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Title: Circulating MicroRNA as Novel Non-Invasive Biomarkers for Breast Cancer
Authors: Shaheen, Jawaria
Keywords: Biological & Medical Sciences
Issue Date: 2019
Publisher: University of the Punjab , Lahore
Abstract: Breast cancer is the second deadly malignancy worldwide. The occurrence rate of breast cancer is more in developing and under-developed. In Pakistan, it is the main cause of death in women which is an alarming indicator as these women are unaware of different diagnostic procedures and fright from their costs as well. So, the discovery of non-invasive and cost effective method of diagnosis and prognosis will provide the opportunity to lessen the misery of those who suffered from breast cancer and timely diagnosis will surely help them to go for treatment and spend healthy, risk free life. Nowadays, many screening procedures are available, but the issue of cost and invasiveness will remain the problem. The solution for this problem was given by researchers in the form of expression profiling of miRNAs. MicroRNAs are small, non protein coding RNA molecules which have role in regulation of cellular mechanisms either by activating or inhibiting target genes. These small molecules are known to be specific to different tissues of the body and it was revealed by research studies that when cancer develops, those tissue specific miRNAs shed in the body fluids i.e., blood as well. In present study, five miRNAs were selected which were reported previously to be involved in oncogenic activities and their deregulated expression was assumed to be highly linked with breast cancer. The use of DNA based primers for the expression analysis of these target miRNAs has given cost effective solution. The present study reports the highly deregulated expression levels of all five target miRNAs in plasma samples of breast cancer vs. healthy control samples, revealing their use as diagnostic biomarkers while their aberrant expression association with stages and tumor grade have made them good candidate for prognostic biomarkers as well. The blood samples were collected from 100 breast cancer patients and 25 age matched healthy controls, to investigate the expression of miRNA panel. Plasma was vii isolated by standard protocol of centrifugation and then isolation of total RNA including miRNA was done. Approximately, 100ng of isolated RNA was converted to cDNA after addition of poly (A) tailing. Expression profiling was done by using qRT-PCR technique in which mir-specific DNA primers were used to increase sensitivity and specificity of reaction. Expression of miRNA panel was normalized by using reference miRNA i.e., mir-16 and differences in expression of miRNA panel in breast cancer plasma samples and healthy control samples were calculated by 2-ΔΔCt method. Further statistical analysis was done by SPSS, medcalc and Graphpad prism softwares. Area under curve analysis was used to get diagnosis outcome/values of miRNA panel. The miRNA-target genes pathway has been proposed by using bioinformatics programs i.e., gene target prediction tools (miRwalk, DIANA, mRBD), gene interactions tool (STRING) and other breast cancer databases. Upregulation of miRNAs i.e., mir-155, mir-376c, mir-10b and mir-17 and downregulation of mir-181b, have been seen in the plasma samples of patient samples relative to normal control samples. The scatter plots, heatmap and other statistical analysis have given differences in expression of miRNA panel with significant p-value (p < 0.001) respectively. Kruskal Walis test and Mann Whitney U test have clearly displayed significance and association of miRNA panel with clinicopathological characteristics of breast cancer, which have revealed their role as diagnostic as well as prognostic biomarkers. AUC curve analysis has also remarked their high sensitivity and specificity and AUC calculated were 1.000 (mir-155), 0.976 (mir-376c), 0.932 (mir-10b), 0.797 (mir-17) and 0.964 (mir-181b) respectively, p < 0.001 was showing statistical significance. The proposed pathway has displayed the importance of these miRNAs having inter-related target genes, which presents these miRNAs as a panel to increase the sensitivity of procedure. viii We have identified that miRNA panel has highly deregulated expression levels in plasma of breast cancer patients relative to healthy controls, confirming their role as diagnostic biomarkers. The high expression fold change of mir-155, mir-376c, mir-10, mir-17 in early stages and tumor grade of disease and low expression of mir-181b in patient samples relative to normal control samples has also made the prognostic value of this panel. Secondly, by the use of mir-specific DNA primers, sensitivity of the qRT-PCR increases and minimizes the cost of procedure.
Gov't Doc #: 22865
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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