Mesenchymal stem cell epigenetic modulation after DNA methyltransferase inhibitor RG108 treatment

Author(s)

Rahyza Inacio Freire de Assis

• Document type: Master's Dissertation
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2017-02-17

Examining board members

Denise Carleto Andia; Rodrigo Augusto da Silva; Karina Gonzales Silverio

• Advisor: Denise Carleto Andia

Abstract

Background: The human bone marrow-derived mesenchymal stem cells (hBMMSCs) have been considered a promising source for cellular therapy, althoug their epigenetic regulation is not well understood. The RG108 is a DNA methyltransferase (DNMT) inhibitor, designed to block the active site of the DNMTs enzymes. Objective: This study was designed to better comprehend the basic biology of the hBMMSCs and the effects of the RG108 and the DNMT1 inhibition, in the epigenetic global levels and in specific genes from epigenetic machinery; in addition, core genes involved in maintaining the hBMMSCs in the undifferentiated state were also evaluated. Methods: The hBMMSCs were distributed in groups and treated during 3 days: DMEM ¿ cells were cultivated in complete culture medium; DMSO (RG108 vehicle) ¿ cells were cultivated in complete culture medium/DMSO; RG108 - cells were cultivated in complete culture medium/RG108 (50 µM) and the RG108 effect was assessed through cell viability, population doubling time and apoptosis assays. The global DNA methylation and hydroxymethylation levels, the demethylases and the DNMTs activities and the DNMT1 protein levels were assessed by colorimetric assays. The DNMT1, TET1 and OCT4 genes transcript levels were quantified by quantitative real-time polymerase chain reaction (qPCR) and the DNMT1/3B/NANOG genes methylation/hydroxymethylation status were evaluated by qPCR, preceded by glucosylation of 5-hydroxymethylcytosine and restriction enzymes digestion. Results: The RG108 did not reduce the cell viability or induce cellular death by apoptosis and the proliferation rate in vitro was not affected. At global levels, the RG108 significantly decreased methylation status (p ? 0.05), without modulation at hydroxymethylation levels. There was a significant decrease in the DNMTs activity levels for DMSO and RG108 treated cells, comparing to the DMEM group (p ? 0.05); in an antagonistic way, the demethylases activity levels were found to be increased for the DMSO and RG108 groups, comparing to the DMEM group (p ? 0.05), without changing in the DNMT1 protein levels. The DNMT1 mRNA levels were decreased for DMSO and RG108 groups (p ? 0.05) and the RG108 treated cells showed an increase in the TET1 mRNA levels (p ? 0.05) and OCT4 mRNA levels (p ? 0.05), comparing to the DMEM group. There was no modulation in the DNMT1 gene methylation/hydroxymethylation levels; however, a significant increase in the methylation levels of DNMT3B was observed for the DMSO and RG108 groups, comparing to the DMEM group (p ? 0.05) and no modulation in the hydroxymethylation levels was found. On the other hand, the NANOG gene have responded significantly decreasing the hydroxymethylation levels for the RG108 group (p ? 0.05), without modulation in its methylation levels. Conclusions: The RG108 is a safe demethylating agent, with the ability of modulating the DNMTs and demethylases activities, in the hBMMSCs. This modulation was able to trigger changes at global status and transcription levels; however, no modulation was found at DNMT1 protein and gene levels; yet it was observed at DNMT3B gene instead, also reflecting at NANOG gene, which was modulated at hydroxymethylation levels. The epigenetic modulator RG108 and the DNMT1 inhibition are capable of promoting changes in the epigenetic machinery genes and in relevant genes for maintaining the hBMMSCs in an undifferentiated state. Interestingly, the DMSO was also capable of triggering modulation, however it was restricted to the epigenetic machinery genes (AU)

• FAPESP's process: 15/02160-0 - Human bone marrow mesenchymal stem cells demethylation: epigenetic regulation
• Grantee: Rahyza Inacio Freire de Assis
• Support Opportunities: Scholarships in Brazil - Master