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Identification of pathogenic virus sequences in pancreatic ductal adenocarcinoma

Amaravadi, Mohanachary

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Abstract

Pancreatic ductal adenocarcinoma (PDAC) is by far the most common type of pancreatic cancer. It constitutes about 90% of tumors of the exocrine pancreas. The aggressive nature of PDAC along with a lack of diagnostic markers contributes to high lethality of this disease, which is nearly identical to its incidence. Studies from malignancies such as hepatocellular carcinoma and cervical cancer, along with the fact that liver and pancreas are in a close proximity, provided a plausible basis for the hypothesis of virus association in PDAC tumor development. However, there have been no established reports about virus(es) associated with pancreatic cancer.

The present study identified a new cancer-associated virus in human PDAC samples, called Meleagrid herpesvirus-1 (MeHV-1), or also known commonly as herpesvirus of turkeys, by two different and independent approaches: experimental genomic subtraction and digital microRNAome subtraction between healthy and PDAC patients. In the first approach, a genome-wide experimental comparison of DNA from PDAC tissues to DNA from tissues of healthy individuals was performed by representational difference analysis (RDA). Using this technique, differences in sequence composition were selectively isolated and amplified with very high sensitivity. Virus sequences associated with the occurrence of pancreatic cancer were detected by this process. The second approach, performed in parallel, involved a sequence analysis of the complete microRNA (miRNA) content of PDAC tissue samples. The sequencing data was digitally compared to databases of human and viral sequences so as to identify viral miRNAs. Because of the limited number of molecules, this analysis form did not need any experimental selection and amplification in order to achieve a sufficiently enough sensitivity to find viral microRNAs. The common results of the two analyses strongly suggested that MeHV-1 plays a crucial role in PDAC tumor progression. One of the viral microRNAs – hvt-miR-H14-3p – was studied in detail at the functional level by both in vitro and in vivo experiments in order to define the molecular mechanism of action with regard to its effect on pancreatic tumor carcinogenesis.

The key findings from this work include:

1. Identification of MeHV-1 DNA sequences in the PCR difference products (DPs) resulting from RDA on genomic DNA from PDAC and healthy tissues. 2. A tumor-specific MeHV-1 signature was also identified in the miRNA sequence analysis of tumor DNA, using an independent methodological approach. 3. RT-qPCR analyses showed that hvt-miR-H14-3p from MeHV-1 was expressed at significantly higher levels in PDAC and chronic pancreatitis (CP) tissues – CP being a chronic inflammation of the pancreas and a well-known risk factor of PDAC – than in healthy tissues. This observation was further verified using independent digital PCR platforms. 4. Metastatic and non-metastatic PDAC cell lines overexpressing hvt-miR-H14-3p showed a significant increase in migration and invasion compared to the respective controls, interestingly, without any significant change in proliferation. 5. Hvt-miR-H14-3p was found targeting cellular p27, down-regulating its expression. 6. The functional consequences of viral sequences identified in vitro could also be confirmed in vivo in NOD scid gamma mice.

In conclusion, this study is very significant in elucidating functional consequences of viral sequences in PDAC for the definition of relevant molecular effects responsible for carcinogenesis.

Item Type: Dissertation
Supervisor: Eils, Prof. Dr. Roland
Place of Publication: Heidelberg
Date of thesis defense: 29 July 2015
Date Deposited: 05 Aug 2015 06:52
Date: 2016
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
570 Life sciences
Controlled Keywords: Pancreatic Cancer, Virus, micro RNA, Genomic Integration
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