干扰组蛋白去甲基化酶KDM3A表达对肝星状细胞增殖和胶原合成的影响

Effects of interfering histone demethylase KDM3A expression on hepatic stellate cell proliferation and collagen synthesis

目的探讨干扰组蛋白去甲基化酶KDM3A表达对肝星状细胞增殖和胶原合成的影响。方法首先乙醛处理大鼠肝星状细胞,用实时荧光定量PCR技术(RT-PCR)检测KDM3A mRNA的表达。接着利用慢病毒转染肝星状细胞72 h,干扰KDM3A的表达,乙醛刺激后,收集肝星状细胞,采用噻唑蓝法检测细胞增殖活性,酶联免疫吸附试验检测Ⅲ型和Ⅰ型胶原蛋白的含量,RT-PCR和蛋白质印迹法检测转化生长因子-β_(2)(TGF-β_(2))的表达水平。结果与对照组比较,乙醛处理组KDM3A mRNA相对表达量升高约4倍(1.00±0.10 vs.4.00±0.20),TGF-β_(2)mRNA相对表达量升高约3倍(1.00±0.40 vs.3.00±0.50),差异均有统计学意义(t=43.201、44.032,P<0.001)。干扰KDM3A后,与对照组比较,转染组的细胞增殖活性下降约43.3%[转染组光密度值(0.38±0.06)低于对照组的(0.67±0.08)],Ⅲ型胶原蛋白含量下降约20.00%(1.00±0.10 vs.0.80±0.04),Ⅰ型胶原蛋白含量下降约30.00%(1.00±0.10 vs.0.70±0.04),TGF-β_(2)蛋白及mRNA水平也明显下降(1.00±0.10 vs.0.70±0.10),差异均有统计学意义(t=12.272、12.621、21.369,P<0.001)。结论干扰组蛋白去甲基化酶KDM3A可一定程度阻止肝星状细胞的增殖和胶原合成,机制可能是通过抑制TGF-β_(2)表达来实现。

Objective To investigate the impact of interfering with the expression of histone demethylase KDM3A on the proliferation and collagen synthesis of hepatic stellate cells(HSCs).Methods Primary rat HSCs were treated with acetaldehyde,and the expression of KDM3A mRNA was detected using real-time quantitative PCR(RT-PCR).Subsequently,HSCs were transfected with lentivirus to interfere with KDM3A expression for 72 h.After acetaldehyde stimulation,the HSCs were collected,and cell proliferation activity was assessed using the thiazolyl blue assay.The content of type Ⅲ and type Ⅰ collagen proteins was determined using enzyme-linked immunosorbent assay,while the expression levels of transforming growth factor-beta 2(TGF-β_(2)) were measured by RT-PCR and Western blotting.Results Compared with the control group,the relative expression levels of KDM3A mRNA in the acetaldehyde-treated group increased approximately 4-fold(1.00±0.10 vs.4.00±0.20),and TGF-β_(2) mRNA expression increased approximately 3-fold(1.00±0.40 vs.3.00±0.50),with both differences being statistically significant(t=43.201,44.032,P<0.001).Following interference with KDM3A,the proliferation activity of transfected cells decreased by approximately 43.3% compared to the control group[transfected group optical density(0.38±0.06) lower than control group(0.67±0.08)].Compared to the control group,the content of type Ⅲ collagen decreased by approximately 20.00%(1.00±0.10 vs.0.80±0.04),and type Ⅰ collagen content decreased by approximately 30.00%(1.00±0.10 vs.0.70±0.04),the protein and mRNA levels of TGF-β_(2) also significantly decreased(1.00±0.10 vs.0.70±0.10) in the transfected group,with all differences being statistically significant(t=12.272,12.621,21.369,P<0.001).Conclusion Interfering with histone demethylase KDM3A can partially inhibit the proliferation and collagen synthesis of hepatic stellate cells,possibly by suppressing TGF-β_(2) expression.