Semester

Fall

Date of Graduation

2011

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Michael Ruppert.

Abstract

Arsenic is a well-studied human carcinogen. The mechanism by which arsenic induces cancer, however, is not fully understood. It is known that as a general stress inducer, arsenic can activate kinases, such as mitogen-activated protein kinases, leading to over activation of transcription factors. These transcription factors, including AP-1, NF-kB and Myc, are known to regulate the expression of early response genes, and likely to regulate miRNAs. The expression of miRNAs is often altered in cancer and other proliferative disorders. It is highly probable that miRNAs whose expressions are altered by arsenic will play a significant role in carcinogenesis. To test this hypothesis, we investigated: (1) the role of arsenic in the generation of miRNAs in the human bronchial epithelial cell line, BEAS-2B, using a microRNA array; (2) detailed the concentration-dependent regulation of the selected individual miRNA, such as miR-190, by arsenic using real-time PCR; (3) co-transcription of the intronic miRNA, miR-190, and its host gene, talin2, by a dual luciferase reporter gene assay and real-time PCR; (4) the potential target genes of miR-190 using in silico analysis, western blot, and 3'UTR reporter assays, and (5) the overall carcinogenic potential or cellular responses to arsenic-induced miR-190 through transient and stable overexpression of miR-190, followed by analytical tests such as western blot, proliferation assay and soft agar assay. The data obtained from this study show that arsenic is capable of inducing expression of several miRNAs, most remarkably miRNA-190 whose expression correlated with that of its host gene talin 2. In silico analysis of possible miR-190 targets indicated that this miRNA may be involved in tumor formation by targeting multiple proteins including PHLPP, an Akt phosphatase, and TP53INP1, a key cell apoptosis regulator. PHLPP is a known tumor suppressor that inactivates Akt by dephosphorylating serine 473, leading to decreased cell growth and enhanced apoptosis. PHLPP reporter assays indicate that miR-190 is a genuine PHLPP repressor that binds to the 3'UTR of the PHLPP mRNA. Kinase activation analysis demonstrated that miR-190 is able to mediate arsenic-induced Akt activation in a PHLPP dependant manner. Furthermore, overexpression of a miR-190 precursor could enhance the expression of VEGF, a growth factor downstream of Akt signaling responsible for enhancing tumor growth through neoangiogenesis and epithelial cell proliferation. Stable over expression of miR-190 led to an increase in colony number and size in a soft agar assay. This increase in colonies was accompanied by an increase in basal Akt phosphorylation and VEGF expression.;Taken together, these data suggest that arsenic is capable of inducing expression of miRNAs that may play critical roles in arsenic-induced carcinogenesis. Specifically, arsenic-induced miR-190 expression led to increased Akt activation and overall proliferation through repression of PHLPP. Accordingly, these findings not only revealed a novel mechanism of arsenic-induced carcinogenesis but also highlight a pathway which may be a good target for therapeutic intervention. Inhibiting miR-190 would lead to an elevated expression of PHLPP which inactivates Akt, and consequently, reduces potential for the malignant transformation of cells or the tumorigenesis of the transformed cells.

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