ANALYSIS OF MICRORNA SIGNATURES AS BIOMARKER TO INVESTIGATE INTERLINK BETWEEN TYPE 2 DIABETES AND BREAST CANCER
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Type 2 diabetes and breast cancer are two heterogeneous, multifactorial, chronic health problems involving several overlapping risk factors. Studies have suggested that type 2 diabetes is associated with 10-20% excessive relative risk of breast cancer. Evidence indicates link between type 2 diabetes and breast cancer, through insulin resistance and hyperinsulinemia. Numerous substantial evidence pointing towards the potential efficacy of antidiabetic metformin as anticancer therapeutics. MicroRNAs are endogenous, small non-coding RNA molecules regulating protein-coding gene expression and participate in nearly all the events of life. These small RNA molecules can have diagnostic or prognostic value, as microRNA expression profiles reflect disease origin, stage and other pathological factors. We hypothesized that there might be several microRNAs which commonly function in the “origin of type 2 diabetes to progression towards breast cancer.” Such common microRNAs can act via the related signalling pathways which may provide the critical insight into the better understanding of these diseases. The present study is aimed to investigate the interlinking between type 2 diabetes and breast cancer through microRNA signatures. Methods: In vitro cell experiments (using breast cancer cell lines MCF-7, MDA-MB-231, & T47D and pancreatic beta insulinoma cell lines MIN6 and RIN-5F) referred as MTT proliferation, trypan blue exclusion test, NBT assay, colony formation analysis, and scratch assay. Reactive oxygen species (ROS) assays (DCFH-DA and DHE) along with fluorescence microscopy (DAPI staining, Acridine orange + Ethidium bromide dual staining, JC1 staining) were used for apoptotic parameters. Insulin release in pancreatic beta cell lines was measured by ELISA. mRNA expression levels of Bax, Bcl-2, MMP-2, MMP-9, SOD 1, SOD 2, SOD 3, were quantified by qRT-PCR. Four common microRNAs- let 7a, miR-21, miR-155, miR-375 expression profiling in both breast cancer cell lines and pancreatic cell lines was performed by relative quantification real time analysis. Results: Insulin acts as a potential mitogenic factor accelerating the proliferation of breast cancer cells. On the other hand, metformin inhibits growth, proliferation and v clonogenic potential of breast carcinoma cells. ROS levels in breast cancer cells were significantly reduced by metformin by up-regulating SOD isoforms expression. Insulin increased the ROS to a very small limit. Metformin activates apoptosis by inducing mitochondrial dysfunction, upregulating Bax and downregulating Bcl-2. Migration is strongly suppressed by metformin by regulating matrix metalloproteinase (MMP-2 and MMP-9). Oncogenic miR-21 and miR-155 were downregulated by metformin, significantly correlated with reduced metastasis. The results of our study suggest that both MIN6 and RIN-5F cells show a significant differential pattern of proliferation, insulin secretion, and microRNA expression pattern. RIN-5F beta cells were found to be highly refractory to glucose-stimulated insulin secretion. However, metformin negatively regulates glucose-stimulated insulin release in both MIN6 and RIN-5F. In MIN6 cells, levels of microRNA-375 and let-7a were significantly up- & down-regulated by metformin at normal-glucose and high glucose culture conditions respectively whereas in RIN-5F both were significantly down-regulated. Conclusions: Our data supports that metformin plays a pivotal role in the modulation of the antioxidant system including SOD machinery. Our results indicate that metformin inhibit breast cancer cell proliferation by inducing apoptosis via mitochondrial signalling. Furthermore, emerging view from this study is that microRNAs (let-7a, mir-21, miR-155 and miR- 375) are involved in the process of disease (type 2 diabetes and breast cancer) development, and there is the potential utility of microRNAs as effective biomarker for diagnostic and prognostic application in type 2 diabetes and breast cancer.
- PhD 
- Plant Sciences-Research Publications