A SNP in a let-7 microRNA complementary site in the KRAS 3' untranslated region increases non-small cell lung cancer risk

Abstract

Lung cancer is the leading cause of cancer deaths worldwide, yet few genetic markers of lung cancer risk useful for screening exist. The let-7 family-of-microRNAs (miRNA) are global genetic regulators important in controlling lung cancer oncogene expression by binding to the 3' untranslated regions of their target mRNAs. The purpose of this study was to identify single nucleotide polymorphisms (SNP) that could modify let-7 binding and to assess the effect of such SNPs on target gene regulation and risk for non-small cell lung cancer (NSCLC). let-7 complementary sites (LCS) were sequenced in the KRAS 3' untranslated region from 74 NSCLC cases to identify mutations and SNPs that correlated with NSCLC. The allele frequency of a previously unidentified SNP at LCS6 was characterized in 2,433 people (representing 46 human populations). The frequency of the variant allele is 18.1% to 20.3% in NSCLC patients and 5.8% in world populations. The association between the SNP and the risk for NSCLC was defined in two independent case-control studies. A case-control study of lung cancer from New Mexico showed a 2.3-fold increased risk (confidence interval, 1.1-4.6; P = 0.02) for NSCLC cancer in patients who smoked <40 pack-years. This association was validated in a second independent case-control study. Functionally, the variant allele results in KRAS overexpression in vitro. The LCS6 variant allele in a KRAS miRANA complementary site is significantly associated with increased risk for NSCLC among moderate smokers and represents a new paradigm for let-7 miRNAs in lung cancer susceptibility.

Figure 1. Identification of a SNP in the KRAS 3′UTR

(A) Location of the putative LCSs in the KRAS 3′UTR. LCS1-LCS8 had been previously identified. LCSs where mutations are found are shown in red. The KRAS 3′UTR is 5016 bp, and the markers are positioned every 1000 bp. (B) The sequence of LCS6 with either the reference allele or the variant allele (red). (C) The variant allele in LCS6 was seen in 20.3% of the primary lung tumors and was present in the adjacent tissue when available (marked with an *).

Figure 2. Frequency of the reference allele across the world

Frequency of the reference allele (T) at the SNP locus was examined in 2433 people. They represent 46 populations from around the world, which were categorized based on geography: Africa (blue), Europe, Southwest Asia and Western Siberia (pink), South central Asia, East Asia and the Pacific (green), and the Americas (orange). The frequency of the reference allele (T) across all chromosomes tested is 97.1% and the frequency of the alternative allele (G) is 2.9%. The frequency of the G allele in African populations is 1.3%, in European populations is 7.6%, in Asian populations is 0.3%, and in Native American populations is 0.3%. The allele frequencies in the individual populations are in ALFRED.

Figure 3. The effect of the variant allele on KRAS and let-7 levels

(A) Luciferase reporter constructs containing the KRAS 3′UTR. Grey boxes signify the 3′UTR used in the reporter constructs. A blue star represents the variant allele in LCS6. (B) Representative graph of luciferase activity of KRAS wild-type and KRAS mLCS6 (P<0.009) in A549 cells. Triplicate repeats were conducted showing similar results. (C) let-7a, b, d and g levels in eight tumors with and eight without the variant allele. Samples were normalized to two non-cancerous patients, whose let-7 levels were similar. Error bars represent variation between PCR reactions for each sample.