Tumor Biology

, Volume 37, Issue 2, pp 2425–2433 | Cite as

miR-16 induction after CDK4 knockdown is mediated by c-Myc suppression and inhibits cell growth as well as sensitizes nasopharyngeal carcinoma cells to chemotherapy

  • Qingping Jiang
  • Yajie Zhang
  • Mengyang Zhao
  • Qiulian Li
  • Ruichao Chen
  • Xiaobing Long
  • Weiyi Fang
  • Zhen Liu
Original Article


Cyclin-dependent kinase 4 (CDK4) is a member of cyclin-dependent kinase family which regulates G1 to S cell cycle transition. CDK4 activity is increased in many tumor types. Here, we report a negative automodulatory feedback loop between CDK4 and miR-16 that regulates cell cycle progression in nasopharyngeal carcinoma (NPC). By miRNA array and real-time PCR, we identified upregulation of tumor suppressor miR-16a, which inhibited cell cycle progression and sensitized NPC cells to chemotherapy. CDK4 knockdown reduced the expression of c-Myc, the latter of which directly suppresses the miR-16 expression by directly binding to the miR-16 promoter. Moreover, we found that miR-16 upregulation could reduce CDK4 expression by repressing CCND1 and thus forms a feedback loop via the CDK4/c-Myc/miR-16/CCND1 pathway. Finally, miR-16 was negatively correlated with CDK4 expression in NPC biopsies. In summary, our results define a double-negative feedback loop involving CDK4 and miR-16 mediated by c-Myc that modulates NPC cell growth and chemotherapy sensitivity.


NPC CDK4 miR-16 c-Myc Feedback loop 



This study was supported by the National Nature Science Fund of China (No. 30971438, 81102061), the New Star Plan of Pearl River Science and Technology from Guangzhou City (No. 2011J2200009), Guangdong Science and Technology Planning Project (No. 2014A020212342), Yangcheng Scholar Research Projects from Universities of Guangzhou (No. 12A011D), The Oustanding Young Teacher Training Project of Collegues and Universities in GuangDong Province (No.Yp2013136), Guangzhou Science and Technology Planning Project (No. 2013J4100035), and the Ph.D. Programs Foundation of Ministry of Education of China (No. 20134423110001)

Conflicts of interest


Authors’ contributions

QJ, YZ, MZ, and XJL coordinated and performed the studies as well as assisted in the editing of manuscript. QL and RC collected the tissue samples. WF and ZL designed this study and wrote this paper.

Supplementary material

13277_2015_3966_Fig8_ESM.gif (9 kb)
Figure S1

The interference efficiency of siCDK4s in mRNA level was examined by qPCR in NPC cells (GIF 9 kb)

13277_2015_3966_MOESM1_ESM.tif (700 kb)
High resolution image (TIFF 699 kb)


  1. 1.
    Poomsawat S, Buajeeb W, Khovidhunkit SO, Punyasingh J. Alteration in the expression of cdk4 and cdk6 proteins in oral cancer and premalignant lesions. J Oral Pathol Med. 2010;39:793–9.CrossRefPubMedGoogle Scholar
  2. 2.
    Lindberg D, Hessman O, Akerström G, Westin G. Cyclin-dependent kinase 4 (CDK4) expression in pancreatic endocrine tumors. Neuroendocrinology. 2007;86:112–8.CrossRefPubMedGoogle Scholar
  3. 3.
    Wikman H, Nymark P, Väyrynen A, Jarmalaite S, Kallioniemi A, Salmenkivi K, et al. CDK4 is a probable target gene in a novel amplicon at 12q13.3-q14.1 in lung cancer. Gene Chromosome Cancer. 2005;42:193–9.CrossRefGoogle Scholar
  4. 4.
    Dobashi Y, Goto A, Fukayama M, Abe A, Ooi A. Overexpression of cdk4/cyclin D1, a possible mediator of apoptosis and an indicator of prognosis in human primary lung carcinoma. Int J Cancer. 2004;110:532–41.CrossRefPubMedGoogle Scholar
  5. 5.
    Fang W, Li X, Jiang Q, Liu Z, Yang H, Wang S, et al. Transcriptional patterns, biomarkers and pathways characterizing nasopharyngeal carcinoma of Southern China. J Transl Med. 2008;6:32.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Jiang Q, Mai C, Yang H, Wu Q, Hua S, Yan C, et al. Nuclear expression of CDK4 correlates with disease progression and poor prognosis in human nasopharyngeal carcinoma. Histopathology. 2014;64(5):722–30.CrossRefPubMedGoogle Scholar
  7. 7.
    Liu Z, Long X, Chao C, Yan C, Wu Q, Hua S, et al. Knocking down CDK4 mediates the elevation of let-7c suppressing cell growth in nasopharyngeal carcinoma. BMC Cancer. 2014;14:274.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    O’Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc regulated microRNAs modulate E2F1 expression. Nature. 2005;435:839–43.CrossRefPubMedGoogle Scholar
  9. 9.
    Chang TC et al. Widespread microRNA repression by Myc contributes to tumorigenesis. Nat Genet. 2008;40:43–50.CrossRefPubMedGoogle Scholar
  10. 10.
    Yu X, Zhen Y, Yang H, Wang H, Zhou Y, Wang E, et al. Loss of connective tissue growth factor as an unfavorable prognosis factor activates miR-18b by PI3K/AKT/C-Jun and C-Myc and promotes cell growth in nasopharyngeal carcinoma. Cell Death Dis. 2013;4:e634.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Zhen Y, Liu Z, Yang H, Yu X, Wu Q, Hua S, et al. Tumor suppressor PDCD4 modulates miR-184-mediated direct suppression of C-MYC and BCL2 blocking cell growth and survival in nasopharyngeal carcinoma. Cell Death Dis. 2013;4:e872.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Zhang X, Chen X, Lin J, Lwin T, Wright G, Moscinski LC, et al. Myc represses miR-15a/miR-16-1 expression through recruitment of HDAC3 in mantle cell and other non-Hodgkin B-cell lymphomas. Oncogene. 2012;31(24):3002–8.CrossRefPubMedGoogle Scholar
  13. 13.
    Dews M et al. Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster. Nat Genet. 2006;38:1060–5.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Cai CK, Zhao GY, Tian LY, Liu L, Yan K, Ma YL, et al. miR-15 and miR-16-1 downregulate CCND1 and induce apoptosis and cell cycle arrest in osteosarcoma. Oncol Rep. 2012;28(5):1764–70.PubMedGoogle Scholar
  15. 15.
    Bonci D, Coppola V, Musumeci M, Addario A, Giuffrida R, Memeo L, et al. The miR-15-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities. Nat Med. 2008;14(11):1271–7.CrossRefPubMedGoogle Scholar
  16. 16.
    Aqeilan RI, Calin GA, Croce CM. miR-15a and miR-16-1 in cancer: discovery, function and future perspectives. Cell Death Differ. 2010;17:215–20.CrossRefPubMedGoogle Scholar
  17. 17.
    Klein U, Lia M, Crespo M, Siegel R, Shen Q, Mo T, et al. The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia. Cancer Cell. 2010;17:28–40.CrossRefPubMedGoogle Scholar
  18. 18.
    Chen F, Hou SK, Fan HJ, Liu YF. miR-15-16 represses Cripto and inhibits NSCLC cell progression. Mol Cell Biochem. 2014;391(1–2):11–9.CrossRefPubMedGoogle Scholar
  19. 19.
    Sun CY, She XM, Qin Y, Chu ZB, Chen L, Ai LS, et al. miR-15 and miR-16 affect the angiogenesis of multiple myeloma by targeting VEGF. Carcinogenesis. 2013;34(2):426–35.CrossRefPubMedGoogle Scholar
  20. 20.
    Cittelly DM, Das PM, Salvo VA, Fonseca JP, Burow ME, Jones FE. Oncogenic HER2{Delta}16 suppresses miR-15/16 and deregulates BCL-2 to promote endocrine resistance of breast tumors. Carcinogenesis. 2010;31(12):2049–57.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Rissland OS, Hong SJ, Bartel DP. MicroRNA destabilization enables dynamic regulation of the miR-16 family in response to cell-cycle changes. Mol Cell. 2011;43:993–1004.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Lerner M, Harada M, Lovén J, Castro J, Davis Z, Oscier D, et al. DLEU2, frequently deleted in malignancy, functions as a critical host gene of the cell cycle inhibitory microRNAs miR-16 and miR-16-1. Exp Cell Res. 2009;315(17):2941–52.CrossRefPubMedGoogle Scholar
  23. 23.
    Wang B, Hsu SH, Wang X, Kutay H, Bid HK, Yu J, et al. Reciprocal regulation of microRNA-122 and c-Myc in hepatocellular cancer: role of E2F1 and transcription factor dimerization partner 2. Hepatology. 2014;59(2):555–66.CrossRefPubMedGoogle Scholar
  24. 24.
    Kong FB, Wang XT, Xie YB, Xiao Q. Inhibitory effect of E2F-1-silencing lentivirus vector on chemoresistance of subcutaneous human gastric cancer in nude mice. Zhonghua Zhong Liu Za Zhi. 2013;35(9):655–9.PubMedGoogle Scholar
  25. 25.
    Wu L, de Bruin A, Wang H, Simmons T, Cleghorn W, Goldenberg LE, et al. Selective roles of E2Fs for ErbB2- and Myc-mediated mammary tumorigenesis. Oncogene. 2015;34(1):119–28.CrossRefPubMedGoogle Scholar
  26. 26.
    Yan LH, Wang XT, Yang J, Kong FB, Lian C, Wei WY, et al. Reversal of multidrug resistance in gastric cancer cells by E2F-1 downregulation in vitro and in vivo. J Cell Biochem. 2014;115(1):34–41.CrossRefPubMedGoogle Scholar
  27. 27.
    Bar J, Gorn-Hondermann I, Moretto P, Perkins TJ, Niknejad N, Stewart DJ, Goss GD, Dimitroulakos J. miR profiling identifies cyclin-dependent kinase 6 downregulation as a potential mechanism of acquired cisplatin resistance in non-small-cell lung carcinoma. Clin Lung Cancer. 2015.Google Scholar
  28. 28.
    Cheng W, Liu T, Wan X, Gao Y, Wang H. MicroRNA-199a targets CD44 to suppress the tumorigenicity and multidrug resistance of ovarian cancer-initiating cells. FEBS J. 2012;279(11):2047–59.CrossRefPubMedGoogle Scholar
  29. 29.
    Hu B, Jiang D, Chen Y, Wei L, Zhang S, Zhao F, et al. High CHMP4B expression is associated with accelerated cell proliferation and resistance to doxorubicin in hepatocellular carcinoma. Tumour Biol. 2015;36(4):2569–81.CrossRefPubMedGoogle Scholar
  30. 30.
    Liu J, Shen W, Tang Y, Zhou J, Li M, Zhu W, et al. Proteasome inhibitor MG132 enhances the antigrowth and antimetastasis effects of radiation in human nonsmall cell lung cancer cells. Tumour Biol. 2014;35(8):7531–9.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Qingping Jiang
    • 1
    • 3
    • 4
  • Yajie Zhang
    • 3
    • 4
  • Mengyang Zhao
    • 2
  • Qiulian Li
    • 1
    • 3
    • 4
  • Ruichao Chen
    • 1
    • 3
    • 4
  • Xiaobing Long
    • 2
  • Weiyi Fang
    • 2
    • 5
  • Zhen Liu
    • 1
    • 3
    • 4
  1. 1.Department of PathologyThird affiliated hospital, Guangzhou Medical UniversityGuangzhouChina
  2. 2.Cancer Research InstituteSouthern Medical UniversityGuangzhouChina
  3. 3.Key Laboratory for Major Obstetric Diseases of Guangdong ProvinceThird Affiliated Hospital of Guangzhou Medical UniversityGuangzhouChina
  4. 4.Department of PathologyGuangzhou Medical UniversityGuangzhouChina
  5. 5.Cancer CenterTraditional Chinese Medicine-Integrated Hospital, Southern Medical UniversityGuangzhouChina

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