A protector sequence against the NOR-1 3’-UTR seed region prevents the regulation by miR-17 and -20.

<p>HUVEC were transfected with precursors of either miR-17 or miR-20a (P17 or P20) or a scramble sequence (Scr) in the presence or in the absence of a miScript target protector that blocks the NOR-1 3’-UTR seed sequence recognized by the miR-17 and -20 (NOR1-Prot). Then cells were stimulated with VEGF (100 ng/ml, 2 h). NOR-1 expression was assessed by real-time PCR <b>(A)</b> and Western-blot <b>(B)</b>. Results are expressed as mean ± SD from at least n = 4. (<i>p</i><0.05: *, <i>vs</i>. untransfected cells [Control, CT] or cells transfected with Scr).</p

miR-17 and -20a regulate the expression of NOR-1.

<p>HUVEC were transfected with precursors of either miR-17 or miR-20a (P17 or P20) or a scramble sequence (Scr) in the presence or in the absence of their corresponding specific antagomirs (A17 or A20). Cells were stimulated with VEGF (100 ng/ml, 2 h). NOR-1 expression was assessed by real-time PCR <b>(A)</b>, Western-blot <b>(B)</b> and immunocytochemistry <b>(C)</b>. Results are expressed as mean ± SD from at least n = 4. (<i>p</i><0.05: *, <i>vs</i>. untransfected cells [Control, CT] or cells transfected with Scr).</p

The NOR-1 protector prevents the down-regulation of VCAM-1 promoted by miR-17 and -20.

<p>HUVEC were transfected with precursors of either miR-17 or miR-20a (P17 or P20) or a scramble sequence (Scr) in the presence or in the absence of a miScript target protector that blocks the NOR-1 3’-UTR seed sequence recognized by the miR-17 and -20a (NOR1-Prot). Then cells were stimulated with VEGF (100 ng/ml, 2 h). VCAM-1 expression was assessed by real-time PCR <b>(A)</b> and Western-blot <b>(B)</b>. Results are expressed as mean ± SD from at least n = 4. (<i>p</i><0.05: *, <i>vs</i>. untransfected cells [Control, CT] or cells transfected with Scr; #, <i>vs</i>. cells transfected with Scr and stimulated with VEGF; †, <i>vs</i>. cells exposed to the same condition but without NOR1-Prot).</p

NOR-1 transcript contains a miR-17/-20 putative binding site in the 3’-UTR.

<p>Schematic representation of the human NOR-1 3’-UTR transcript (NM_006981) showing the highly conserved miR-17/-20 binding site among species (positions 2894–2900 in NM_006981 sequence). The seed sequence of miR-17/-20 (nt 2–10) is indicated in bold.</p

KEGG pathways involving miRNA29, miRNA433 and miRNA25.

<p>Pathways involving gene targets for miRNA29, miRNA433 and miRNA25, as according to miRSystem software (ver. 20150312—mirsystem.cgm.ntu.edu.tw/). In order to draw inferences on potential functional interactions between miRNA and their gene targets, pathways identified are listed according to the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway map. Relevant nomenclature consists of a molecular network in terms of the KEGG Orthology (KO) groups. Genes are listed in each box according to their ability to serve as targets of each of the three miRNA considered in the upmost shaded headings. The miRNA targets involved in each specific pathway are reported according to a rank list where the first preferentially listed members, in each corresponding, box are common targets to more than one miRNAs.</p

VCAM1 expression in endothelial cells treated with N-homocysteinylated albumin.

<p>Panel A: Time course of induction of VCAM1 transcripts, in EAhy926 endothelial cells, by treatment with 1 µmol/L homocysteinylated albumin. Panel B: cytofluorimetric analysis of ICAM1 time course surface expression by EAhy926 endothelial cells treated with homocysteinylated albumin. (C: unmodified albumin negative control; Tnf-α: positive control). Panel C: Time course of ICAM1 release in the culture medium, quantitated by ELISA assay. C: negative control (untreated cells); A: unmodified albumin; AH: 1 µmol/L homocysteinylated albumin. (p<0.001).</p

Amplification conditions and primer pairs for PCR experiments.

<p>All conditions are relevant to real time PCR except for <i>VCAM1</i>, where tradition PCR has been employed. 35 amplification cycles have been performed.</p

Differential miRNA levels in the two outcome-related MPM patient groups.

<p>Fold change level for each miRNA is represented. Color code to each miRNA has been assigned in order to uniquely identify each miRNA within individual serum samples. Analyses were performed and calculation accomplished as described under “Material and Methods”. Bars, standard deviations derived from at least three different calculations.</p

Time-dependent, increased expression of ADAM17, MCP1 and Hsp60 in endothelial cells upon homocysteinylated albumin treatment.

<p>Panel A: Real time PCR evaluation during time course of <i>ADAM17</i>, <i>MCP1</i> and <i>Hsp60</i> mRNA. Panel B: ELISA assay of MCP1 released in the culture medium of treated cells. Panel C: Western blotting analysis of intracellular levels of ADAM17, and Hsp60, and analysis of Hsp60 released in the medium by immunoprecipitation and western blotting (Hsp60 IP). A: unmodified albumin control; AH: homocysteinylated albumin treatment. Levels of transcripts or proteins in the homocysteinylated albumin sample group were significantly increased compared to control (p<0.001).</p

Validation by Q-PCR of transcriptome results relevant to upregulated gene involved in endothelial dysfunction.

<p>A: unmodified albumin; AH: homocysteinylated albumin. Gene expression in the AH sample group was significantly increased with respect to the corresponding genes in the A sample group (p<0.001).</p