Cong Min, Liu Tianhui, Wang Ping, Xu Yong, Tang Shuzhen, Wang Baoen, Jia Jidong, Liu Yong, Hermonat Paul L, You Hong
Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, PR China.
Int J Mol Med. 2009 Nov;24(5):685-92. doi: 10.3892/ijmm_00000280.
Elevated tissue inhibitor of metalloproteinase (TIMP)-1 expression contributes to excess production of extracellular matrix in liver fibrosis. However, there are few studies on sustained suppression of TIMP-1. We aimed to construct a recombinant adeno-associated virus (AAV) carrying small interfering RNAs (siRNAs) of TIMP-1 and investigate the long-term effects of RNA interference upon the TIMP-1 gene in rat hepatic stellate cells (HSCs). Five siRNA oligomers targeting rat TIMP-1 were designed and transfected into HSCs. A U6 promoter followed by the siRNA which had the strongest suppression effect was cloned into the AAV vector and packed into 293 cells to construct the recombinant AAV/siRNA-TIMP-1/neo. After infecting HSCs with this recombinant AAV, the transcription and expression levels of the TIMP-1 and matrix metalloproteinase-13 (MMP-13) genes were detected at 4 and 12 weeks. Three of the five designed siRNA oligomers had a suppressing effect on TIMP-1 expression in rat HSCs within 72 h. The transcription and expression levels of TIMP-1 were suppressed significantly (P<0.05) following recombinant AAV/siRNA1-TIMP-1/neo infection and lasted 12 weeks. TIMP-1 expression in rAAV/siRNA1-TIMP-1/neo-infected HSCs was suppressed by 60% after four weeks and 90% after twelve weeks when compared to the control recombinant AAV/neo and uninfected HSCs. Furthermore, the transcription and protein expression levels of MMP-13, the main substrate of TIMP-1, were elevated by approximately 40% at twelve weeks in rAAV/siRNA-TIMP-1/neo-infected HSCs. RNA interference exerts suppressive effect on the TIMP-1 gene in cultured HSCs for a longer time when a recombinant AAV is utilized as the gene delivery vector.
金属蛋白酶组织抑制剂（TIMP）-1表达升高会导致肝纤维化中细胞外基质过度产生。然而，关于TIMP-1持续抑制的研究较少。我们旨在构建携带TIMP-1小干扰RNA（siRNA）的重组腺相关病毒（AAV），并研究RNA干扰对大鼠肝星状细胞（HSC）中TIMP-1基因的长期影响。设计了5种靶向大鼠TIMP-1的siRNA寡聚物并转染到HSC中。将具有最强抑制作用的siRNA后面接上U6启动子，克隆到AAV载体中并包装到293细胞中，构建重组AAV/siRNA-TIMP-1/neo。用这种重组AAV感染HSC后，在4周和12周时检测TIMP-1和基质金属蛋白酶-13（MMP-13）基因的转录和表达水平。5种设计的siRNA寡聚物中有3种在72小时内对大鼠HSC中TIMP-1的表达有抑制作用。重组AAV/siRNA1-TIMP-1/neo感染后，TIMP-1的转录和表达水平受到显著抑制（P<0.05），并持续12周。与对照重组AAV/neo和未感染的HSC相比，rAAV/siRNA1-TIMP-1/neo感染的HSC中TIMP-1的表达在4周后被抑制60%，12周后被抑制90%。此外，在rAAV/siRNA-TIMP-1/neo感染的HSC中，TIMP-1的主要底物MMP-13的转录和蛋白表达水平在12周时升高了约40%。当使用重组AAV作为基因递送载体时，RNA干扰对培养的HSC中的TIMP-1基因有较长时间的抑制作用。
