RHABDOMYOSARCOMA 2 ASSOCIATED TRANSCRIPT (RMST) AS A NEGATIVE REGULATOR OF MAMMALIAN TARGET OF RAPAMYCIN (MTOR) IN CANCER CELLS

Author

Entkhab Alanisi, Southern Illinois University CarbondaleFollow

Date of Award

12-1-2019

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Nie, Daotai

Abstract

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides that do not code for known proteins. These lncRNAs were originally thought as non-functional. However, loss and/or gain-of-function studies of these transcripts suggest that lncRNAs have crucial roles in many biological functions like apoptosis, cell cycle, translation control, epigenetic regulation, splicing regulation and many other processes in the cells. Lovastatin is a FDA-approved drug for treatment of hypercholesterolemia. Lovastatin can cause apoptosis in a number of tumor cells, but the mechanisms remain poorly understood.In this study, we set out to identify and determine lncRNAs that can regulate tumor cell responses towards lovastatin. We hypothesized that certain lncRNAs are stimulated by lovastatin treatment and these lncRNAs may serve as effectors for lovastatin to inhibit tumor cell proliferation and/or induce apoptosis. For our studies, PC-3, HCT116 and most other cell lines were obtained from ATCC. The cytotoxic effects of lovastatin on these cell lines were evaluated by proliferation assay, MTS assays and morphological changes at different doses and time intervals. The qPCR array was used to identify lncRNAs whose levels are changed by lovastatin treatment. The regulation of lncRNAs by lovastatin was further confirmed in multiple cell lines. Western blot was also used to determine the effects of lovastatin on the cellular signaling pathway. The ability of the cancer cells to proliferate was measured by colony forming assay. Loss- and gain-of-function experiments by using CRISPR-Cas9 approach and over expression experiments (sequentially) were used to determine the role of lncRNA identified in the cells. The lncRNA RMST promoter was analyzed using the UCSC genome and Genomatix browser websites. Chromatin immunoprecipitation (ChIP) assay was used to confirm the binding of SOX2 with RMST promoter (protein-DNA interaction). Finally, fluorescence in-situ hybridization (FISH) assay (to detect the localization and quantification of RMST in both cancer and normal prostate and colon human tissues) and Immunohistochemistry study (to detect the p-mTOR levels in both cancer and normal prostate and colon human tissues) were performed and then their levels were compared in both the normal and cancer human prostate and colon tissue studied.The lovastatin treatment has cytotoxic effects in multiple cell lines we evaluated. Lovastatin reduced mTOR signaling in cancer cells in a dose and time dependent manner. Through profiling array, we have identified a number of lncRNAs, including RMST, as responders to lovastatin. The lncRNAs identified were stimulated at RNA level after lovastatin treatment in a dose and time dependent manner (5 and 20 µM for 24 and 48 hrs). Over-expression of RMST attenuated mTOR signaling and reduced colony formation in tumor cells, phenocopying the cellular effects of lovastatin. Through heterozygous knock-out of RMST expression, we found that RMST is an important mediator for lovastatin to downregulate mTOR activities and reduce colony formation of tumor cells. Through ChIP analyses, we found the transcription factor SOX2 binding to RMST promoter and both SOX2 and RMST levels were affected differentially by lovastatin treatment. Moreover, we found that RMST and p-mTOR were inversely correlated in human prostate and colon tissues.Taken together, our data suggest that RMST lncRNA can be stimulated by lovastatin to elicit cellular responses including downregulation of mTOR signaling. Our data also suggest a possible role of SOX2 in regulating RMST expression. Furthermore, and most importantly, it has been found that RMST lncRNA and p-mTOR are inversely correlated in human normal and cancer (prostate, colon, and lymph nodes) tissues, suggesting RMST as a negative regulator of mTOR signaling.