TSS-targeted siRNA abolishes the heparanase expression in cancer cells.

<p><b>A</b>, scheme of siRNAs targeting TSS (locating at 101 bp upstream of the translation start site), upstream promoter and encoding regions of heparanase, including siH1 (−174/−155 bp), siH2 (−134/−115 bp), siH3 (−9/+10 bp) and siH4 (+1496/+1515 bp). Automated nucleotide basic local alignment search tool (BLAST) searches demonstrated that there was no sequence similar to siH1, siH2, or siH3 in the transcribed sequence of heparanase. <b>B</b>, 72 hrs post-transfection of siRNAs into PC-3, EJ and SGC-7901 cells, 100 nmol/L siH3 or siH4, but not of siH1, siH2 or siNC, attenuated the heparanase expression in cancer cells. <b>C</b>, 72 hrs post-transfection of siH3, but not of siNC or siScb, resulted in abolished heparanase expression of cancer cells in a dose-dependent manner. <b>D</b>, siH3 (100 nmol/L)-induced gene silencing of heparanase in cancer cells lasted up to 5 days, and partially restored at day 7. <b>E</b>, 72 hrs post-transfection of 100 nmol/L DNA duplexes analogous to siH3, siM31 or siM32 (two mismatched siRNAs of siH3), did not abolish the heparanase expression in cancer cells. <b>F</b>, dual-luciferase reporter assay indicated that 72 hrs post-transfection of siH3, but not of siScb, siM31 or siM32, suppressed the heparanase promoter activity and transcription in cancer cells. The symbols (# and Δ) indicate a significant decrease from untransfected control (mock) and a significant increase from pGL3/Basic, respectively.