Expression of Receptor for Advanced Glycation End Product mRNA and Protein in Cultured Hepatic Stellate Cells Inhibited by Specific Small Interfering RNA

Abstract: Three pair of small interfering RNA (siRNA) sequences (21 nt) directing to RAGE mRNA 417， 221 and 534 targets were designed by utilizing two RNA design softwares on line to stimulate secondary structure of receptor for advanced glycation end product (RAGE) mRNA in SD rats， and were converted into cDNA coding expression of small hairpin RNAs (shRNA) of siRNA for RAGE gene. These DNA sequences were synthesized and separately cloned into pGCsi-U6/Neo/GFP vectors and identified by digestion with restriction enzymes and sequence analysis. Specific recombinant siRNA expressive vectors were differently transfected into hepatic stellate cell (HSC)-T6 cell line with lipofectamine， HSC-T6 untreated and transfected unspecific recombinant siRNA expressive vector as control. The expression of RAGE genes and proteins in the cells were determined by real-time fluorescence quantitative PCR and Western blot. The results showed that specific recombinant siRNA expressive vectors targeting 417， 221 and 534 sites of RAGE mRNA coding domain (pGCsi-R1， pGCsi -R2 and pGCsi -R3) as well as unspecific recombinant siRNA expressive vector (pGCsi-C) were constructed successfully. Compared with blank control， the expression of RAGE at mRNA level was remarkably down-regulated in specific recombinant siRNA expressive vectors-transfeted HSC- T6， degree of down-regulation in RAGE mRNA increased in a concentration-dependent manner within 0.25?.0 nmol/L， especially pGCsi-R1 at 1.0 nmol/L， the expression of RAGE at mRNA level was not found any significant change in pGCsi-C-transfected HSC-T6. The expression of RAGE at mRNA level down-regulated 79.46%±3.5% (F=71.38， P<0.01)，78.96%±7.94% (F=61.82， P<0.01) and 73.11%±6.89% (F=61.98， P<0.01)， respectively， in PGCsi-R1- transfected HSC-T6 compared with blank control， extent of down-regulation in RAGE mRNA decreased in a time-dependent manner within 24-72 h. The expressions of 50 kDa and 46 kDa RAGE protein， alpha-smooth muscle actin (α-SMA) mRNA and protein in pGCsi-R1-transfected HSCs were markedly down-regulated by 43.91%±1.18% (F=365.19， P<0.01)， 36.33%±0.78% (F=386.07， P<0.01)，57.53%±3.25% (F=20.91， P<0.05) and 58.48%±3.08% (F= 56.59， P<0.05) of that in blank control cells. The results indicated that RAGE specific siRNA expressed by pGCsi-R1 could effectively inhibit RAGE gene and protein expression and activation in HSCs.

CSTR: 32200.14.cjcb.2007.05.0020