DataSheet_1_Cross-reactivity between histone demethylase inhibitor valproic acid and DNA methylation in glioblastoma cell lines.pdf

Currently, valproic acid (VPA) is known as an inhibitor of histone deacetylase (epigenetic drug) and is used for the clinical treatment of epileptic events in the course of glioblastoma multiforme (GBM). Which improves the clinical outcome of those patients. We analyzed the level of 5-methylcytosine, a DNA epigenetic modulator, and 8-oxodeoxyguanosine, an cellular oxidative damage marker, affected with VPA administration, alone and in combination with temozolomide (TMZ), of glioma (T98G, U118, U138), other cancer (HeLa), and normal (HaCaT) cell lines. We observed the VPA dose-dependent changes in the total DNA methylation in neoplastic cell lines and the lack of such an effect in a normal cell line. VPA at high concentrations (250-500 μM) induced hypermethylation of DNA in a short time frame. However, the exposition of GBM cells to the combination of VPA and TMZ resulted in DNA hypomethylation. At the same time, we observed an increase of genomic 8-oxo-dG, which as a hydroxyl radical reaction product with guanosine residue in DNA suggests a red-ox imbalance in the cancer cells and radical damage of DNA. Our data show that VPA as an HDAC inhibitor does not induce changes only in histone acetylation, but also changes in the state of DNA modification. It shows cross-reactivity between chromatin remodeling due to histone acetylation and DNA methylation. Finally, total DNA cytosine methylation and guanosine oxidation changes in glioma cell lines under VPA treatment suggest a new epigenetic mechanism of that drug action.</p

DataSheet_2_Cross-reactivity between histone demethylase inhibitor valproic acid and DNA methylation in glioblastoma cell lines.pdf

Currently, valproic acid (VPA) is known as an inhibitor of histone deacetylase (epigenetic drug) and is used for the clinical treatment of epileptic events in the course of glioblastoma multiforme (GBM). Which improves the clinical outcome of those patients. We analyzed the level of 5-methylcytosine, a DNA epigenetic modulator, and 8-oxodeoxyguanosine, an cellular oxidative damage marker, affected with VPA administration, alone and in combination with temozolomide (TMZ), of glioma (T98G, U118, U138), other cancer (HeLa), and normal (HaCaT) cell lines. We observed the VPA dose-dependent changes in the total DNA methylation in neoplastic cell lines and the lack of such an effect in a normal cell line. VPA at high concentrations (250-500 μM) induced hypermethylation of DNA in a short time frame. However, the exposition of GBM cells to the combination of VPA and TMZ resulted in DNA hypomethylation. At the same time, we observed an increase of genomic 8-oxo-dG, which as a hydroxyl radical reaction product with guanosine residue in DNA suggests a red-ox imbalance in the cancer cells and radical damage of DNA. Our data show that VPA as an HDAC inhibitor does not induce changes only in histone acetylation, but also changes in the state of DNA modification. It shows cross-reactivity between chromatin remodeling due to histone acetylation and DNA methylation. Finally, total DNA cytosine methylation and guanosine oxidation changes in glioma cell lines under VPA treatment suggest a new epigenetic mechanism of that drug action.</p

Selected human miRNAs contained immunostimulatory motifs.

<p>The table presents the miRNA sequences with at least one UGUGU motif (highlighted in bold).</p

Pyrimidines- and purines-rich sequences of miRNAs.

<p>The table shows the content of the pyrimidines or purines nucleotides according to the sequence of miRNAs. The total contents of purines or pyrimidines of the miRNAs presented in the table is above 90%.</p

Occurrence and relative count of SSRs in mature miRNAs.

<p>Occurrence and relative count of all SSRs in mature miRNAs:mononucleotides, dinucleotides and the list of the longest tetra-and pentanucleotides.</p

The KEGG pathway analysis for GU-rich miRNAs.

<p>A. The table illustrating the: list of GU-rich miRNAs (first column); the IDs and KEGG pathways names (second and third column); the number of genes and GU-rich miRNAs involved associated with the pathways (fourth and sixth column). P-value was given in fifth column as a result of statistical analysis. P-value threshold is considered 0.05. B. GU-rich miRNAs in predicted pathway heat map. Significant miRNA-pathway interaction p<0.001.</p

Mature MiRNAs Form Secondary Structure, which Suggests Their Function beyond RISC

<div><p>The generally accepted model of the miRNA-guided RNA down-regulation suggests that mature miRNA targets mRNA in a nucleotide sequence-specific manner. However, we have shown that the nucleotide sequence of miRNA is not the only determinant of miRNA specificity. Using specific nucleases, T1, V1 and S1 as well as NMR, UV/Vis and CD spectroscopies, we found that miR-21, miR-93 and miR-296 can adopt hairpin and/or homoduplex structures. The secondary structure of those miRNAs in solution is a function of RNA concentration and ionic conditions. Additionally, we have shown that a formation of miRNA hairpin is facilitated by cellular environment.Looking for functional consequences of this observation, we have perceived that structure of these miRNAs resemble RNA aptamers, short oligonucleotides forming a stable 3D structures with a high affinity and specificity for their targets. We compared structures of anti-tenascin C (anti-Tn-C) aptamers, which inhibit brain tumor glioblastoma multiforme (GBM, WHO IV) and selected miRNA. A strong overexpression of miR-21, miR-93 as well Tn-C in GBM may imply some connections between them. The structural similarity of these miRNA hairpins and anti-Tn-C aptamers indicates that miRNAs may function also beyond RISC and are even more sophisticated regulators, that it was previously expected. We think that the knowledge of the miRNA structure may give a new insight into miRNA-dependent gene regulation mechanism and be a step forward in the understanding their function and involvement in cancerogenesis. This may improve design process of anti-miRNA therapeutics.</p></div

Nucleotide occupancy position in human mature miRNAs.

<p>A Web logo with the distinct nucleotides at all positions of the mature miRNA sequences. B. Nucleotides representation at the first position of miRNAs mature strand at 5’ and 3’ end, in context to their length. The number under the name of nucleotide (e.g. 108 for A) reflects 108 sequences with A as a first nucleoside at 5’ end, and 27 at 3’, respectively.</p

The KEGG pathway analysis for pyrimidine/purine (P/P)-rich miRNAs.

<p>A. The table illustrating the: list of P/P-rich miRNAs (first column); the IDs and KEGG pathways names (second and third column); the number of genes and P/P-rich miRNAs involved associated with the pathways (fourth and sixth column). P-value was given in fifth column as a result of statistical analysis. P-value threshold is considered 0.05. B. P/P-rich miRNAs in predicted pathway heat map. Significant miRNA-pathway interaction p<0.001.</p

Enzymatic probing (A, B, E, F) and NMR analysis (C, D, G, H, I) of miR-21 (A-D), miR-93 (E-H) and miR-296 (I).

<p><b>A, B, E, F</b>. Cleavage patterns obtained for limited hydrolysis of 5'-end labeled miR-21 (<b>A, B</b>) and miR-93 (<b>E, F</b>) with RNase V1, nuclease S1 and RNase T1 in native conditions. Lanes: C - reaction control; L – OH ladder; T1 - limited hydrolysis by RNase T1 (0.025u/µl) in denaturing condition. <b>A</b>. Lines V1 - limited hydrolysis with RNase V1 (0, 0.03125, 0.0625 or 0.125 u/µl). <b>B</b>. Lanes T1(N) - limited hydrolysis with RNase T1 (0, 0.04, 0.02 or 0.01 u/µl) in native conditions. <b>E</b>. Line V1 - limited hydrolysis with RNase V1 (0.125 u/µl). <b>F</b>. Lines S1 - limited hydrolysis with nuclease S1 (0.00475 u/µl). The increasing concentrations of RNase V1 and RNase T1 are indicated by arrows. Cleavage sites are indicated in autoradiogram. <b>C, D, G, H, I</b>. NMR analysis of miR-21 (<b>C, D</b>) miR-93 (<b>G, H</b>) and miR-296 (<b>I</b>) structures. <b>C</b>. The imino region of ¹H NMR spectrum of miR-21 (0.7 mM) recorded at 7°C in H₂O:D₂O (90%:10%) with 150 mM sodium chloride, 10 mM phosphate buffer and 0.1 mM EDTA. Resonances arising from the hairpin form are indicated with *. <b>D</b>. Imino region of the ¹H-¹H 2D NOESY spectrum of miR-21 at 15°C in H₂O:D₂O (90%:10%) with 150 mM sodium chloride, 10 mM phosphate buffer and 0.1 mM EDTA. The lines indicate the imino proton connectivity. <b>G</b>. The imino region of ¹H NMR spectrum of miR-93 (0.75 mM) recorded at 25°C in H₂O:D₂O (90%:10%) with 150 mM sodium chloride (top) or 50 mM sodium chloride (bottom), 10 mM phosphate buffer and 0.1 mM EDTA. Resonances arising from the hairpin form are indicated with *. <b>H</b>. Imino region of the ¹H-¹H 2D NOESY spectrum of miR-93 at 15°C in H₂O:D₂O (90%:10%) with 150 mM sodium chloride, 10 mM phosphate buffer and 0.1 mM EDTA. The lines indicate the imino proton connectivity. <b>I</b>. The imino region of ¹H NMR spectrum of miR-296 (0.3 mM) recorded at 25°C in H₂O:D₂O (90%:10%) with 150 mM sodium chloride, 10 mM phosphate buffer and 0.1 mM EDTA.</p