miRNA-21通过对血小板源性生长因子B和血管内皮生长因子的调控促进肝星状细胞活化的研究

miRNA-21 promotes the activation of hepatic stellate cells by regulating platelet-derived growth factor beta and vascular endothelial growth factor

目的研究miRNA-21通过对血小板源性生长因子B(PDGF-B)和血管内皮生长因子(VEGF)的调控促进肝星状细胞(HSC)活化的分子机制。方法将大鼠肝星状细胞HSC-T6分为正常组、酒精组、miRNA-21模拟物组和miRNA-21抑制剂组。正常组给予含有10%胎牛血清的DMEM培养基正常培养48 h;酒精组给予含有100 mmol·L^(-1)乙醇和10%胎牛血清的DMEM培养基培养48 h;miRNA-21模拟物组将miRNA-21模拟物用Lip3000转染试剂瞬时转染入细胞,再用含有100 mmol·L^(-1)乙醇和10%胎牛血清的DMEM培养基培养48 h;miRNA-21抑制剂组将miRNA-21抑制剂用Lip3000转染试剂瞬时转染入细胞,再用含有100 mmol·L^(-1)乙醇和10%胎牛血清的DMEM培养基培养48 h。用CCK-8实验检测细胞的增殖情况,用蛋白质印迹法检测PDGF-B和VEGF因子的表达情况。结果正常组、酒精组、miRNA-21模拟物组和miRNA-21抑制剂组的细胞增殖率分别为0.17±0.01,0.27±0.01,0.49±0.03和0.14±0.01。PDGF-B在酒精组的表达量(0.45±0.04)均显著高于正常组(0.21±0.03,P<0.05)和miRNA-21抑制剂组(0.20±0.03,P<0.01),而显著低于miRNA-21模拟物组的表达量(0.88±0.05,P<0.01)。VEGF在各组的表达变化则与PDGF-B的表达变化相同。结论miRNA-21能够通过对PDGF-B和VEGF的调控来促进HSC的增殖与活化,促进HSC成纤维化的进展。

Objective To study the molecular mechanism that miRNA-21 promotes the activation of hepatic stellate cell(HSC)through the regulation of platelet-derived growth factor beta(PDGF-B)and vascular endothelial growth factor(VEGF).Methods Passage HSC-T6 rat hepatic stellate cells were divided into normal,alcohol,miRNA-21 mimic and miRNA-21 inhibitor groups.Among them,the normal group was not treated;the miRNA-21 mimic group transiently transfected the miRNA-21 mimic into HSC;the miRNA-21 inhibitor group transiently transfected the miRNA-21 inhibitor into HSC.Then,change the cell culture medium to a medium containing 100 mmol·L^(-1) ethanol to induce the activation of HSC in the alcohol,miRNA-21 mimic and miRNA-21 inhibitor groups.Four groups were treated for 48 hours.Cell proliferation was detected by CCK-8 experiment.The expressions of PDGF-B and VEGF factors were detected by Western blot.Results The cell proliferation rates of the normal,alcohol,miRNA-21 mimic and miRNA-21 inhibitor groups were 0.17±0.01,0.27±0.01,0.49±0.03 and 0.14±0.01,respectively.The expressions of PDGF-B in the alcohol group(0.45±0.04)were significantly higher than those in normal group(0.21±0.03,P<0.05)and miRNA-21 group(0.20±0.03,P<0.01),while significantly lower than that in miRNA-21 mimic group(0.88±0.05,P<0.01).The expression change of VEGF in each group was the same as that of PDGF-B.Conclusion miRNA-21 can promote the proliferation and activation of HSC by regulating PDGF-B and VEGF,and promote the progress of HSC fibrosis.