Genetic variant rs1058240 at the microRNA-binding site in the GATA3 gene may regulate its mRNA expression

The GATA binding protein 3 (GATA3) is a member of a family of 6 GATA dual zinc finger transcription factors (GATA1-6), which are required for the development and morphogenesis of the mammary gland. GATA3 is considered to play a dual role in oncogenesis and cancer development, whereas somatic GATA3 mutations have been reported in breast cancer. Variants of the GATA3 genetic 3′ untranslated region (3′UTR) microRNA (miRNA) binding sites have been associated with breast cancer risk. However, the roles of genetic variants in the GATA3 gene 3′UTR and its post-transcriptional regulation have not been fully elucidated. We discovered that rs1058240 in the GATA3 3′UTR displayed potential miRNA binding sites and this variant was found to be significantly associated with GATA3 mRNA expression (P=2.36E-07), suggesting that rs1058240 may be a putative variant mediating the post-transcriptional regulation of the GATA3 target gene. Further studies investigating the regulatory mechanism of GATA3 transcriptional activity are required to design novel strategies against breast cancer cell growth and differentiation.


Introduction
The GATA binding protein 3 (GATA3) is a member of the GATA family of zinc finger transcription factors that bind to the consensus 5'-(A/T)GATA(A/G)-3' motif (1). Human GATA3 exhibits 85% amino acid homology with human GATA1 in the DNA-binding domain, with no homology elsewhere in the protein, located on the 10p15 band of the human genome (2). GATA3, similar to other GATA family members, plays important roles in vertebrate embryonic organogenesis, including the mammary gland, sympathetic nervous system, parathyroid gland, kidney, inner ear, skin and T-cell lineages (3)(4)(5)(6)(7)(8)(9). Each GATA family member exhibits a distinctive pattern of expression in tissues and cell lines. The GATA3 protein is highly expressed in T-lymphoid cells and was suggested to be involved in the regulation of T-cell receptor α-and β-chain genes (2). GATA3 was identified in the luminal cells of mammary ducts and the body cells of terminal end buds, suggesting that GATA3 actively maintains luminal epithelial differentiation in the adult mammary gland, which suggests important implications in the pathogenesis of breast cancer (10). The majority of breast cancers arise from luminal epithelial cells; therefore, GATA3 appears to control a set of genes involved in the differentiation and proliferation of breast cancer cells. The expression of GATA3 is strongly associated with estrogen receptor (ER) expression in breast cancer (11) and there is accumulating evidence that GATA3 may be used as a clinical marker to determine response to hormonal therapy and refine the prognosis of breast cancer patients (12,13). The GATA3 gene was recently identified as a potential tumor marker and putative tumor suppressor gene in breast cancer, whose expression may be associated with a more fvorable prognosis and prolonged disease-free survival in breast cancer patients (14). A meta-analysis reported that GATA3 was one of the most significant genes exhibiting low expression in invasive carcinomas of the breast with poor clinical outcome, whereas low GATA3 expression was associated with a higher histological grade, positive nodes, larger tumor size, negative ER and progesterone receptor and HER2-neu overexpression (15).
To the best of our knowledge, microRNAs (miRNAs) may act as tumor suppressors and oncogenes by genetic variations in the 3' untranslated region (3'UTR) binding sites, regulating the target-gene expression post-transcriptionally (16). Chou et al (17) demonstrated that GATA3 increased the level of expression of miRNA (miR)-29b, which in turn repressed a network of prometastatic microenvironmental components, including angiopoietin-like 4, lysyl oxidase, matrix metalloproteinase 9 and vascular endothelial growth factor A, through binding to specific sequence motifs in their 3'UTR. The realisation that the GATA3-miR-29b axis regulates the tumor microenvironment and inhibits metastasis may open up novel possibilities for therapeutic intervention in breast cancer. However, the role of genetic variations in the miRNA binding sites of GATA3 has not been fully elucidated. Therefore, we Genetic variant rs1058240 at the microRNA-binding site in the GATA3 gene may regulate its mRNA expression tested our hypothesis that the GATA3 3'UTR variants may be associated with its mRNA expression by performing a bioinformatics analysis and genotype-phenotype association analysis based on the HapMap database.

Materials and methods
Bioinformatics and selection of polymorphisms. We identified the single-nucleotide polymorphisms (SNPs) in the GATA3 gene and coding region by searching the National Center for Biotechnology (Table I). As presented in Table I, rs1058240 has three potential mRNA binding sites, including hsa-miR-1299, hsa-miR-182 and hsa-miR-95. We listed the genotype frequencies of 3 SNPs among different populations. rs58582188 was excluded, as it was not found in the database (Table II). We calculated the pairwise LD values of all the SNPs in the GATA3 gene with a MAF of >0.05 and selected the SNPs not in LD (r 2 <0.8) to plot LD maps with the online SNP Function Prediction bioinformatics tool. The color of each SNP spot changing from red to white reflects the decrease in its D' value ( Fig. 1).   The GATA3 mRNA expression levels of the cell lines with rs2229360 and rs1058240 are illustrated in Fig. 2. There was no significant difference in GATA3 mRNA expression among the cell lines carrying rs2229360 variants (P=0.6012; Fig. 2A). The AA genotype of rs1058240 exhibited a significantly higher GATA3 mRNA expression level compared to the GG and GA genotypes (P=2.36E-07; Fig. 2B).

Discussion
The zinc finger transcription factor GATA3 was first identified in the early 1990s and is considered to be a marker of luminal breast cancers. GATA3 was confirmed to be necessary for the differentiation and maintenance of the luminal epithelium in the adult mammary gland (10). Usary et al (21) demonstrated that mutations in the second zinc finger may affect DNA binding, indicating its crucial role in the development and progression of breast cancer. GATA3 was identified as one of the most significantly mutated genes in breast cancer by whole-exome sequencing and GATA3 gene mutations were identified in 4 patients with luminal tumors, including 3 previously unknown frameshift mutations near the 3'-end of the coding sequence (22). GATA3 mutations in breast cancer may be associated with loss of DNA binding, aberrant nuclear localization, decrease in transactivational activity and alterations in invasiveness, but not cell proliferation (23). It is well known that miRNAs may function as tumor suppressors and oncogenes through interactions with the 3'UTR of their mRNA targets and may control the target-gene expression post-transcriptionally (16). For example, GATA3 may promote differentiation, suppress metastasis and alter the tumor microenvironment in breast cancer by inducing miR-29b  expression (17). It was also demonstrated that miR-30c was transcriptionally regulated by GATA3 in breast tumors (24). However, the roles of genetic variants in GATA3 gene 3'UTR and its post-transcriptional regulation has not been fully elucidated. Our data demonstrated that rs1058240 located in the GATA3 3'UTR displays 3 putative miRNA binding sites by using bioinformatics analysis; this SNP is significantly associated with the mRNA expression level, suggesting it may be partly involved in GATA3 post-transcriptional regulation.
Our findings may enable a better understanding of the roles miRNA variants in GATA3 3'UTR play in its mRNA expression and open up novel possibilities for therapeutic intervention in breast cancer.
In conclusion, our results indicated the vital role of GATA3 variants in 3'UTR in the post-transcriptional regulation of mRNA expression. However, the association of the regulation of GATA3 transcription with variations in the 3'UTR requires further validation to facilitate the development of novel therapeutic strategies.