摘要
目的探讨HCV核心蛋白对Wnt信号通路抑制分子SFRP1启动子活性的影响。方法以人肝癌细胞系Huh7基因组为模板,扩增SFRP1启动子区不同片段(-407～-27bp,-837～-27 bp,-1 202～-27 bp,-1 619～-27 bp,-2 029～-27 bp),以pGL3-Basic为载体分别构建SFRP1启动子区截短报告质粒。将构建好的质粒与pRL-TK共转染HEK293细胞,确定启动子区活性最强区域;分别用编码HCV核心蛋白的腺病毒或GFP对照腺病毒感染已转染重组报告质粒的SK-Hep1细胞,观察AdCore对SFRP1启动子活性的调控作用。结果 SFRP1启动子区域-407～-27 bp片段活性最强;与pGL3-Basic对照组相比较,相对荧光素酶活性增加了37.31±4.45倍(P<0.01),pGL3-S2～S5的活性分别增加了28.74±2.47、13.56±2.52、12.97±0.87和8.29±0.09倍(P<0.01);HCV Core蛋白能够抑制SFRP1基因启动子区活性,其对pGL3-S1的抑制作用最强,与GFP对照组相比较,抑制率为63.8%±1.0%(P<0.01)。结论 HCV核心蛋白通过抑制SFRP1启动子区活性下调SFRP1基因的表达,可能参与Wnt/β-catenin信号通路的活化,并与原发性肝癌的发生密切相关。
Objective To investigate the effects of HCV Core on SFRP1 gene promoter activity. Methods Differ- ent fragments of 5＇-flanking region of SFRP1 promoter were amplified from hepatoma cell line Huh7 genome DNA by PCR methods. The products were cloned into pGL-Basic vector. The recombinant reporter plasmids and control plasmid pRL-TK were cotransfected into HEK293 to determine the strongest activity area of SFRP1 promoter. To examine whether HCV Core could inhibit SFRP1 promoter activity, hepatoma cell line SK-Hepl were transfected with reporter plasmids and then infected with AdCore or AdGFP control. The promoter activity was measured by lu- ciferase activity. Results The most active area of SFRP1 promoter was -407 - -27 nt. Compared with pGL3- Basic negative control, the relative luciferase activity increased by 37.31 ±4. 45 folds （P 〈0. 01 ）. Other reporters also increased by28.74 ±2. 47, 13.56 ±2.52, 12.97±0. 87and 8.29 ±0.09 folds respectively（P 〈0.0 Core protein inhibited SFRP1 promoter activity. The inhibitory effect on pGL3-S1 was the strongest, with 1 ）. HCV ibitoryrate 63.8% ±1. 0% （P 〈0. 01 ）. Conclusions HCV Core protein inhibits SFRP1 promoter activity, resulting in down-regulation of SFRP1 mRNA expression level, thus participating in HCC carcinogenesis.
出处
《基础医学与临床》
CSCD
北大核心
2013年第4期406-411,共6页
Basic and Clinical Medicine
基金
国家自然科学基金(31171307
30972586)
