Sense Single-stranded Phosphorothioate Oligodeoxy-nucleotide Conciding With Promoter Genes Inhibits The Promoter Activity Of Human α1(Ⅰ) Collagen Gene Induced By Insulin

Objective:(1)To investigate the effect of sense single-strandphosphorothioate oligodeoxynucleotide(ssPTODN) conciding with promotergenes on promoter activity of human COL1A1 gene induced by insulin.(2)Tolocate the response element of insulin on human COL1A1 gene moreprecisely.Methods: (1) The construct of pCOLH₁2.5 containing –2483 to +42 bp ofthe promoter and CAT gene as a reporter was stably transfected into WI-38cells.The cell clone integrated with pCOLH₁2.5 were treated with insulinof various dosages.(2) Four different ssPTODNs were synthesized. Variousdosage's ssPTODN1 coinciding with position -129 to -105 of COL1A1 genewere transitently transfected into the cell clone, consequently treatedwith insulin of 2.5μmol/ml.Results: (1) the WI-38 cell clone named PN1 containing pCOLH12.5 wasSuccessfully constructed. (2) At 0,0.25,2.5,10μmol/ml insulin,the CATvalues(pg/ml)were 920±94,1021±98,1595±101(P<0.01) and 1711±117a(P<0.01) respectively. But it showed no significance between 2.5 and10μmol/ml insulin (P>0.05).(3) At 0,40,80 ,120,160nM ssPTODN1, theCAT values(pg/ml)were 1593±87,1535±49,1370±54,1147±93(P<0.01)and 899±85(P<0.01) respectively in the presence of 2.5μmol/ml insulin.At 120nM ssPTODN1, insulin-induced increase in CAT value was abrogatedto basal level.Conclusions: (1)The ssPTODN coinciding with position -129 to -105 ofCOL1A1 gene can inhibit the promoter activity of human α1(I) collagen geneinduced by insulin.(2)The location of insulin response element on humanCOL1A1 gene is suggested between -129 and -105 in the promoter.