Regulation of Chromatin Modifier Genes by Microrna Vis-À-Vis Regulation of Microrna by DNA Methylation and Histone Modifications in Human Cancer

Abstract

This thesis deals with the role of microRNA (miRNA) regulating other epigenetic modifiers like DNA methyltransferase 1 (DNMT1), and histone methyltransferase myeloid/lymphoid or mixed-lineage leukaemia (MLL1) also known as Histone-lysine N-methyltransferase 2A. It also divulges the reason for aberrant expression of miRNAs (miR-152, miR-148a, and miR-193a) in breast and prostate cancer. Silencing of the miR-152 gene due to promoter DNA methylation alter the expression pattern of several other genes. E-cadherin (CDH1) forms the core of adherent junctions between surrounding epithelial cells, link with the actin cytoskeleton and affects cell signalling. CDH1 gene is downregulated by promoter DNA methylation during cancer progression. In this investigation, we attempt to elucidate the correlation of miR-152 and CDH1 function, as it is well known that the loss of CDH1 function is one of the primary reasons for cancer metastasis and aggressiveness of spreading. For the first time here it has been shown that loss of CDH1 expression is directly proportional to the loss of miR-152 function in breast cancer cells. mRNA and protein expression profile of DNMT1 implicate that miR-152 targets DNMT1 mRNA and inhibits its protein expression. Tracing the molecular marks on DNA and histone 3 for understanding the mechanism of gene regulation by ChIP analyses leads to a paradoxical result that shows DNA methylation adjacent to active histone marking (enrichment of H3K4me3) silence miR- 152 gene.
This thesis also demonstrated that miR-148a remains downregulated in hormone-refractory prostate cancer compared to other healthy cells and its upregulation induce apoptosis in hormone-refractory and metastatic prostate cancer cells. Here for the first time, it was analyzed the role of miR-148a in the regulation of DNMT1 in prostate cancer cells. The ectopic expression of miR-148a shows a noticeable amount of programmed cell death and repression of cancer cell proliferation. It also revealed the silencing of miR-148a in prostate cancer cells was done by DNMT1. This finding gives a new avenue to targeting prostate cancer cells and proved the role of miR-148a as a therapeutic. Moreover, other experiments also demonstrate the regulation of MLL1 by miR-193a. MiR-193a has been downregulated in prostate cancer by DNA methylation and help in MLL1 overexpression during prostate cancer progression. Most importantly it was found by inhibiting MLL1 it changes the global H3K4 methylation pattern increasing the monomethylation and decreasing trimethylation at H3K4 positions. H3K4 trimethylation is an active gene mark present in various oncogenes during cancer progression. By inhibiting H3K4, tri-methylation cancer progression can be repressed. Ectopic expression of miR- 193a results in cell death, inhibition of cellular migration, and anchorage-independent growth of cancer cells. All together this thesis supports that miR-152, miR-148a, and miR-193a are regulated by DNA methylation, and they affect the expression of the various epigenetic modifiers. Hence these can be targeted for therapeutic intervention for breast and prostate cancer.