热休克蛋白47(HSP47)-短发夹RNA的构建及其细胞水平对HSP47表达的影响

Construction of HSP47-shRNA and its Impact on HSP47 at Cellular Level

目的针对肝纤维化发病关键基因热休克蛋白47(HSP47)基因构建HSP47-shRNA,初步验证其在NIH/3T3细胞系干预HSP47基因的表达,影响HSP47 mRNA及蛋白水平表达,并观察该细胞功能学变化。方法设计HSP47-shRNA模板,并将其分别设计于U6启动子的下游引物,将U6启动子及其下游HSP47-shRNA的模板双链DNA连接入载体,构建HSP47-pGCsi-U6-shRNA重组质粒(HSP47-1-pGCsi-U6-shRNA、HSP47-2-pGCsi-U6-shRNA和HSP47-3-pGCsi-U6-shRNA)。以非相关干扰质粒作为对照,通过脂质体介导转染HSP47-shRNA至小鼠胚胎成纤维细胞(NIH/3T3)中,分别于12、24、48和72h在倒置荧光显微镜下观察细胞转染效率和荧光表达强度;同时于转染后0、24和48h收集细胞,采用RT-PCR和蛋白质印迹(Western blotting)分析HSP47 mRNA和蛋白表达情况;并观察干预后该细胞分泌胶原功能和小鼠转化生长因子(TGF-β1)表达的变化。结果成功构建HSP47-shRNA载体,通过脂质体介导转染入NIH/3T3细胞,各干扰质粒转染效率均约为60.0%,各干扰质粒转染效率间差异无统计学意义(P>0.05)。转染12 h,可见少量绿色荧光细胞,随转染时间的延长,细胞荧光表达量逐渐增加,各干扰质粒于转染72h荧光表达最强。shRNA干扰显著抑制HSP47蛋白的表达,以转染HSP47-1-shRNA 24h后对蛋白表达抑制效果最佳,对HSP47 mRNA的相对沉默效率为(25.83±1.79)%,与空白组及非相关对照组比较,差异有统计学意义(P<0.05)。HSP47-1-shRNA转染24 h,I、III型胶原mRNA相对表达量分别下调为(56.52±1.64)%和(53.48±2.54)%,转染48 h分别下调为(54.17±2.63)%和(50.21±2.34)%,明显低于空白组和非相关对照组(P<0.05),但各实验组间差异无统计学意义(P>0.05)。各组HSP47-shRNA干扰质粒对TGF-β1 mRNA的抑制效率以转染24 h效果最佳,相对表达分别为(63.23±2.18)%、(64.53±3.17)%和(75.19±4.20)%,均低于空白组和非相关对照组(P<0.01),但干预组间对TGF-β1的抑制率差异无统计学意义(P>0.05)。各HSP47-shRNA干扰质粒组细胞上清中TGF-β1的抑制率以转染24h为最佳,分别为(51.79±3.12)%、(66.67±2.13)%和(69.61±3.65)%,与空白组相比,HSP47-1-shRNA组与HSP47-2-shRNA组对TGF-β1均有显著抑制作用,且以HSP47-1-shRNA组抑制效率更佳(P<0.05),HSP47-3-shRNA组与空白对照组间差异则无统计学意义(P>0.05)。结论构建了HSP47-shRNA干扰质粒,初步证实其对HSP47的表达干预有效,并引起NIH/3T3细胞功能学的变化。

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 PCRbased methods. The PCR product was digested with Nhe Ⅰ/Hind Ⅲ 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 e fficiency 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 at24 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 andⅢ 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 at24 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.