关键词: 热休克蛋白47, RNA干扰, shRNA, NIH/3T3细胞, I/III型胶原, 转染

Abstract:

Objective  To construct a short hairpin RNA（shRNA） against HSP47 gene， assess the expression level of HSP47 gene in NIH/3T3 cells， and observe the influence on cell function.  Methods  The HSP47-shRNA sequence presented at the downstream of the U6 promoter. The shRNA expression constructs were created using PCR-based methods. The PCR product was digested with NheⅠ/HindⅢ and ligated into pGCsi/U6/Neo vector to produce HSP47-pGCsi-U6-shRNA（HSP47-1-pGCsi-U6-shRNA，HSP47-2-pGCsi-U6-shRNA and HSP47-3-pGCsi-U6-shRNA）. The non-interference vector and non-related interference vector served as control. The vectors were transfected into NIH/3T3 fibroblast cells by liposome mediated gene transfection method. Transfection efficiency and fluorescence intensity were determined by fluorescence microscopy at 12， 24， 48， and 72 hours after transfection， respectively. Cells were collected before transfection, and at 24， and 48 hours post-transfection， respectively， HSP47 mRNA and protein expression levels were assessed by real-time PCR and Western blotting. The mRNA expression of TGF-β1， collagen types Ⅰ and Ⅲ in NIH/3T3 cells， and TGF-β1 levels in cell culture supernatant were determined.  Results  HSP47-shRNA vector was transfected into NIH/3T3 cells by liposome-mediated transfection. The transfection efficiency in each HSP47-shRNA plasmid interference group was about 60.0%， and there is no statistical difference among the interference groups（P>0.05）. A small amount of green fluorescent cells were found at 12 h post-transfection. The number of green fluorescent cells increased with the transfection time， and reached strongest at 72 h after transfection. shRNA interference significantly inhabited HSP47 expression in NIH/3T3 cells. At 24 h after transfection with HSP47-1-shRNA， the inhibition effect was the strongest， and the relative silence efficiency of HSP47 mRNA was（25.83±1.79）%， lower than that of control group and non-related group（P＜0.05）. Collagen synthesis and secretion by NIH/3T3 cells reduced significantly at 24 and 48 hours post-transfection with HSP47-1-shRNA； and there was a significant difference between HSP47-1-shRNA intervention group and non-related controls. After transfection for 24 and 48 h， mRNA expression of collagen types Ⅰ and Ⅲ decreased to （56.52±1.64）% and （53.48±2.54）%， （54.17±2.63）% and （50.21±2.34）%， respectively， significantly lower than that of the control group and non-related group （P<0.05）； however， no significant difference was found among the interference groups（P>0.05）. In each HSP47-shRNA plasmid interference group， TGF-β1 mRNA expression was the lowest at 24 h post-transfection. The relative mRNA expression level was（63.23±2.18）%， （64.5±3.17）%， and（75.19±4.20）% in HSP47-1-shRNA， HSP47-2-shRNA， HSP47-3-shRNA groups（P>0.05）， respectively， lower than that of the control group and non-related group（P<0.01）. At 24 and 48 h post-transfection， TGF-β1 expression was the lowest at 24 h post-transfection， and the relative expression level in HSP47-1-shRNA， HSP47-2-shRNA， HSP47-3-shRNA groups was（51.79±3.12）%， （66.67±2.13）%， and （69.61±3.65）%， respectively. Compared with control group， the expression of TGF-β1 in HSP47-1-shRNA and HSP47-2-shRNA2 groups was significant inhibited， but there was no significantly difference between control group and HSP47-3-shRNA group（P＞0.05）.  Conclusion  HSP47-shRNA interference plasmid is constructed. HSP47-shRNA effectively inhibits protein expression of HSP47， and results in changes of cell function.

Key words: Heat shock protein 47, RNA interference, short hairpin RNA, NIH/3T3 cell, Collagen Ⅰ/Ⅲ, Transfection