Abstract A23: Allele specific regulation of esophageal cancer related gene 2 by miR-1322 in esophageal squamous cell carcinoma

Abstract

Abstract Background and Aim: TCA3/TCA4 Short Tandem Repeat (STR) polymorphism in 3’ untranslated regions of esophageal cancer related gene 2 (ECRG2) was reported to be associated with esophageal squamous cell carcinoma (ESCC) incidences and the prognosis of ESCC patients. This study is to explore the mechanisms of TCA3 allele as a risk factor and poor prognosis indicator rather than TCA4 allele mainly in the functional relevant of microRNAs regulation in ESCC. Methods: Two genotypes of luciferase reporter plasmids containing ECRG2 3’UTR (TCA3 allele and TCA4 allele respectively) were constructed and expression were compared between the two alleles in three esophageal cancer cell lines: EC9706, EC109 and KYSE150. Bioinformatics predictions of microRNAs binding were performed by software TargetScan5.1, MicroInspector, RNA22 and RNAcofold. Dual-Luciferase reporter assay with or without microRNAs mimics or inhibitors were conducted to verify the effects of predicted microRNAs on expression of different alleles. The different expression of identified microRNAs in cancer tissues and the adjacent normal esophageal tissues from 44 ESCC patients were determined by Real-Time PCR. Results: Dual luciferase reporter assays indicated that expression level of TCA4 allele is 2-4 folds higher than TCA3 allele in all the three esophageal cancer cell lines. Bioinformatics analysis by software TargetScan5.1, MicroInspector, RNA22 and RNAcofold showed 4 potential microRNAs (miR-580, miR-1182, miR-1272 and miR-1322) binding at the TCA polymorphism site of the 3’UTR of ECRG2. Dual luciferase reporter assays indicated that miR-1182 and miR-1272 is not able to regulate either TCA3 or TCA4 allele in all three cell lines, however, miR-1322 and miR-580 dramatically down-regulated TCA3 allele expression (P <0.01) whereas slightly regulated TCA4 allele (P>0.05). The expression of miR-580 were under the detection threshold of real-time PCR analysis in most of the 44 paired tissue samples but the expression of miR-1322 were detectable, and 28 cases (63.6%) had at least 2-fold up regulation in their cancer tissues. No statistical differences were found between metastasis and non-metastasis and in different stage of disease. Conclusions: The differential regulation of TCA3 and TCA4 allele by miR-1322 might explain the difference in cancer risk and prognosis for different genotype carriers. MiR-1322 has an important role in human ESCC by regulating tumor suppressor gene ECRG2 and miR-1322 was up regulated in esophageal cancer, which itself might severs as a tumor promoter and potential marker and therapeutic target for esophageal squamous cell carcinoma. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A23.