Date of Award

7-11-2021

Degree Type

Thesis

Degree Name

MPhil in Biological and Biomedical Sciences

First Advisor

Dr. Syed Hani Abidi

Second Advisor

Dr. Kulsoom Ghias

Third Advisor

Dr. Muhammad Nouman Mughal

Department

Biological and Biomedical Sciences

Abstract

Introduction: Globally prostate cancer is the second most commonly diagnosed cancer amongst all the cancers afflicting men. Despite being extensively studied, several molecular events involved in the onset and progression of neoplastic changes within the prostate gland remain poorly understood. Onset and/progression of prostate cancer can be attributed to host (including genetic) and environmental factors. In addition to these, pathogens, such as viruses, are often associated with prostate cancer. However, little is known about how these viral agents infect and exploit the host micro-environment to initiate or drive oncogenesis. Epstein Bar Virus (EBV) is one such virus that has been associated with prostate cancer, however, very little is known about how EBV contributes to the onset or progression of prostate cancer. Several studies have documented EBV-encoded proteins and miRNAs as drivers to neoplasia in non-prostate EBV-associated cancers, however, the role of these host miRNAs in prostate cancer has not been evaluated. The aim of this study was to detect EBV in prostate cancer tissues and perform differential expression analysis of several key prostate-related miRNAs, and target genes of differentiallyexpressed miRNAs in EBV-positive and -negative prostate cancer tissues.
Methodology: For this study, a total of 109 retrospectively collected formalin-fixed paraffin-embedded (FFPE) prostate cancer samples were deparaffinized and homogenized. Subsequently, from each sample, the RNA was extracted using the TRIzol method and converted into cDNA. The cDNA was initially used to determine the presence of EBV in the samples, using conventional PCR employing gene-specific primers against the EBV’s EBNA-2 gene. Subsequently, the cDNA from the EBV positive samples was used to determine the expression of EBNA-1, and - 2 (the hallmark of EBV infection) genes, as well as EBV-encoded miRNAs EBER-1, -2 using the quantitative real-time PCR employing gene-specific primers. The resulting EBV positive and negative samples were then grouped according to their Gleason scores and were randomly selected to include high, intermediate, and low Gleason score EBV-negative and -positive prostate cancer samples. The selected samples were then subjected to quantitative real-time PCR, to determine the differential relative expression of 25 host-specific miRNAs, as well as target genes (related to autophagy and chromatin remodeling complexes) of differentially expressed miRNA in EBV-negative and -positive prostate cancer samples. Finally, the Spearman’s correlation test was applied to determine the correlation between the expression of host miRNAs and autophagy and chromatin remodeling genes.
Results: Out of 109 samples, 39 (35.77%) were found positive for EBV. Among these EBV[1]positive samples, 38, 37, 36, and 26 samples exhibited expression of EBNA-1, EBNA-2, EBER-1, and EBER-2 genes, respectively. Analysis of the host-specific miRNAs in EBV-positive- and -negative samples revealed a significant difference (p< 0.05) in the relative expression of miR-152-3p, miR-21-5p, miR-452, and miR-146b in the EBV[1]positive and -negative samples. Analysis of correlation between the miRNA expression and the autophagy-associated markers showed a strong positive correlation between miR[1]146b and SQSTM-1, while a strong negative correlation between miR-21-5p and LAMP[1]2, ATG-5, and RFLP-34 in EBV-positive cancer samples. Similarly, an analysis of the correlation between the miRNA expression and the chromatin remodeling-associated genes showed a positive correlation between the miR-425 and DNMT-1, DNMT-2, and DNMT-3L, whereas negative correlation to HMGA-1 and Tet-3.
Conclusion: Detection of EBV in prostate cancer (especially those with high Gleason score) and down-modulation of some important key miRNAs and chromatin complex genes, such as HMGA-1, may suggest a contributory role of EBV in the onset/progression of prostate carcinoma. Furthermore, the differentially expressed miRNAs, especially in EBV[1]positive samples, may be considered as a therapeutic target for further exploration.

First Page

1

Last Page

101

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