Journal of Biosciences

, Volume 40, Issue 5, pp 873–883 | Cite as

Regulatory single nucleotide polymorphisms at the beginning of intron 2 of the human KRAS gene

  • Elena V Antontseva
  • Marina Yu MatveevaEmail author
  • Natalia P Bondar
  • Elena V Kashina
  • Elena Yu Leberfarb
  • Leonid O Bryzgalov
  • Polina A Gervas
  • Anastasia A Ponomareva
  • Nadezhda V Cherdyntseva
  • Yury L Orlov
  • Tatiana I Merkulova


There are two regulatory single nucleotide polymorphisms (rSNPs) at the beginning of the second intron of the mouse K-ras gene that are strongly associated with lung cancer susceptibility. We performed functional analysis of three SNPs (rs12228277: T>A, rs12226937: G>A, and rs61761074: T>G) located in the same region of human KRAS. We found that rs12228277 and rs61761074 result in differential binding patterns of lung nuclear proteins to oligonucleotide probes corresponding two alternative alleles; in both cases, the transcription factor NF-Y is involved. G>A substitution (rs12226937) had no effect on the binding of lung nuclear proteins. However, all the nucleotide substitutions under study showed functional effects in a luciferase reporter assay. Among them, rs61761074 demonstrated a significant correlation with allele frequency in non-small-cell lung cancer (NSCLC). Taken together, the results of our study suggest that a T>G substitution at nucleotide position 615 in the second intron of the KRAS gene (rs61761074) may represent a promising genetic marker of NSCLC.


K-RAS lung cancer NF-Y binding regulatory SNPs (rSNP) 



The research has been carried out with support of the grant from Russian Academy of Science Program Fundamental Science for Medicine #23, and Federal Targeted Programmer for Research and Development in Priority Areas of Development of the Russian Scientific and Technological Complex for 2014-2020, Development of Molecular Signatures for Early Detection of Lung Cancer (No. 14.575.21.0064 from 05.08.2014) and State Budget Project N VI.58.1.2.

Supplementary material

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  1. Alj Y, Georgiakaki M, Savouret JF, Mal F, Attali P, et al. 2004 Hereditary persistence of alpha-fetoprotein is due to both proximal and distal hepatocyte nuclear factor-1 site mutations. Gastroenterology 126 308–317Google Scholar
  2. Bryzgalov LO, Antontseva EV, Matveeva MY, Shilov AG, Kashina EV, Mordvinov VA and Merkulova TI 2013 Detection of regulatory SNPs in human genome using ChIP-seq ENCODE data. PLoS One 8 e78833Google Scholar
  3. Chen B, Johanson L, Wiest JS, Anderson MW and You M 1994 The second intron of the K-ras gene contains regulatory elements associated with mouse lung tumor susceptibility. Proc. Natl. Acad. Sci. USA 91 1589–1593Google Scholar
  4. Chin LJ, Ratner E, Leng S, Zhai R, Nallur S, Babar I, Muller RU, Straka E, et al. 2008 A SNP in a let-7 microRNA complementary site in the KRAS 3' untranslated region increases non-small cell lung cancer risk. Cancer Res. 68 8535–8540PubMedCentralPubMedCrossRefGoogle Scholar
  5. Comings DE, Gade R, Muhleman D, Chiu C, Wu S, et al. 1996 Exon and intron variants in the humman tryptophan 2,3-dioxygenase gene: potentional association with Tourette sindrome, substans abuse and other disorders. Pharmacogenetics 6 307–318Google Scholar
  6. Cooper DN, Chen JM, Ball EV, Howells K, Mort M, et al. 2010 Genes, mutations, and human inherited disease at the dawn of the age of personalized genomics. Hum. Mutat. 31 631–655PubMedCrossRefGoogle Scholar
  7. Davison LJ, Wallace C, Cooper JD, Cope NF, Wilson NK, Smyth DJ, Howson JM, Saleh N, et al. 2012 2012 Long-range DNA looping and gene expression analyses identify DEXI as an autoimmune disease candidate gene. Hum. Mol. Genet. 21 322–333PubMedCentralPubMedCrossRefGoogle Scholar
  8. Drachkova I, Savinkova L, Arshinova T, Ponomarenko M, Peltek S and Kolchanov N 2014 The mechanism by which TATA-box polymorphisms associated with human hereditary diseases influence interactions with the TATA-binding protein. Hum. Mutat. 35 601–608PubMedCrossRefGoogle Scholar
  9. Epstein DJ 2009 Cis-regulatory mutations in human disease. Brief. Funct. Genomic Proteomic 8 310–316PubMedCentralPubMedCrossRefGoogle Scholar
  10. Fehrmann RS, Jansen RC, Veldink JH, Westra HJ, Arends D, Bonder MJ, Fu J, Deelen P, et al. 2011 PLoS Genet. 7 e1002197PubMedCentralPubMedCrossRefGoogle Scholar
  11. Fei YJ, Stoming TA, Efremov GD, Efremov DG, Battacharia R, et al. 1988 Beta-thalassemia due to a T–A mutation within the TATA box. Biochem. Biophys. Res. Commun. 153 741–747PubMedCrossRefGoogle Scholar
  12. Fleming JD, Pavesi G, Benatti P, Imbriano C, Mantovani R and Struhl K 2013 NF-Y coassociates with FOS at promoters, enhancers, repetitive elements, and inactive chromatin regions, and is stereo-positioned with growth-controlling transcription factors. Genome Res. 23 1195–1209PubMedCentralPubMedCrossRefGoogle Scholar
  13. Gorski K, Carneiro M and Schibler U 1986 Tissue-specific in vitro transcription from the mouse albumin promoter. Cell 47 767–776PubMedCrossRefGoogle Scholar
  14. Gorshkova EV, Kaledin VI, Kobzev VF and Merkulova TI 2006 Lung cancer-associated SNP at the beginning of mouse k-ras gene intron 2 is essential for transcription factor binding. Bull. Exp. Biol. Med. 141 731–733PubMedCrossRefGoogle Scholar
  15. Henley JS, Richards TB, Underwood MJ, Sunderam CR, Plescia M and McAfee TA 2014 Lung cancer incidence trends among men and women - United States, 2005-2009. MMWR Morb. Mortal. Wkly Rep. 63 1–5PubMedGoogle Scholar
  16. Huncharek M, Muscat J and Geschwind J-F 1999 K-ras oncogene mutation as a prognostic marker in non-small cell lung cancer: a combined analysis of 881 cases. Carcinogenesis 20 1507–1510PubMedCrossRefGoogle Scholar
  17. Johnson JL, Pillai S and Chellappan SP 2012 Genetic and biochemical alterations in non-small cell lung cancer. Biochem. Res. Int. 2012 940405PubMedCentralPubMedCrossRefGoogle Scholar
  18. Kohanbash G, Ishikawa E, Fujita M, Ikeura M, McKaveney K, Zhu J, Sakaki M, Sarkar SN, et al. 2012 Differential activity of interferon-α8 promoter is regulated by Oct-1 and a SNP that dictates prognosis of glioma. Oncoimmunology 1 487–492Google Scholar
  19. Kohno T, Kunitoh H, Suzuki K, Yamamoto S, Kuchiba A, Matsuno Y, Yanagitani N and Yokota J 2008 Association of KRAS polymorphisms with risk for lung adenocarcinoma accompanied by atypical adenomatous hyperplasias. Carcinogenesis 29 957–963PubMedCrossRefGoogle Scholar
  20. Kundu ST, Nallur S, Paranjape T, Boeke M, Weidhaas JB and Slack FJ 2012 KRAS alleles: the LCS6 3'UTR variant and KRAS coding sequence mutations in the NCI-60 panel. Cell Cycle 11 361–366PubMedCrossRefGoogle Scholar
  21. Larsen JE and Minna JD 2011 Molecular biology of lung cancer: clinical implications. Clin. Chest Med. 32 703–740PubMedCentralPubMedCrossRefGoogle Scholar
  22. Lin YC, Chen YN, Lin KF, Wang FF, Chou TY and Chen MY 2014 Association of p21 with NF-YA suppresses the expression of Polo-like kinase 1 and prevents mitotic death in response to DNA damage. Cell Death Dis. 5, e987PubMedCentralPubMedCrossRefGoogle Scholar
  23. Liu P, Wang Y, Vikis H, Maciag A, Wang D, Lu Y, Liu Y and You M 2006 Candidate lung tumor susceptibility genes identified through whole-genome association analyses in inbred mice. Nat. Genet. 38 888–895PubMedCrossRefGoogle Scholar
  24. Lowry OH, Rosebrough NY, Farr AL and Randall RJ 1951 Protein measurement with Folin phenol reagent. J. Biol. Chem. 193 265–275PubMedGoogle Scholar
  25. Ly LL, Yoshida H and Yamaguchi M 2013 Nuclear transcription factor Y and its roles in cellular processes related to human disease. Am. J. Cancer Res. 3 339–346PubMedCentralPubMedGoogle Scholar
  26. Malumbres M and Barbacid M 2003 RAS oncogenes: the first 30 years. Nat. Rev. Cancer. 3 459–465PubMedCrossRefGoogle Scholar
  27. Manenti G, Galbiati F, Giannì-Barrera R, Pettinicchio A, Acevedo A and Dragani TA 2004 Haplotype sharing suggests that a genomic segment containing six genes accounts for the pulmonary adenoma susceptibility 1 (Pas1) locus activity in mice. Oncogene 23 4495–4504PubMedCrossRefGoogle Scholar
  28. Manolio TA, Collins FS, Cox NJ, Goldstein DB, Hindorff LA, Hunter DJ, McCarthyMI REM, Cardon LR, et al. 2009 Finding the missing heritability of complex diseases. Nature 461 747–753PubMedCentralPubMedCrossRefGoogle Scholar
  29. Maurano MT, Humbert R, Rynes E, Thurman RE, Haugen E, Wang H, Reynolds AP, Sandstrom R, et al. 2012 Systematic localization of common disease‐associated variation in regulatory DNA. Science 337 1190–1195PubMedCentralPubMedCrossRefGoogle Scholar
  30. Meuwissen R, Linn SC, van der Valk M, Mooi WJ and Berns A 2001 Mouse model for lung tumorigenesis through Cre/lox controlled sporadic activation of the K-ras oncogene. Oncogene 206 551–558Google Scholar
  31. Milton AC, Packard AV, Clary L and Okkema PG 2013 The NF-Y complex negatively regulates Caenorhabditiselegans tbx-2 expression. Dev. Biol. 382 38–47PubMedCentralPubMedCrossRefGoogle Scholar
  32. Ponomarenko JV, Orlova GV, Frolov AS, Gelfand MS and Ponomarenko MP 2002a SELEX_DB: a database on in vitro selected oligomers adapted for recognizing natural sites and for analyzing both SNPs and site-directed mutagenesis data. Nucleic Acids Res. 30 195–199PubMedCentralPubMedCrossRefGoogle Scholar
  33. Ponomarenko JV, Orlova GV, Merkulova TI, Gorshkova EV, Fokin ON, Vasiliev GV, Frolov AS and Ponomarenko MP 2002b rSNP_Guide: an integrated database-tools system for studying SNPs and site-directed mutations in transcription factor binding sites. Hum. Mutat. 20 239–248PubMedCrossRefGoogle Scholar
  34. Ramakrishna G, Biakovska A, Perella C, Birely L, Fornwald LW, Diwan BA, Schiao Y-H and Anderson LM 2000 Ki-ras and characteristics of mouse lung tumors. Mol. Carcinog. 28 156–167PubMedCrossRefGoogle Scholar
  35. Rodenhuis S and Slebos RJ 1992 Clinical significance of ras oncogene activation in human lung cancer. Cancer Res. 52 2665s–2669sGoogle Scholar
  36. Raney BJ, Cline MS, Rosenbloom KR, Dreszer TR, Learned K, Barber GP, Meyer LR, Sloan CA, et al. 2011 ENCODE whole-genome data in the UCSC genome browser (2011 update). Nucleic Acids Res. 39 D871–D875PubMedCentralPubMedCrossRefGoogle Scholar
  37. Romier C, Cocchiarella F, Mantovani R and Moras D 2003 The NF-YB/NF-YC structure gives insight into DNA binding and transcription regulation by CCAAT factor NF-Y. J. Biol. Chem. 278 1336–1345PubMedCrossRefGoogle Scholar
  38. Ronchi A, Bellorini M, Mongelli N and Mantovani R 1995 CCAAT-box binding protein NF-Y (CBF, CP1) recognizes the minor groove and distorts DNA. Nucleic Acids Res. 23 4565–4572PubMedCentralPubMedCrossRefGoogle Scholar
  39. Ryan J, Barker PE, Nesbitt MN and Ruddle FH 1987 KRAS2 as a genetic marker for lung tumor susceptibility in inbred mice. J. Natl. Cancer Inst. 79 1351–1357Google Scholar
  40. Siegel R, Ward E, Brawley O and Jemal A 2011 Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J. Clin. 61 212–236PubMedCrossRefGoogle Scholar
  41. Sinnott R, Winters L, Larson B, Mytsa D, Taus P, Cappell KM and Whitehurst AW 2014 Mechanisms promoting escape from mitotic stress-induced tumor cell death. Cancer Res. 74 3857–3869PubMedCentralPubMedCrossRefGoogle Scholar
  42. Shapiro DJ, Sharp PA, Wahli WW and Keller MJ 1988 A high-efficiency HeLa cell nuclear transcription extract. DNA 7 47–55PubMedCrossRefGoogle Scholar
  43. Schubbert S, Shannon K and Bollag G 2007 Hyperactive Ras in developmental disorders and cancer. Nat. Rev. Cancer. 7 295–308PubMedCrossRefGoogle Scholar
  44. Stenson PD, Mort M, Ball EV, Howells K, Phillips AD, et al. 2009 The human gene mutation database: 2008 update. Genome Med. 1 13Google Scholar
  45. Timofeeva OA, Gorshkova EV, Levashova ZB, Kobzev VF, Filipenko ML, Kaledin VI and Merkulova TI 2002 Pulmonary carcinogenesis susceptibility-associated single-nucleotide polymorphisms in K-ras intron 2 affect the binding of factor Gata-6 but not gene expression. Mol. Biol. (Mosk). 36 817–824CrossRefGoogle Scholar
  46. Timofeeva OA, Filipenko ML and Kaledin VI 1999 The study of correlation between the K-ras genotype and murine susceptibility to chemically induced lung neoplasms. Genetika 35 1309–1312PubMedGoogle Scholar
  47. Van den Boogaard M, Wong LY, Tessadori F, Bakker ML, Dreizehnter LK, Wakker V, Bezzina CR, Hoen PA, et al. 2012 Genetic variation in T-box binding element functionally affects SCN5A/SCN10A enhancer. J. Clin. Invest. 122 2519–2530Google Scholar
  48. Vasiliev GV, Merkulov VM, Kobzev VF, Merkulova TI, Ponomarenko MP and Kolchanov NA 1999 Point mutations within 663-666 bp of intron 6 of the human TDO2 gene, associated with a number of psychiatric disorders, damage the YY-1 transcription factor binding site. FEBS Lett. 462 85–88PubMedCrossRefGoogle Scholar
  49. Warburton D, Wuenschell C, Flores-Delgado G and Anderson K 1998 Commitment and differentiation of lung cell lineages. Biochem. Cell Biol. 76 971–995PubMedCrossRefGoogle Scholar
  50. Wenhu P, Xingguo Z, Min W, Yongchao W, Sadanand F, Ali E, Jianhua L and Dorothy T 2010 Long-range function of an intergenic retrotransposon. Proc. Natl. Acad. Sci. USA 107 12992–12997Google Scholar

Copyright information

© Indian Academy of Sciences 2015

Authors and Affiliations

  • Elena V Antontseva
    • 1
  • Marina Yu Matveeva
    • 1
    Email author
  • Natalia P Bondar
    • 1
  • Elena V Kashina
    • 1
  • Elena Yu Leberfarb
    • 1
  • Leonid O Bryzgalov
    • 1
  • Polina A Gervas
    • 3
  • Anastasia A Ponomareva
    • 3
    • 4
  • Nadezhda V Cherdyntseva
    • 3
  • Yury L Orlov
    • 1
    • 2
  • Tatiana I Merkulova
    • 1
    • 2
  1. 1.Institute of Cytology and Genetics, Siberian BranchRussian Academy of SciencesNovosibirskRussian Federation
  2. 2.Novosibirsk State UniversityNovosibirskRussian Federation
  3. 3.Tomsk Cancer Research InstituteTomskRussian Federation
  4. 4.Tomsk Polytechnical UniversityTomskRussian Federation

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