UBE2N and SMARCA4 as novel targets of miR-101.

<p>(a) Bio-informatics revealing the presence of miR-101 binding site at 3′UTR of UBE2N and SMARCA4, (b) activity of Luc gene showing a significant decrease in Luciferase activity when miR-101 was over-expressed in cells expressing Luc with 3′UTR of either (i & ii) UBE2N or (iii & iv) SMARCA4, in MCF7 and Hela cells. (c & d) Real-Time PCR showing a change in expression using Sybergreen and UBE2N and SMARCA4 specific primers, (c) under miR-101 and anti-miR-101 transfected conditions, showing decrease & increase, respectively, and (d) after 12 hrs exposure to 1uM, 5 uM and 10 uM etoposide, in MCF7 cells, confirming UBE2N and SMARCA4 as targets of miR-101, which is induced to 10 fold with 1 uM etoposide. At the higher concentrations of etoposide miR-101 expression is reduced leading to increased expression of UBE2N and SMARCA4, again confirming the relationship between miR-101 and the two novel targets.</p

Halo assay performed in MCF7 cells.

<p>(a) NDF-nuclear diffusion factor-values showing the extent of DNA damage with increasing concentrations of etoposide (p Value <0.001), (b) Percentage of senescent cell population (using β-gal assay) and Annexin-V positive-apoptotic-cell population (through FACS analysis): showing decrease in senescence and increase in Apoptosis (*p Value <0.05), (c) miR-101 expression (using Real-Time PCR): showing decrease with increasing concentration of etoposide (1 µM, 5 µM and 10 µM) for 12 hrs (**p Value <0.005).</p

miR-101 induces senescence and not apoptosis in MCF7 cells.

<p>(a) Original Flow-cytometry dot-blot of PI Staining Vs Annexin-V Staining, (b) Percentage of Annexin-V positive (apoptotic) and negative cells, (c) β-gal positively stained (senescent) cells, (d) Fold change in number of senescent cells, under control (mock-pEP-Null-transfected, mock-DMSO treated & mock negative with scrambled primers) and experimental conditions (pEP-miR-101 transfection, Etoposide 1 µM treatment & anti-miR-101 transfection). Time dependent increase in senescence in miR-101 transfected cells is also shown (d).</p

DNA methyltransferase 3 alpha and TET methylcytosine dioxygenase 2 restrain mitochondrial DNA-mediated interferon signaling in macrophages

Deleterious somatic mutations in DNA methyltransferase 3 alpha (DNMT3A) and TET mehtylcytosine dioxygenase 2 (TET2) are associated with clonal expansion of hematopoietic cells and higher risk of cardiovascular disease (CVD). Here, we investigated roles of DNMT3A and TET2 in normal human monocyte-derived macrophages (MDM), in MDM isolated from individuals with DNMT3A or TET2 mutations, and in macrophages isolated from human atherosclerotic plaques. We found that loss of function of DNMT3A or TET2 resulted in a type I interferon response due to impaired mitochondrial DNA integrity and activation of cGAS signaling. DNMT3A and TET2 normally maintained mitochondrial DNA integrity by regulating the expression of transcription factor A mitochondria (TFAM) dependent on their interactions with RBPJ and ZNF143 at regulatory regions of the TFAM gene. These findings suggest that targeting the cGAS-type I IFN pathway may have therapeutic value in reducing risk of CVD in patients with DNMT3A or TET2 mutations