeting high mobility group box 1 in emergent cancer therapies. Am J Cancer Res. 2013;3:1–20.PubMedPubMedCentralGoogle Scholar
  21. 21.
    Tang D, Kang R, Zeh HJ, Lotze MT. High-mobility group box 1 and cancer. Biochim Biophys Acta. 2010;1799:131–40.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E, et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A. 2002;99:15524–9.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Volinia S, Calin GA, Liu C-G, Ambs S, Cimmino A, Petrocca F, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A. 2006;103:2257–61.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Pereira PM, Marques JP, Soares AR, Carreto L, Santos M. A S. Microrna expression variability in human cervical tissues. PLoS One. 2010;5:e11780.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Hu X, Schwarz JK, Lewis JS, Huettner PC, Rader JS, Deasy JO, et al. A microRNA expression signature for cervical cancer prognosis. Cancer Res. 2010;70:1441–8.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Hur K, Toiyama Y, Schetter AJ, Okugawa Y, Harris CC, Boland CR, et al. Identification of a metastasis-specific MicroRNA signature in human colorectal cancer. JCNI. 2015;107:1–11.Google Scholar
  27. 27.
    Chen X, Shi K, Wang Y, Song M, Zhou W. Clinical value of integrated-signature miRNAs in colorectal cancer : miRNA expression profiling analysis and experimental validation. 2015;6.Google Scholar
  28. 28.
    Hu Y, Chen H-Y, C-Y Y, Xu J, Wang J-L, Qian J, et al. A long non-coding RNA signature to improve prognosis prediction of colorectal cancer. Oncotarget. 2014;5:2230–42.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    He L, He X, Lim LP, de Stanchina E, Xuan Z, Liang Y, et al. A microRNA component of the p53 tumour suppressor network. Nature. 2007;447:1130–4.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Li B, Hu Y, Ye F, Li Y, Lv W, Xie X. Reduced miR-34a expression in normal cervical tissues and cervical lesions with high-risk human papillomavirus infection. Int J Gynecol Cancer. 2010;20:597–604.CrossRefPubMedGoogle Scholar
  31. 31.
    Aherne ST, Madden SF, Hughes DJ, Pardini B, Naccarati A, Levy M, et al. Circulating miRNAs miR-34a and miR-150 associated with colorectal cancer progression. BMC Cancer. 2015;15:1–13.CrossRefGoogle Scholar
  32. 32.
    Gao J, Li N, Dong Y, Li S, Xu L, Li X, et al. miR-34a-5p suppresses colorectal cancer metastasis and predicts recurrence in patients with stage II/III colorectal cancer. Oncogene. 2015;34:4142–52.CrossRefPubMedGoogle Scholar
  33. 33.
    Tazawa H, Tsuchiya N, Izumiya M, Nakagama H. Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells. Proc Natl Acad Sci U S A. 2007;104:15472–7.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Geng D, Song X, Ning F, Song Q, Yin H. MiR-34a inhibits viability and invasion of human papillomavirus-positive cervical cancer cells by targeting E2F3 and regulating Survivin. Int J Gynecol Cancer. 2015;25:707–13.CrossRefPubMedGoogle Scholar
  35. 35.
    Pang RTK, Leung CON, Ye TM, Liu W, Chiu PCN, Lam KKW, et al. MicroRNA-34a suppresses invasion through downregulation of Notch1 and Jagged1 in cervical carcinoma and choriocarcinoma cells. Carcinogenesis. 2010;31:1037–44.CrossRefPubMedGoogle Scholar
  36. 36.
    Beg M, Brenner A, Sachdev J, Borad M, Cortes J, Tibes R, et al. 4LBA a phase 1 study of first-in-class microRNA-34 mimic, MRX34, in patients with hepatocellular carcinoma or advanced cancer with liver metastasis. Eur J Cancer. 2014;50:196.CrossRefGoogle Scholar
  37. 37.
    Yao S, Zhao T, Jin H. Expression of MicroRNA-325-3p and its potential functions by targeting HMGB1 in non-small cell lung cancer. Biomed Pharmacother. 2015;70:72–9.CrossRefPubMedGoogle Scholar
  38. 38.
    Li X, Wang S, Chen Y, Liu G, Yang X. miR-22 targets the 3′ UTR of HMGB1 and inhibits the HMGB1-associated autophagy in osteosarcoma cells during chemotherapy. Tumor Biol. 2014;35:6021–8.CrossRefGoogle Scholar
  39. 39.
    Guo S, Bai R, Liu W, Zhao A, Zhao Z, Wang Y, et al. miR-22 inhibits osteosarcoma cell proliferation and migration by targeting HMGB1 and inhibiting HMGB1-mediated autophagy. Tumor Biol. 2014;35:7025–34.CrossRefGoogle Scholar
  40. 40.
    Dormoy-Raclet V, Cammas A, Celona B, Lian XJ, van der Giessen K, Zivojnovic M, et al. HuR and miR-1192 regulate myogenesis by modulating the translation of HMGB1 mRNA. Nat Commun. 2013;4:2388.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Dahlhaus M, Schult C, Lange S, Freund M, Junghanss C. MicroRNA 181a influences the expression of HMGB1 and CD4 in acute Leukemias. Anticancer Res. 2013;33:445–52.PubMedGoogle Scholar
  42. 42.
    Saito K, Oku T, Ata N, Miyashiro H, Hattori M, Saiki I. A modified and convenient method for assessing tumor cell invasion and migration and its application to screening for inhibitors. Biol Pharm Bull. 1997;20:345–8.CrossRefPubMedGoogle Scholar
  43. 43.
    Suzuki HI, Katsura A, Matsuyama H, Miyazono K. MicroRNA regulons in tumor microenvironment. Oncogene. 2014;34:3085–94.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Chou J, Shahi P, Werb Z. microRNA-mediated regulation of the tumor microenvironment. Cell Cycle. 2013;12:3262–71.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Kuninty PR, Schnittert J, Storm G, Prakash J. MicroRNA targeting to modulate tumor microenvironment. Front Oncologia. 2016;6:3.Google Scholar
  46. 46.
    Esquela-Kerscher A, Slack FJ. Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer. 2006;6:259–69.CrossRefPubMedGoogle Scholar
  47. 47.
    Savita U, Karunagaran D. MicroRNA-106b-25 cluster targets β-TRCP2, increases the expression of snail and enhances cell migration and invasion in H1299 (non small cell lung cancer) cells. Biochem Biophys Res Commun. 2013;434:841–7.CrossRefPubMedGoogle Scholar
  48. 48.
    Subramanian M, Rao SR, Thacker P, Chatterjee S, Karunagaran D. MiR-29b downregulates canonical Wnt signaling by suppressing coactivators of β-catenin in human colorectal cancer cells. J Cell Biochem. 2014;115:1974–84.PubMedGoogle Scholar
  49. 49.
    Bommer GT, Gerin I, Feng Y, Kaczorowski AJ, Kuick R, Love RE, et al. p53-mediated activation of miRNA34 candidate tumor-suppressor genes. Curr Biol. 2007;17:1298–307.CrossRefPubMedGoogle Scholar
  50. 50.
    Misso G, Di Martino MT, De Rosa G, Farooqi AA, Lombardi A, Campani V, et al. Mir-34: a new weapon against cancer? Mol Ther Nucleic Acids. 2014;3:e194.CrossRefPubMedGoogle Scholar
  51. 51.
    Yunqing L, Guessous F, Ying Z, DiPierro C, Kefas B, Johnson E, et al. MicroRNA-34a inhibits glioblastoma growth by targeting multiple oncogenes. Cancer Res. 2009;69:7569–76.CrossRefGoogle Scholar
  52. 52.
    Yan K, Gao J, Yang T, Ma Q, Qiu X, Fan Q, et al. MicroRNA-34a inhibits the proliferation and metastasis of osteosarcoma cells both in vitro and in vivo. PLoS One. 2012;7:e33778.CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Li N, Fu H, Tie Y, Hu Z, Kong W, Wu Y, et al. miR-34a inhibits migration and invasion by down-regulation of c-met expression in human hepatocellular carcinoma cells. Cancer Lett. 2009;275:44–53.CrossRefPubMedGoogle Scholar
  54. 54.
    Yamamura S, Saini S, Majid S, Hirata H, Ueno K, Chang I, et al. Microrna-34a suppresses malignant transformation by targeting c-myc transcriptional complexes in human renal cell carcinoma. Carcinogenesis. 2012;33:294–300.CrossRefPubMedGoogle Scholar
  55. 55.
    Gnanasekar M, Thirugnanam S, Ramaswamy K. Short hairpin RNA (shRNA) constructs targeting high mobility group box-1 (HMGB1) expression leads to inhibition of prostate cancer cell survival and apoptosis. Int J Oncol. 2009;34:425–31.PubMedGoogle Scholar
  56. 56.
    Chen RC, Yi PP, Zhou RR, Xiao MF, Huang ZB, Tang DL, et al. The role of HMGB1-RAGE axis in migration and invasion of hepatocellular carcinoma cell lines. Mol Cell Biochem. 2014;390:271–80.CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Liu K, Huang J, Xie M, Yu Y, Zhu S, Kang R, et al. MIR34A regulates autophagy and apoptosis by targeting HMGB1 in the retinoblastoma cell. Autophagy. 2014;10:442–52.CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Ribeiro J, Marinho-Dias J, Monteiro P, Loureiro J, Baldaque I, Medeiros R, et al. miR-34a and miR-125b expression in HPV infection and cervical cancer development. Biomed Res Int 2015;2015:1–6.Google Scholar
  59. 59.
    Zhang JG, Wang JJ, Zhao F, Liu Q, Jiang K, Yang GH. MicroRNA-21 (miR-21) represses tumor suppressor PTEN and promotes growth and invasion in non-small cell lung cancer (NSCLC). Clin Chim Acta. 2010;411:846–52.CrossRefPubMedGoogle Scholar
  60. 60.
    Xu LF, Wu ZP, Chen Y, Zhu QS, Hamidi S, Navab R. MicroRNA-21 (miR-21) regulates cellular proliferation, invasion, migration, and apoptosis by targeting PTEN, RECK and Bcl-2 in lung squamous carcinoma, Gejiu City, China. PLoS One. 2014;9.Google Scholar
  61. 61.
    Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian MicroRNA targets. Cell. 2003;115:787–98.CrossRefPubMedGoogle Scholar
  62. 62.
    Krek A, Grün D, Poy MN, Wolf R, Rosenberg L, Epstein EJ, et al. Combinatorial microRNA target predictions. Nat Genet. 2005;37:495–500.CrossRefPubMedGoogle Scholar
  63. 63.
    Gebhardt C, Riehl A, Durchdewald M, Németh J, Fürstenberger G, Müller-Decker K, et al. RAGE signaling sustains inflammation and promotes tumor development. J Exp Med. 2008;205:275–85.CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Kusume A, Sasahira T, Luo Y, Isobe M, Nakagawa N, Tatsumoto N, et al. Suppression of dendritic cells by HMGB1 is associated with lymph node metastasis of human colon cancer. Pathobiology. 2009;76:155–62.CrossRefPubMedGoogle Scholar
  65. 65.
    van Beijnum JR, Nowak-Sliwinska P, van den Boezem E, Hautvast P, Buurman WA, Griffioen AW. Tumor angiogenesis is enforced by autocrine regulation of high-mobility group box 1. Oncogene. 2012;32:363–74.CrossRefPubMedGoogle Scholar
  66. 66.
    Michaltros, Ozaki T, Bačíková A, Kageyama H, Nakagawara A. HMGB1 and HMGB2 cell-specifically down-regulate the p53- and p73-dependent sequence-specific transactivation from the human Bax gene promoter. J Biol Chem. 2002;277:7157–64.CrossRefGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  1. 1.Department of Biotechnology, Bhupat and Jyoti Mehta School of BiosciencesIndian Institute of Technology MadrasChennaiIndia

Personalised recommendations