Role of microRNAs in Cancer

Author

Resha Bajracharya, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2012

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Mo, Yin-Yuan

Abstract

MicroRNAs are well known for their role in translational inhibition or mRNA degradation by base pairing to the 3' untranslated region (3'UTR) of the target mRNAs. Till date thousands of microRNAs have been reported, and the function of most microRNAs is yet to be determined. MicroRNAs have been described as "OncomiRs" or "Tumor suppressors" as some microRNAs are reported to be up-regulated whereas some are down-regulated in different types of tumors. To better understand the role of microRNAs in cancer, we were involved in two different projects. In our first project, the main objective was to identify the functionally important microRNA species that play role in the activation of Akt and study the mechanism through which these microRNAs mediate Akt activation. An extensive screening against LY294002 (PI3K inhibitor) was performed on the microRNA-library consisting of more than 600 microRNA precursors. LY29 concentration was optimized such that the drug killed most of the vector control MCF7 cells. Presence of some microRNAs assists in cell survival even in the presence of PI3K inhibitor due to their involvement in Akt activation. Based on this screening, we were able to identify 14 different microRNAs that helped cell survive the LY29 treatment. Western blots demonstrated that miR-9 and miR-943 consistently activated Akt which was further confirmed with cytotoxic assay that demonstrated increased cell survival in presence of miR-9 and miR-943. Online prediction tools identified PTEN as one of the potential target and luciferase reporter assay with 3'UTR of PTEN showed decreased luciferase intensity in presence of miR-9 and miR-943. Therefore, miR-9 and miR-943 were identified as Akt activators and a better understanding on their mechanism of action could help indicate their significance as a novel biomarker in tumorigenesis. In our second project, the major aim was to study the post transcriptional regulation of microRNAs by ADARs (Adenosine deaminases acting on RNA). The processing of microRNAs has been shown to be affected through A-to-I editing presumably because the stem-loop structure of the microRNA precursors makes them ideal targets for ADARs. Several microRNAs are known to be subject to ADAR mediated editing. However, the role of microRNA editing in cancer has not yet been studied. We performed IHC in breast tissue microarray which indicated that the level of ADAR1 is significantly higher in breast tumor tissue as compared to the normal breast tissue. Also, the expression of ADAR1 was tissue specific as there was not much difference in ADAR1 expression between the colon tumor vs. normal colon specimen. The increased expression of ADAR1 in breast tumor possibly alters microRNA editing and this deregulation in microRNA editing might be one of the potential causative of tumor. Although, due to some technical difficulty in amplifying pre-microRNAs we were unable to relate altered ADAR1 expression in breast tumor to deregulation in microRNA editing, none the less we were able to demonstrate a differential cell growth when ADAR was over-expressed and a significant increase of ADAR1 expression in breast tumor.