title: Targeted knock-in of the polymorphism rs61764370 does not affect KRAS expression but reduces let-7 levels.
abstract: Understanding the role of single-nucleotide polymorphisms (SNPs) in the pathological process represents a unique experimental challenge especially when the variants occur outside of coding regions. The noncoding SNP rs61764370 located in the 3'-untranslated region of Kirsten rat sarcoma viral oncogene homolog (KRAS) has been implicated as a risk factor for the development of cancer and the response to targeted therapies. This cancer-associated variant is thought to affect the binding of the microRNA let-7, which allegedly modulates KRAS expression. Using site-specific homologous recombination, we inserted the rs61764370:T>G KRAS gene variant in the colorectal cancer cell line SW48 (SW48 +SNP) and assessed the cellular and biochemical phenotype. We observed a significant increase in cellular proliferation, as well as a reduction in the levels of the microRNA let-7a, let-7b, and let-7c. Transcriptional and biochemical analysis showed no concomitant change in the KRAS protein expression or modulation of the downstream mitogen activated kinase or PI3K/AKT signaling. These results suggest that the cancer-associated rs61764370 variant exerts a biological effect not through transcriptional modulation of KRAS but rather by tuning the expression of the microRNA let-7.
authors.affiliation: IRCC, Institute for Cancer Research at Candiolo, SP142 Km 3.95, Torino, Italy.
authors.forename: Emily Hannah
authors.initials: EH
authors.lastname: Crowley
authors.forename: Sabrina
authors.initials: S
authors.lastname: Arena
authors.forename: Simona
authors.initials: S
authors.lastname: Lamba
authors.forename: Federica
authors.initials: F
authors.lastname: Di Nicolantonio
authors.forename: Alberto
authors.initials: A
authors.lastname: Bardelli
mesh_terms: D020413:3' Untranslated Regions; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D055879:Gene Knock-In Techniques; D011905:Genes, ras; D014644:Genetic Variation; D057809:HEK293 Cells; D059765:Homologous Recombination; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras); D015398:Signal Transduction; D018631:ras Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; C117307:KRAS protein, human; D035683:MicroRNAs; D011518:Proto-Oncogene Proteins; C487931:mirnlet7 microRNA, human; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
keywords: 3′ untranslated region; KRAS; cancer; genetics; let-7; miRNA; single-nucleotide polymorphism