rs12416605:C>T in MIR938 associates with gastric cancer through affecting the regulation of the CXCL12 chemokine gene

Abstract

Background: MicroRNAs are small regulatory RNAs with important roles in carcinogenesis. Genetic variants in these regulatory molecules may contribute to disease. We aim to identify allelic variants in microRNAs as susceptibility factors to gastric cancer using association studies and functional approaches.

Methods: Twenty-one single nucleotide variants potentially functional, because of their location in either the seed, mature or precursor region of 22 microRNAs, were selected for association studies. Genetic association with gastric cancer in 365 cases and 1,284 matched controls (European Prospective Investigation into Cancer and Nutrition Cohort) was analysed using logistic regression. MicroRNA overexpression, transcriptome analysis, and target gene validation experiments were performed for functional studies.

Results: rs3746444:T>C, in the seed of MIR499A and mature MIR499B, associated with the cardia adenocarcinoma location; rs12416605:C>T, in the seed of MIR938, associated with the diffuse subtype; and rs2114358:T>C, in the precursor MIR1206, associated with the noncardia phenotype. In all cases, the association was inverse, indicating a protective affect against gastric cancer of the three minor allelic variants. MIR499 rs3746444:T>C and MIR1206 rs2114358:T>C are reported to affect the expression of these miRNAs, but the effect of MIR938 rs12416605:C>T is unknown yet. Functional approaches showed that the expression of MIR938 is affected by rs12416605:C>T and revealed that MIR938 could regulate a subset of cancer-related genes in an allele-specific fashion. Furthermore, we demonstrated that CXCL12, a chemokine participating in gastric cancer metastasis, is specifically regulated by only one of the rs12416605:C>T alleles.

Conclusion: rs12416605 appears to be involved in gastric cancer by affecting the regulatory function of MIR938 on genes related to this cancer type, particularly on CXCL12 posttranscriptional regulation.

Keywords: functional SNV; gastric cancer; gene regulation; genetic susceptibility; microRNA.

Figure 1

Effect of rs12416605:C>T on MIR938 expression. (a) Outline of the miRNA co‐transfection experiments in HeLa cells using the pmR‐ZsGreen1 vector (Clontech) containing either one of the two MIR938 rs12416605 alleles or a nonhuman control miRNA used for normalization of transfection. (b) Box‐plot showing differences in the expression levels (ΔCt), measured by qPCR for the C and T MIR938 rs12416605 alleles after co‐transfection experiments, related to the control reference miRNA. Each experiment was done in triplicate and four independent experiments were performed. Data reported here are the means ± SEM of all experiments performed. A significant reduction between the expression of the C and T alleles was found (p < 0.05, Student's t test)

Figure 2

Allele‐specific regulation of the chemokine CXCL12 by MIR938. (a) Sequence alignment between the MIR938 rs12416605 C and T alleles and the CXCL12 mRNA showing the PITA predicted target site and ΔΔG PITA scores for both alleles on this site. (b) Results of the luciferase‐reporter assay testing the interaction between MIR938 rs12416605 C and T alleles and the 3′UTR of the CXCL12 in HeLa cells. Ratios of the Firefly and Renilla luciferase luminescence are presented after normalization to the empty plasmid pGL4.13. Each experiment sswas done in triplicate and six independent experiments were performed. Data reported here are the means ± SEM of all experiments performed. A significant reduction in the luciferase activity between both the alleles and between the C allele and the control miRNA was found (*, p < 0.01, Student's t‐test)