Master's thesis in Biological chemistry.Breast cancer is a heterogeneous disease. It is the leading cause of death due to cancer among women in the western world. With the increase in incidence of breast cancer and mostly the lymph node negative breast cancer, accurate prognosis is necessary to make the treatment effective and to avoid under and over treatment. In attempt to provide reliable diagnosis, and to find out which therapy is most suitable for a particular patient various prognostic biomarkers have been developed. microRNA is one among the potential biomarkers. These small non- coding RNA molecules exerts its effect through mRNA either by binding directly and specifically to 3’-UTR region (imperfect match) and silencing them or by enabling the degradation of target mRNA (perfect match).
In this study, expression level of nine different miRNAs (let-7b, miR-18b, miR-21, miR-25, miR-29c, miR-106b, miR375, miR505 and miR-150) was investigated using quantitative real time PCR (qPCR). One hundred and twenty-three formaldehyde fixed paraffin embedded (FFPE) tissues from the MMMCP project were used to isolate total RNA. Tumor infiltrating lymphocytes (TILs) were assessed from the samples to observe its prognostic value. Among the miRNAs studied, the biological function of let-7b was examined in ER negative (MDA- MB-231) and positive (MCF-7) breast cancer cell lines. For this, PNATM inhibitor was transfected and the corresponding miRNA was analyzed by using qPCR. The rate of proliferation and migration potential of the transfected cells was studied by wound healing assay. A immunohistochemical screening was performed to see the expression of markers in the let-7b inhibited. Furthermore, metabolic functions of the cells were studied by mitochondrial mito-stress test using a Seahorse XF analyzer. Independent t-test was used to detect significant correlations between miRNA expression and different clinical features of breast cancer.
Kaplan- Meier survival analysis showed that high MAI (>10) and high histological grade (≥2) were the most important prognostic factors for distance metastasis free survival. Among the nine miRNAs studied, miR-18b, miR-21, miR-25 and miR-150 are associated with high grade while miR-18b and miR-106b are associated with higher proliferation in breast tumor cells. Similarly let-7b and miR-150 were found to be associated with low and high TILs respectively while let-7b, miR-375 and miR-150 were found to be associated with older age (>50yrs). The expression of let-7b in both MCF-7 and MDA-MB-231 cells was successfully knocked out with an inhibitor concentration of 0.5μM. However, a wound healing assay performed in order to see the rate of proliferation, in absence of let-7b, did not provide any conclusive results. No distinct difference in the expression of markers was obtained except for Cyclin D1. Cyclin D1 was seen to be expressed higher in the cells transfected with inhibitor in both MCF-7 and MDA- MB-231 cells. Mitochondrial mito stress test done to study the cellular metabolism showed that, the mitochondrial parameters as well as ATP production was lowered in MCF-7 cell transfected with let-7b in comparison to the control. While MDA-MB-cell do not show any significant change the the mitochondrial respiration in absence of let-7b inhibitor. our results confirm the role of let7b as a tumor suppressor in breast cancer. Furthermore, we confirm the strong prognostic value of proliferation in lymph node negative breast cancer, also the prognostic
value of TILs is confirmed
