Decreased Expression of Bioinformatically Predictedpiwil2-targetting microRNAs, miR-1267 and miR-2276in Breast Cancer

Abstract Purpose: Human Piwil2, a member of Piwi subfamily of Argonaute proteins, is primarily expressed in testis, where it regulates self-renewal of germ cells. However, its ectopic expression has been reported with several tumors, including breast cancer. The upregulation of piwil2 in various stages of breast cancer suggested its suitability as a novel tumor biomarker. Considering the vital role of microRNAs (miRNAs) in regulating the expression of most human genes, we hypothesized a concomitant downregulation of the bioinformatically-predicted piwil2-targetting microRNAs in breast cancer. Method: We employed different bioinformatic tools to predict piwil2-targeting miRNAs. Then, from the list of predicted miRNAs, we chose two less studied miRNAs (miR-1267 and miR-2276) for experimental validation. Using a real-time RT-PCR approach, we 􀁔􀁘􀁄􀁑􀁗􀁌􀂿􀁈􀁇 the relative expression of the miRNAs in 31 pairs of formalin-􀂿􀁛􀁈􀁇 􀁓􀁄􀁕􀁄􀁉􀂿􀁑-embedded tumor/non-tumor tissue samples. Results: Our data revealed a noticeable but not statistically 􀁖􀁌􀁊􀁑􀁌􀂿􀁆􀁄􀁑􀁗 (P = 0.133) downregulation of miR-1267 in tumor samples, compared to non-tumor samples obtained from the same patients. Downregulation of miR-1267 was more 􀁖􀁌􀁊􀁑􀁌􀂿􀁆􀁄􀁑􀁗 in higher grades of malignancies (fold change = 2.39, P = 0.033) and also in lymph nodes containing high-grade tumor cells (fold change = 6.66, P = 0.02). Interestingly, a 􀁖􀁌􀁊􀁑􀁌􀂿􀁆􀁄􀁑􀁗 upregulation of miR-1267 was observed in tumors at high stages (stage 3a, 3b), compared to low stages (stage 2a, 2b) (fold change = 8.05, P = 0.048). 􀀶􀁌􀁐􀁌􀁏􀁄􀁕 patterns of expression alteration were also observed for miR-2276. Conclusion: Altogether, our 􀂿􀁑􀁇􀁌􀁑􀁊􀁖 suggest a probable tumor suppressor role for miR-1267 and miR-2276 in breast tumor initiation and progression, but a probable promoting role for them in invasion and metastasis. Abstract Purpose: Human Piwil2, a member of Piwi subfamily of Argonaute proteins, is primarily expressed in testis, where it regulates self-renewal of germ cells. However, its ectopic expression has been reported with several tumors, including breast cancer. The upregulation of piwil2 in various stages of breast cancer suggested its suitability as a novel tumor biomarker. Considering the vital role of microRNAs (miRNAs) in regulating the expression of most human genes, we hypothesized a concomitant downregulation of the bioinformatically-predicted piwil2-targetting microRNAs in breast cancer. Method: We employed different bioinformatic tools to predict piwil2-targeting miRNAs. Then, from the list of predicted miRNAs, we chose two less studied miRNAs (miR-1267 and miR-2276) for experimental validation. Using a real-time RT-PCR approach, we 􀁔􀁘􀁄􀁑􀁗􀁌􀂿􀁈􀁇 the relative expression of the miRNAs in 31 pairs of formalin-􀂿􀁛􀁈􀁇 􀁓􀁄􀁕􀁄􀁉􀂿􀁑-embedded tumor/non-tumor tissue samples. Results: Our data revealed a noticeable but not statistically 􀁖􀁌􀁊􀁑􀁌􀂿􀁆􀁄􀁑􀁗 (P = 0.133) downregulation of miR-1267 in tumor samples, compared to non-tumor samples obtained from the same patients. Downregulation of miR-1267 was more 􀁖􀁌􀁊􀁑􀁌􀂿􀁆􀁄􀁑􀁗 in higher grades of malignancies (fold change = 2.39, P = 0.033) and also in lymph nodes containing high-grade tumor cells (fold change = 6.66, P = 0.02). Interestingly, a 􀁖􀁌􀁊􀁑􀁌􀂿􀁆􀁄􀁑􀁗 upregulation of miR-1267 was observed in tumors at high stages (stage 3a, 3b), compared to low stages (stage 2a, 2b) (fold change = 8.05, P = 0.048). 􀀶􀁌􀁐􀁌􀁏􀁄􀁕 patterns of expression alteration were also observed for miR-2276. Conclusion: Altogether, our 􀂿􀁑􀁇􀁌􀁑􀁊􀁖 suggest a probable tumor suppressor role for miR-1267 and miR-2276 in breast tumor initiation and progression, but a probable promoting role for them in invasion and metastasis. Keywords: Biomarker, breast cancer, gene expression, microRNAs, piwil2