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

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

DNA methylation regulates Microtubule-associated tumor suppressor 1 in human non-small cell lung carcinoma

Microtubule associated tumor suppressor 1 (MTUS1) has been recognized as a tumor suppressor gene in multiple cancers. However, the molecular mechanisms underlying the regulation of MTUS1 are yet to be investigated. This study aimed to clarify the significance of DNA methylation in silencing MTUS1 expression. We report that MTUS1 acts as tumor suppressor in non-small cell lung carcinoma (NSCLC). Analysis of in silica database and subsequent knockdown of DNMT1 suggested an inverse correlation between DNMT1 and mars]. function. Interestingly, increased methylation at MTUS1 promoter is associated with low expression of MTUS1. Treatment with DNA methyltransferases (DNMTs) inhibitor, 5-aza-2'-deoxycytidine (AZA) leads to both reduced promoter methylation accompanied with enrichment of H3K9Ac and enhanced MTUS1 expression. Remarkably, knockdown of MTUS1 showed increased proliferation and migration of NSCLC cells in contrast to diminished proliferation and migration, upon treatment with AZA. We concluded that low expression of MTUS1 correlates to DNA methylation and histone deacetylation in human NSCLC

Epigenetic silencing of genes enhanced by collective role of reactive oxygen species and MAPK signaling downstream ERK/Snail axis: Ectopic application of hydrogen peroxide repress CDH1 gene by enhanced DNA methyltransferase activity in human breast cancer

Loss of E-cadherin and epithelial to mesenchymal transition (EMT) are key steps in cancer progression. Reactive oxygen species (ROS) play significant roles in cellular physiology and homeostasis. Roles of E-cadherin (CDH1), EMT and ROS are intriguingly illustrated in many cancers without focusing their collective concert during cancer progression. We report that hydrogen peroxide (H2O2) treatment modulate CDH1 gene expression by epigenetic modification(s). Sublethal dosage of H2O2 treatment decrease E-cadherin, increase DNMT1, HDAC1, Snail, Slug and enrich H3K9me3 and H3K27me3 in the CDH1 promoter. The effect of H2O2 was attenuated by ROS scavengers; NAC, lupeol and beta-sitosterol. DNMT inhibitor, AZA prevented the H2O2 induced promoter-CpG-island methylation of CDH1. Treatment of cells with U0126 (inhibitor of ERK) reduced the expression of DNMT1, Snail and Slug, increased CDHL This implicates that CDH1 is synergistically repressed by histone methylation, DNA methylation and histone deacetylation mediated chromatin remodelling and activation of Snail and Slug through ERK pathway. Increased ROS leads to activation of epigenetic machineries and EMT activators Snail/Slug which in their course of action inactivates CDH1 gene and lack of E-cadherin protein promotes EMT in breast cancer cells. ROS and ERK signaling facilitate epigenetic silencing and support the fact that subtle increase of ROS above basal level act as key cell signaling molecules. Free radical scavengers, lupeol and beta-sitosterol may be tested for therapeutic intervention of breast cancer. This work broadens the amplitude of epigenome and open avenues for investigations on conjoint effects of canonical and intrinsic metabolite signaling and epigenetic modulations in cancer