Effects and action mechanism of KLF4 gene silence on collagen metabolism in hepatic stellat e cells of rats

doi:10.3969/j.issn.1002-7386.2016.20.009

Abstract：Objective To investigate the effects of silencing KLF4 expressions on collagen metabolism in hepatic stellate cells-T6 (HSC-T6) of rats.Methods The three interference plasmids (pGPU6-1,pGPU6-2,pGPU6-3) targeting at KLF4 gene of rats and negative control plasmid were designed and established.The expression levels of KLF4 mRNA and protein were detected by real time fluorescent quantitation RT-PCR ( Real-time RT-PCR) and Western Blot to select out the interference plasmid that had the best silencing effects.Then the interference plasmid ( pGPU6-3) was transfected into HSC-T6 cells of rats.On 24h and 48h after transfection, the expression levels of typeⅠcollagen, type Ⅲ collagen, matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA and protein were detected by Real-time RT-PCR and Western Blot,respectively.Real-time RT-PCR data were analyzed and calculated by 2-ΔΔCt method by taking GAPDH as an internal reference, however, Western Blot examination results were analyzed by using β-actin as internal reference protein.Results The examination results by Real-time RT-PCR showed that after HSC-T6 cells of rats were transfected by pGPU6-3, the expression levels of typeⅠcollagen,type Ⅲ collagen and TIMP-1 were decreased by 0.358 ± 0.038,0.298 ±0.041, 0.478 ±0.048, respectively, however, the expression levels of MMP-13 mRNA were increased by 1.712 ±0.034.The Western Blot showed that after HSC-T6 cells of rats were transfected by pGPU6-3, the expression levels of typeⅠcollagen, type Ⅲ collagen and TIMP-1 were decreased by 0.326 ±0.051, 0.283 ±0.074, 0.331 ±0.039, respectively,however, the expression levels of MMP-13 were increased by 1.273 ±0.038.Conclusion The KLF4 gene silencing can significantly decrease the expression levels of typeⅠcollagen and typeⅢcollagen in HSC-T6 cells of rats,and its action mechanism may be correlated to increasing the expressions of MMP-13 and inhibiting the expressions of TIMP-1.