Cancer Causes & Control

, Volume 24, Issue 6, pp 1099–1109

Association of germline microRNA SNPs in pre-miRNA flanking region and breast cancer risk and survival: the Carolina Breast Cancer Study

  • Jeannette T. Bensen
  • Chiu Kit Tse
  • Sarah J. Nyante
  • Jill S. Barnholtz-Sloan
  • Stephen R. Cole
  • Robert C. Millikan
Original paper



Common germline variation in the 5′ region proximal to precursor (pre-) miRNA gene sequences is evaluated for association with breast cancer risk and survival among African Americans and Caucasians.


We genotyped nine single nucleotide polymorphisms (SNPs) within six miRNA gene regions previously associated with breast cancer, in 1,972 cases and 1,776 controls. In a race-stratified analysis using unconditional logistic regression, odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated to evaluate SNP association with breast cancer risk. Additionally, hazard ratios (HRs) for breast cancer-specific mortality were estimated.


Two miR-185 SNPs provided suggestive evidence of an inverse association with breast cancer risk (rs2008591, OR = 0.72 (95 % CI = 0.53–0.98, p value = 0.04) and rs887205, OR = 0.71 (95 % CI = 0.52–0.96, p value = 0.03), respectively) among African Americans. Two SNPs, miR-34b/34c (rs4938723, HR = 0.57 (95 % CI = 0.37–0.89, p value = 0.01)) and miR-206 (rs6920648, HR = 0.77 (95 % CI = 0.61–0.97, p value = 0.02)), provided evidence of association with breast cancer survival. Further adjustment for stage resulted in more modest associations with survival (HR = 0.65 [95 % CI = 0.42–1.02, p value = 0.06] and HR = 0.79 [95 % CI = 0.62–1.00, p value = 0.05, respectively]).


Our results suggest that germline variation in the 5′ region proximal to pre-miRNA gene sequences may be associated with breast cancer risk among African Americans and breast cancer-specific survival generally; however, further validation is needed to confirm these findings.


MicroRNA Breast cancer Germline Single nucleotide polymorphism Risk Survival 



3′-Untranslated region


Ancestry informative markers


Carolina Breast Cancer Study


Complementary DNA made from an mRNA template


Confidence interval


Breast carcinoma in situ


Hazard ratio




Linkage disequilibrium


Minor allele frequency

miRNA or miR



Messenger RNA




Odds ratio

Pol II

Polymerase II


Polymerase III


Precursor microRNA


Primary miRNA transcript


Single nucleotide polymorphism

Supplementary material

10552_2013_187_MOESM1_ESM.pdf (95 kb)
Supplementary material 1 (PDF 95 kb)
10552_2013_187_MOESM2_ESM.pdf (77 kb)
Supplementary material 2 (PDF 77 kb)
10552_2013_187_MOESM3_ESM.pdf (178 kb)
Supplementary material 3 (PDF 178 kb)
10552_2013_187_MOESM4_ESM.pdf (69 kb)
Supplementary material 4 (PDF 69 kb)
10552_2013_187_MOESM5_ESM.pdf (146 kb)
Supplementary material 5 (PDF 146 kb)
10552_2013_187_MOESM6_ESM.pdf (123 kb)
Supplementary material 6 (PDF 122 kb)


  1. 1.
    Bentwich I, Avniel A, Karov Y et al (2005) Identification of hundreds of conserved and nonconserved human microRNAs. Nat Genet 37:766–770. doi:10.1038/ng1590 PubMedCrossRefGoogle Scholar
  2. 2.
    Schanen BC, Li X (2011) Transcriptional regulation of mammalian miRNA genes. Genomics 97:1–6. doi:10.1016/j.ygeno.2010.10.005 PubMedCrossRefGoogle Scholar
  3. 3.
    Lee Y, Jeon K, Lee JT et al (2002) MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 21:4663–4670PubMedCrossRefGoogle Scholar
  4. 4.
    Heneghan HM, Miller N, Lowery AJ et al (2009) MicroRNAs as novel biomarkers for breast cancer. J Oncol 2009:950201. doi:10.1155/2010/950201 PubMedGoogle Scholar
  5. 5.
    Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120:15–20. doi:10.1016/j.cell.2004.12.035 PubMedCrossRefGoogle Scholar
  6. 6.
    Lee Y, Kim M, Han J et al (2004) MicroRNA genes are transcribed by RNA polymerase II. EMBO J 23:4051–4060. doi:10.1038/sj.emboj.7600385 PubMedCrossRefGoogle Scholar
  7. 7.
    Borchert GM, Lanier W, Davidson BL (2006) RNA polymerase III transcribes human microRNAs. Nat Struct Mol Biol 13:1097–1101. doi:10.1038/nsmb1167 PubMedCrossRefGoogle Scholar
  8. 8.
    Song Gao J, Zhang Y, Li M et al (2010) Atypical transcription of microRNA gene fragments. Nucleic Acids Res 38:2775–2787. doi:10.1093/nar/gkp1242 PubMedCrossRefGoogle Scholar
  9. 9.
    Marson A, Levine SS, Cole MF et al (2008) Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell 134:521–533. doi:10.1016/j.cell.2008.07.020 PubMedCrossRefGoogle Scholar
  10. 10.
    Baskerville S, Bartel DP (2005) Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. RNA 11:241–247. doi:10.1261/rna.7240905 PubMedCrossRefGoogle Scholar
  11. 11.
    Duan S, Mi S, Zhang W et al (2009) Comprehensive analysis of the impact of SNPs and CNVs on human microRNAs and their regulatory genes. RNA Biol 6:412–425PubMedCrossRefGoogle Scholar
  12. 12.
    Sethupathy P, Borel C, Gagnebin M et al (2007) Human microRNA-155 on chromosome 21 differentially interacts with its polymorphic target in the AGTR1 3′ untranslated region: a mechanism for functional single-nucleotide polymorphisms related to phenotypes. Am J Hum Genet 81:405–413. doi:10.1086/519979 PubMedCrossRefGoogle Scholar
  13. 13.
    Mishra PJ, Humeniuk R, Mishra PJ et al (2007) A miR-24 microRNA binding-site polymorphism in dihydrofolate reductase gene leads to methotrexate resistance. Proc Natl Acad Sci USA 104:13513–13518. doi:10.1073/pnas.0706217104 PubMedCrossRefGoogle Scholar
  14. 14.
    Landi D, Gemignani F, Naccarati A et al (2008) Polymorphisms within micro-RNA-binding sites and risk of sporadic colorectal cancer. Carcinogenesis 29:579–584. doi:10.1093/carcin/bgm304 PubMedCrossRefGoogle Scholar
  15. 15.
    Brendle A, Lei H, Brandt A et al (2008) Polymorphisms in predicted microRNA-binding sites in integrin genes and breast cancer: ITGB4 as prognostic marker. Carcinogenesis 29:1394–1399. doi:10.1093/carcin/bgn126 PubMedCrossRefGoogle Scholar
  16. 16.
    O’Day E, Lal A (2010) MicroRNAs and their target gene networks in breast cancer. Breast Cancer Res 12:201. doi:10.1186/bcr2484 PubMedCrossRefGoogle Scholar
  17. 17.
    Yu Z, Baserga R, Chen L et al (2010) MicroRNA, cell cycle, and human breast cancer. Am J Pathol 176:1058–1064. doi:10.2353/ajpath.2010.090664 PubMedCrossRefGoogle Scholar
  18. 18.
    Hoffman AE, Zheng T, Yi C et al (2009) MicroRNA miR-196a-2 and breast cancer: a genetic and epigenetic association study and functional analysis. Cancer Res 69:5970–5977. doi:10.1158/0008-5472.CAN-09-0236 PubMedCrossRefGoogle Scholar
  19. 19.
    Hu Z, Liang J, Wang Z et al (2009) Common genetic variants in pre-microRNAs were associated with increased risk of breast cancer in Chinese women. Hum Mutat 30:79–84. doi:10.1002/humu.20837 PubMedCrossRefGoogle Scholar
  20. 20.
    Kontorovich T, Levy A, Korostishevsky M et al (2010) Single nucleotide polymorphisms in miRNA binding sites and miRNA genes as breast/ovarian cancer risk modifiers in Jewish high-risk women. Int J Cancer 127:589–597. doi:10.1002/ijc.25065 PubMedCrossRefGoogle Scholar
  21. 21.
    Akkiz H, Bayram S, Bekar A et al (2011) A functional polymorphism in pre-microRNA-196a-2 contributes to the susceptibility of hepatocellular carcinoma in a Turkish population: a case–control study. J Viral Hepat 18:e399–e407. doi:10.1111/j.1365-2893.2010.01414.x PubMedCrossRefGoogle Scholar
  22. 22.
    Mittal RD, Gangwar R, George GP et al (2011) Investigative role of pre-microRNAs in bladder cancer patients: a case–control study in North India. DNA Cell Biol 30:401–406. doi:10.1089/dna.2010.1159 PubMedCrossRefGoogle Scholar
  23. 23.
    Okubo M, Tahara T, Shibata T et al (2010) Association between common genetic variants in pre-microRNAs and gastric cancer risk in Japanese population. Helicobacter 15:524–531. doi:10.1111/j.1523-5378.2010.00806.x PubMedCrossRefGoogle Scholar
  24. 24.
    Jedlinski DJ, Gabrovska PN, Weinstein SR et al (2011) Single nucleotide polymorphism in hsa-mir-196a-2 and breast cancer risk: a case control study. Twin Res Hum Genet 14:417–421. doi:10.1375/twin.14.5.417 PubMedCrossRefGoogle Scholar
  25. 25.
    Permuth-Wey J, Thompson RC, Burton Nabors L et al (2011) A functional polymorphism in the pre-miR-146a gene is associated with risk and prognosis in adult glioma. J Neurooncol. doi:10.1007/s11060-011-0634-1 PubMedGoogle Scholar
  26. 26.
    Liang D, Meyer L, Chang DW et al (2010) Genetic variants in MicroRNA biosynthesis pathways and binding sites modify ovarian cancer risk, survival, and treatment response. Cancer Res 70:9765–9776. doi:10.1158/0008-5472.CAN-10-0130 PubMedCrossRefGoogle Scholar
  27. 27.
    Hu Z, Shu Y, Chen Y et al (2011) Genetic polymorphisms in the precursor MicroRNA flanking region and non-small cell lung cancer survival. Am J Respir Crit Care Med 183:641–648. doi:10.1164/rccm.201005-0717OC PubMedCrossRefGoogle Scholar
  28. 28.
    Newman B, Moorman PG, Millikan R et al (1995) The Carolina Breast Cancer Study: integrating population-based epidemiology and molecular biology. Breast Cancer Res Treat 35:51–60PubMedCrossRefGoogle Scholar
  29. 29.
    Millikan R, Eaton A, Worley K et al (2003) HER2 codon 655 polymorphism and risk of breast cancer in African Americans and whites. Breast Cancer Res Treat 79:355–364PubMedCrossRefGoogle Scholar
  30. 30.
    Millikan RC, Newman B, Tse CK et al (2008) Epidemiology of basal-like breast cancer. Breast Cancer Res Treat 109:123–139. doi:10.1007/s10549-007-9632-6 PubMedCrossRefGoogle Scholar
  31. 31.
    Weinberg CR, Sandler DP (1991) Randomized recruitment in case–control studies. Am J Epidemiol 134:421–432PubMedGoogle Scholar
  32. 32.
    Cannell IG, Bushell M (2010) Regulation of Myc by miR-34c: a mechanism to prevent genomic instability? Cell Cycle 9:2726–2730PubMedCrossRefGoogle Scholar
  33. 33.
    Iorio MV, Ferracin M, Liu CG et al (2005) MicroRNA gene expression deregulation in human breast cancer. Cancer Res 65:7065–7070. doi:10.1158/0008-5472.CAN-05-1783 PubMedCrossRefGoogle Scholar
  34. 34.
    Verghese ET, Hanby AM, Speirs V et al (2008) Small is beautiful: microRNAs and breast cancer-where are we now? J Pathol 215:214–221. doi:10.1002/path.2359 PubMedCrossRefGoogle Scholar
  35. 35.
    Tavazoie SF, Alarcon C, Oskarsson T et al (2008) Endogenous human microRNAs that suppress breast cancer metastasis. Nature 451:147–152. doi:10.1038/nature06487 PubMedCrossRefGoogle Scholar
  36. 36.
    Barnholtz-Sloan JS, Shetty PB, Guan X et al (2010) FGFR2 and other loci identified in genome-wide association studies are associated with breast cancer in African-American and younger women. Carcinogenesis 31:1417–1423. doi:10.1093/carcin/bgq128 PubMedCrossRefGoogle Scholar
  37. 37.
    Tian C, Hinds DA, Shigeta R et al (2006) A genomewide single-nucleotide-polymorphism panel with high ancestry information for African American admixture mapping. Am J Hum Genet 79:640–649. doi:10.1086/507954 PubMedCrossRefGoogle Scholar
  38. 38.
    Barnholtz-Sloan JS, McEvoy B, Shriver MD et al (2008) Ancestry estimation and correction for population stratification in molecular epidemiologic association studies. Cancer Epidemiol Biomarkers Prev 17:471–477. doi:10.1158/1055-9965.EPI-07-0491 PubMedCrossRefGoogle Scholar
  39. 39.
    Lin DY, Zeng D, Millikan R (2005) Maximum likelihood estimation of haplotype effects and haplotype-environment interactions in association studies. Genet Epidemiol 29:299–312. doi:10.1002/gepi.20098 PubMedCrossRefGoogle Scholar
  40. 40.
    Lin D, Zeng D (2006) Likelihood-based inference on haplotype effects in genetic association studies. J Am Stat Assoc 101:89–104CrossRefGoogle Scholar
  41. 41.
    Hu YJ, Lin DY, Zeng D (2010) A general framework for studying genetic effects and gene-environment interactions with missing data. Biostatistics 11:583–598. doi:10.1093/biostatistics/kxq015 PubMedCrossRefGoogle Scholar
  42. 42.
    O’Brien KM, Cole SR, Tse CK et al (2010) Intrinsic breast tumor subtypes, race, and long-term survival in the Carolina Breast Cancer Study. Clin Cancer Res 16:6100–6110. doi:10.1158/1078-0432.CCR-10-1533 PubMedCrossRefGoogle Scholar
  43. 43.
    Hinske LC, Galante PA, Kuo WP et al (2010) A potential role for intragenic miRNAs on their hosts’ interactome. BMC Genom 11:533. doi:10.1186/1471-2164-11-533 CrossRefGoogle Scholar
  44. 44.
    Takahashi Y, Forrest AR, Maeno E et al (2009) MiR-107 and MiR-185 can induce cell cycle arrest in human non small cell lung cancer cell lines. PLoS ONE 4:e6677. doi:10.1371/journal.pone.0006677 PubMedCrossRefGoogle Scholar
  45. 45.
    Liu H, Cao YD, Ye WX et al (2010) Effect of microRNA-206 on cytoskeleton remodelling by downregulating Cdc42 in MDA-MB-231 cells. Tumori 96:751–755PubMedGoogle Scholar
  46. 46.
    Liu M, Lang N, Chen X et al (2011) miR-185 targets RhoA and Cdc42 expression and inhibits the proliferation potential of human colorectal cells. Cancer Lett 301:151–160. doi:10.1016/j.canlet.2010.11.009 PubMedCrossRefGoogle Scholar
  47. 47.
    Corcoran C, Friel AM, Duffy MJ et al (2011) Intracellular and extracellular microRNAs in breast cancer. Clin Chem 57:18–32. doi:10.1373/clinchem.2010.150730 PubMedCrossRefGoogle Scholar
  48. 48.
    Buffa FM, Camps C, Winchester L et al (2011) MicroRNA-associated progression pathways and potential therapeutic targets identified by integrated mRNA and microRNA expression profiling in breast cancer. Cancer Res 71:5635–5645. doi:10.1158/0008-5472.CAN-11-0489 PubMedCrossRefGoogle Scholar
  49. 49.
    Wu H, Zhu S, Mo YY (2009) Suppression of cell growth and invasion by miR-205 in breast cancer. Cell Res 19:439–448. doi:10.1038/cr.2009.18 PubMedCrossRefGoogle Scholar
  50. 50.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297PubMedCrossRefGoogle Scholar
  51. 51.
    Vogt M, Munding J, Gruner M et al (2011) Frequent concomitant inactivation of miR-34a and miR-34b/c by CpG methylation in colorectal, pancreatic, mammary, ovarian, urothelial, and renal cell carcinomas and soft tissue sarcomas. Virchows Arch 458:313–322. doi:10.1007/s00428-010-1030-5 PubMedCrossRefGoogle Scholar
  52. 52.
    Tarasov V, Jung P, Verdoodt B et al (2007) Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle 6:1586–1593PubMedCrossRefGoogle Scholar
  53. 53.
    Chang TC, Wentzel EA, Kent OA et al (2007) Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell 26:745–752. doi:10.1016/j.molcel.2007.05.010 PubMedCrossRefGoogle Scholar
  54. 54.
    Raver-Shapira N, Marciano E, Meiri E et al (2007) Transcriptional activation of miR-34a contributes to p53-mediated apoptosis. Mol Cell 26:731–743. doi:10.1016/j.molcel.2007.05.017 PubMedCrossRefGoogle Scholar
  55. 55.
    He L, He X, Lim LP et al (2007) A microRNA component of the p53 tumour suppressor network. Nature 447:1130–1134. doi:10.1038/nature05939 PubMedCrossRefGoogle Scholar
  56. 56.
    Bommer GT, Gerin I, Feng Y et al (2007) p53-mediated activation of miRNA34 candidate tumor-suppressor genes. Curr Biol 17:1298–1307. doi:10.1016/j.cub.2007.06.068 PubMedCrossRefGoogle Scholar
  57. 57.
    Corney DC, Flesken-Nikitin A, Godwin AK et al (2007) MicroRNA-34b and MicroRNA-34c are targets of p53 and cooperate in control of cell proliferation and adhesion-independent growth. Cancer Res 67:8433–8438. doi:10.1158/0008-5472.CAN-07-1585 PubMedCrossRefGoogle Scholar
  58. 58.
    Hermeking H (2010) The miR-34 family in cancer and apoptosis. Cell Death Differ 17:193–199. doi:10.1038/cdd.2009.56 PubMedCrossRefGoogle Scholar
  59. 59.
    Beviglia L, Matsumoto K, Lin CS et al (1997) Expression of the c-Met/HGF receptor in human breast carcinoma: correlation with tumor progression. Int J Cancer 74:301–309PubMedCrossRefGoogle Scholar
  60. 60.
    Gastaldi S, Comoglio PM, Trusolino L (2010) The met oncogene and basal-like breast cancer: another culprit to watch out for? Breast Cancer Res 12:208. doi:10.1186/bcr2617 PubMedCrossRefGoogle Scholar
  61. 61.
    Adams BD, Furneaux H, White BA (2007) The micro-ribonucleic acid (miRNA) miR-206 targets the human estrogen receptor-alpha (ERalpha) and represses ERalpha messenger RNA and protein expression in breast cancer cell lines. Mol Endocrinol 21:1132–1147. doi:10.1210/me.2007-0022 PubMedCrossRefGoogle Scholar
  62. 62.
    Leivonen SK, Makela R, Ostling P et al (2009) Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines. Oncogene 28:3926–3936. doi:10.1038/onc.2009.241 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Jeannette T. Bensen
    • 1
    • 2
  • Chiu Kit Tse
    • 1
  • Sarah J. Nyante
    • 3
  • Jill S. Barnholtz-Sloan
    • 4
  • Stephen R. Cole
    • 1
  • Robert C. Millikan
    • 1
    • 2
  1. 1.Department of EpidemiologyUniversity of North Carolina at Chapel HillChapel HillUSA
  2. 2.Lineberger Comprehensive Cancer Center of the University of North Carolina at Chapel HillChapel HillUSA
  3. 3.Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of HealthDepartment of Health and Human ServicesBethesdaUSA
  4. 4.Case Comprehensive Cancer CenterCase Western Reserve UniversityClevelandUSA

Personalised recommendations