干扰肝细胞核因子-1α对HepG2细胞载脂蛋白M及胆固醇相关代谢酶表达的影响

目的 检测肝细胞癌和癌旁组织中肝细胞核因子(HNF)-1α、肝受体同系物(LRH)-1与载脂蛋白(apo)M表达的相关性,探讨HNF-1α对apoM、apoA-Ⅰ和胆固醇合成与转化关键酶表达的影响.方法 RT-PCR检测人肝细胞癌组织和癌旁组织中HNF-1α、LRH-1、apoM的表达水平.采用小干扰RNA(SiRNA)在人肝癌细胞株HepG2细胞中干扰HNF-1 α表达后,RT-PCR检测apoM、apoA-Ⅰ及羟甲基戊二酰辅酶A还原酶(HMGCR)、胆固醇7 α-羟化酶1(CYP7A1)、法尼醇X受体(FXR)、小异源二聚体伴侣(SHP)-1的变化情况;Western blot检测apoM在蛋白水平的变化情况.对不同数据进行单因素方差分析、配对t检验或直线相关分析.结果 HNF-1 α、LRH-1、apoM mRNA在人肝细胞癌组织中明显高于癌旁组织(t值分别为-7.075,-8.803,-7.167,P值均＜0.01);HNF-1α和LRH-1与apoM的mRNA表达水平呈正相关(r=0.353,P＜0.01;r=0.523,P＜0.01).干扰HNF-1α后,与阴性对照相比,apoM、FXR、SHP-1 mRNA水平分别下降47.4%(F=77.627,P＜0.01)、47.9%(F=76.363,P＜0.01)、65.2%(F=365.772,P＜0.01),apoM蛋白水平下调54.3%(F=43.482,P＜0.01);HMGCR、CYP7A1 mRNA水平分别升高101.1%(F=218.666,P＜0.01)、138.5%(F=171.926,P＜0.01);而对apoA-Ⅰ无明显影响(F=0.170,P＞0.05).结论 HNF-1 α可提高apoM的表达水平,并通过促进胆固醇代谢关键酶的表达和抑制胆固醇代谢相关抑制因子的生成而促进胆固醇的转化,对心血管疾病具有保护作用.

Abstract：

Objective To determine wether there were connections among hepatocyte nuclear factor1 alfa (HNF-1α),liver receptor homolog-1 (LRH-1),apolipoprotein M (apoM) and to investigate the effects of HNF-1 αt in HepG2 on the expressions of apoM,apolipoprotein A-Ⅰ (apoA-Ⅰ) and the key enzymes in cholesterol metabolism and biotransformation.Methods The mRNA expressions of apoM,LRH- 1 and HNF- 1 α were detected by RT-PCR.HNF- 1 α was interfered and RT-PCR was used to detect the changes of apo M,apo A-Ⅰ,Cyp7Al,farnesoid X receptor (FXR) and small heterodimer partner-1 (SHP-1).Western blot was used to detect the change of apo M protein.Results The expressions of apoM,LRH-1 and HNF-1αmRNA were obviously higher in HCC tissue than that in para-cancer tissue (the vaule of tis -7.167,-7.075,-8.803,P ＜0.01 respectively).HNF-1 α and LRH-1 positively correlated with the expression of apoM (r = 0.353,P ＜0.01;r = 0.523,P ＜ 0.01 respectively);RT-PCR and western blot results showed that the expressions of apoM,FXR and SHP-1 mRNA,could be obviously suppressed by HNF-1 α interfering as compared to the negative controls by 47.4%,47.9%and 65.2% (P ＜ 0.01) respectively,and the expression of apoM protein also decreased by 54.3% (F = 43.482,P ＜ 0.01).The expressions of HMGCR and CYP7A mRNA increased by 101.1% and 138.5% (P ＜ 0.01) respectively as compared to the negative control.But there is no effect on expression of apoA-Ⅰ mRNA (F = 0.170,P ＞ 0.05).Conclusions HNF-1 α could promote cholesterol biotransformation by increasing the expression of apoM and the key enzymes in cholesterol metabolism and decreasing inhibiting factor.So HNF-1 α provided protection against cardiovascular disease.     

siRNA干扰沉默HIF-1α基因在舌癌Tca8113细胞侵袭和迁移中的作用机制

目的探讨利用siRNA技术特异性阻断缺氧诱导因子(HIF)-1α基因表达对舌癌Tca8113细胞侵袭和迁移的影响。方法取指数生长期细胞消化计数后按2×105个/孔接种于6孔细胞培养板中,待细胞融合至40%~50%时,将针对HIF-1α的siRNA序列应用LipofectamineTM2000转染入Tca8113细胞(siRNA HIF-1α组,干扰组)。半定量RT-PCR及Western印迹法检测HIF-1α的m RNA及蛋白,四甲基偶氮唑蓝比色法(MTT)检测细胞增殖,流式细胞仪检测细胞凋亡率,细胞侵袭实验和划痕实验检测细胞的侵袭和迁移能力。结果 siRNA可以有效沉默Tca8113细胞HIF-1α基因;干扰组细胞增殖较对照组明显受到抑制,且与细胞凋亡有关,凋亡率显著高于对照组(P0.05);,siRNA HIF-1α基因转染后的Tca8113细胞黏附能力降低,与对照组比较差异有统计学意义(P0.05);侵袭能力也下降。结论 siRNA能够有效沉默HIF-1α基因,抑制舌癌Tca8113细胞侵袭和迁移,在某种程度上可以改善舌癌细胞的恶性表型,为舌癌的治疗提供思路。