A study of microRNA complementary sites in the 3' UTRs of lung cancer-associated oncogenes

microRNAs (miRNAs) are ∼22 nucleotide-long non-coding RNAs. let-7 is one of the first miRNAs discovered and important for later stages in development. let-7 has been shown to be misregulated in lung cancer; reduced let-7 levels are associated with poor outcome in lung cancer patients. let-7 is thought to behave as a tumor suppressor and repress gene expression of several known human oncogenes, such as KRAS, through let-7 complementary sites (LCSs) in their 3' untranslated regions (UTRs).;The primary cause of cancer deaths in the United States is lung cancer. Through sequencing the KRAS 3' UTR from 74 non-small cell lung cancer (NSCLC) patients, I have identified a single nucleotide polymorphism (SNP) in one of the LCSs. This SNP is significantly more prevalent in lung cancer patients than in controls. Two independent primary lung cancer case-control studies confirm that moderate smokers with the variant allele of this SNP had an increased risk of developing non-small cell lung cancer. Therefore, this SNP can be used to screen for people at greater risk of developing lung cancer. Furthermore, this LCS is just one of ten putative LCSs in the KRAS 3' UTR. To determine which of the LCSs are most important for let-7-mediated repression, luciferase reporters with segments of the KRAS 3' UTR or mutated LCSs were transfected into human cells. The data suggest that there are multiple LCSs and let-7s involved in regulating KRAS expression. However, these results are complicated by the fact that my constructs with LCS1 actually have a variant SNP allele in LCS1. A more recent analysis of the mutations in the KRAS 3' UTR reveals that there has been an update to the 3' UTR sequence. This variant allele appears to affect KRAS expression and does not appear together with the variant LCS6 allele. Ultimately, gaining a better understanding of miRNA targets will serve to help further elucidate how miRNAs, and more specifically let-7, interact with their targets and are involved in human cancers. Finally, the discovery of a SNP associated with NSCLC in the KRAS 3' UTR suggests that there are other lung-cancer associated SNPs in the 3' UTRs of other oncogenes. Based on the parts of the MYC, EGFR, cyclin D2, and cyclin D1 3' UTRs sequenced from the Yale cohort of NSCLC patients, there are no polymorphisms of interest, suggesting that lung cancer-associated SNPs are not in all oncogenes and that the one identified in KRAS is significant.