LIN28B polymorphisms influence susceptibility to epithelial ovarian cancer

Abstract

Defective microRNA (miRNA) biogenesis contributes to the development and progression of epithelial ovarian cancer (EOC). In this study, we examined the hypothesis that single nucleotide polymorphisms (SNP) in miRNA biogenesis genes may influence EOC risk. In an initial investigation, 318 SNPs in 18 genes were evaluated among 1,815 EOC cases and 1,900 controls, followed up by a replicative joint meta-analysis of data from an additional 2,172 cases and 3,052 controls. Of 23 SNPs from 9 genes associated with risk (empirical P < 0.05) in the initial investigation, the meta-analysis replicated 6 SNPs from the DROSHA, FMR1, LIN28, and LIN28B genes, including rs12194974 (G>A), an SNP in a putative transcription factor binding site in the LIN28B promoter region (summary OR = 0.90, 95% CI: 0.82-0.98; P = 0.015) which has been recently implicated in age of menarche and other phenotypes. Consistent with reports that LIN28B overexpression in EOC contributes to tumorigenesis by repressing tumor suppressor let-7 expression, we provide data from luciferase reporter assays and quantitative RT-PCR to suggest that the inverse association among rs12194974 A allele carriers may be because of reduced LIN28B expression. Our findings suggest that variants in LIN28B and possibly other miRNA biogenesis genes may influence EOC susceptibility.

Figure 1

Forest Plots for the Six SNPs Associated with Epithelial Ovarian Cancer Risk in the Joint Meta-analysis. Boxes denote OR point estimates, their areas being proportional to the inverse variance weight of the estimate. Horizontal lines represent 95% CIs. The diamond represents the summary OR computed under a fixed-effects model. The vertical line is at the null value (OR=1.0).

Figure 2

Functional Analysis of the LIN28B rs12194974 (-727 G/A) Polymorphism in vitro. A. Schematic representation of the LIN28B luciferase Reporter which contains an 1.4-kb promoter and 3 exons of LIN28B following IRES and luciferase gene. B. LIN28B SNP Promoter Activity in Ovarian Cancer Cell Lines. The -727-G and -727-A allele constructs were transfected into OV420, OV432, CAOV3, SKOV3, OV8, A2780CP, and A2780S ovarian adenonocarcinoma cell lines and T80 human immortalized ovarian surface epithelial cell line (HIOSE). β-galactosidase was used to standardize transfection efficiency. Relative luciferase activity (RLA) was measured. RLA units are presented as means, and the T bars represent standard deviations (SD). Mean ± SD from triplicates. Statistical analysis was conducted using Student’s t test. C. LIN28B SNP mRNA Expression in Ovarian Cancer Cell Lines and Immortalized Cell Line. The -727-G and -727-A allele constructs were transfected into A2780CP and A2780S and the T80 human cells. LIN28B SNP mRNA expression was determined by real-time PCR, and normalized with β-actin.