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Breast cancer (BC) is the most frequently reported and diagnosed cancer worldwide and is one of the leading causes of mortality in women. Pakistan has the highest breast cancer incidence rate in Asia and majority of the patients present with advanced stage disease with poor survival. This situation accentuates towards the need of a safe and reliable method for early diagnosis of BC to improve the survival rate. MicroRNAs (miRNAs) are small non-coding RNAs. Abnormal expression of miRNA is a sign of various diseases, including cancer. They are remarkably stable in serum and other body fluids and can serve as reliable marker for early diagnosis of BC. The present study has included miRNA 497 and 195 which have not been studied in detail and correlated with clinico-pathological parameters in breast cancer till date. Hereditary breast cancer occurs in 5–10% of women, but those carrying BRCA1 and BRCA2 gene mutations have 40 to 84% life time high risk. Therefore, evaluation of BRCA genes mutations is helpful in predicting BC development on contralateral side and in siblings. The objectives of the present study were to assess the level of expression of miRNAs 497 and 195 in BC patients and to detect mutations in BRCA1 and BRCA 2 genes in BC patients. Additionally, to determine the association of grade and stage of BC with expression status of miRNAs. The samples and relative information of the patients were collected from various hospitals of Hyderabad and Karachi. A total of 209 females including 139 diagnosed BC cases and 70 controls matched for age were studied. Peripheral blood samples were used for extraction of total RNA. cDNA for miR-497 and miR-195 were prepared by using miR-497 antisense primer /Oligo(dT)18 Primer and miR-195 stem loop RT primers / gene specificantisense primer. GAPDH and U6 IT were used as control. The levels of expression of miRNA were recorded through amplification by qPCR. For detection of any mutation in BRCA 1 & 2 genes, DNA bands were identified by gel electrophoresis and visualized under Ultravioletilluminator. The ages of patients ranged from 25 to 65 years and majority of them belonged to 5th decade of life (71.22%). Only 15 % of the patients had history of BC in first degree relative. All of these patients (100%) presented with unilateral breast lump and out of these, 91.3% had breast lump larger than 5.0 cm. Axillary lymph node involvement was present in 64% of these patients. More than half of the patients (66.1%) were in grade II followed by 19.4% in grade I differentiation. The majority of BC patients (55.39 %) were in stage III. In total, out of 139 cases, 76.97% of BC patients and 8.57% of the controls showed downregulation of miRNA 497, while 72.66% of the BC patients and 15.71% of the controls showed downregulation of miRNA 195. Out of 89 patients who were axillary lymph node positive, 76.4% and 71.91% showed downregulation of miRNA 497 and 195 respectively. In contrast, out of the rest of 50 cases, who were axillary lymph nodes negative, 78% and 74%showed downregulation of miRNA 497 and 195 respectively. The results were highly significant at p < 0.001 in both groups. Results for correlation of both miRNAs with histological grade and clinical stage revealed that out of 27 cases of grade I, 92 cases of grade II and 20 cases of grade III BC, 55.55% (p=0.01), 85.86% (p=0.0001) and 65% (p=0.001) showed downregulation of miRNA 497 respectively, while 62.96% (p=0.001), 75% (p=0.0001) and 75% (p=0.001) revealed downregulation of miRNA 195 respectively. Almost similar results were found when correlation of miRNA 497 and 195 were done with clinical staging. It showed that, out of 21 cases of stage I, 39 cases of stage II, 77 cases of stage III and 2 cases of stage IV BC, 85.71%(p=0.01), 79.48%(p=0.001), 72.72%(p=0.001) and 100%(p=0.00001) of the cases showed downregulation of miRNA 497 respectively, whereas 80.95%(p=0.001), 64.1%(p=0.002), 74%(p=0.0001) and 100%(p=0.00001) of the cases showed downregulation of miRNA 195 in the above mentioned stages, respectively. The miRNAs 497 and 195 have been reported to be downregulated in BC. Few studies have been done regarding the level of expression of these miRNAs and their correlation with axillary lymph node involvement, grade and stage of BC. The results of the present study are highly significant and showed that levels of expression of both of these miRNAs are markedly downregulated in BC patients irrespective of lymph node involvement and grades and stages of tumor. All of these BC cases were also evaluated for selected mutations in BRCA 1 and BRCA 2 genes and the results showed the appearance of bands for wild-type alleles in all cases of BC. None of the case showed presence of bands for mutant alleles in any of the three selected founder mutations. In conclusion, the present study reports a significant association of miR-497- and miR-195 with histological grading, lymph node involvement and clinical staging. Both of these miRNAs are significantly down regulated in all grades and stages of BC, even in lymph node negative patients hence, they may be used asreliable, non-invasive biomarkers for the diagnosis and early detection of BC.
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