siRNA induces transcription initiation arrest, but not DNA methylation or histone deacetylation, of heparanase promoter.

<p><b>A</b> and <b>B</b>, bisulfite sequencing and MSP revealed that transfection of siRNAs (100 nmol/L), siH1, siH2 and siH3, did not induce the methylation of CpG islands of heparanase promoter in cancer cells. <b>C</b>, 72 hrs post-transfection, ChIP with distinct primer sets indicated that there were no PCR products for “no-antibody” (No Ab) control, and the binding of H3K9me2, H3K27me3 and AcH3 on the heparanase promoter did not change after transfection of siH2, siH3, siH4 or siScb in cancer cells. <b>D</b>, The binding of H3K9me2 and H3K27me3 was enriched on the promoter of p16 and RARβ2 compared to that on heparanase promoter in PC-3 cells, respectively. <b>E,</b> treatment of siH3- or siScb-transfected cancer cells with TSA (200 nmol/L), resulted in a significant increase in the binding of AcH3 on the heparanase promoter, when compared to those treated with DMSO solvent control. <b>F</b>, cancer cells were transfected with siRNAs and treated with 5-Aza-CdR (5 µmol/L) or TSA (200 nmol/L), resulting in no changes in siH3-induced heparanase silencing. <b>G</b>, 72 hrs post-transfection, ChIP assay indicated that the binding of Sp1 and EGR1 on the heparanase promoter, did not change after transfection of siH2, siH3, siH4 or siScb in cancer cells. <b>H</b>, ChIP assay with distinct primer sets indicated the decreased binding of RNA Pol II and TFIIB on heparanase promoter in siH3-transfected cancer cells, but not in siH2- or siH4-transfected cells. <b>I</b>, ChIP assay indicated that the decreased RNA Pol II or TFIIB binding was seen reproducibly and up to 7 days post-transfection of siH3 in cancer cells. <b>J</b>, qRT-PCR detection demonstrated that the relative inclusion-to-exclusion ratio of the first exon of heparanase did not change after transfection of siH2, siH3, siH4 or siScb in cancer cells. The symbol (#) indicates a significant decrease from untransfected control (mock) or siScb. The symbol (▴) indicates a significant increase from the binding on heparanase promoter. The symbol (Δ) indicates a significant increase from DMSO control.