中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2013年
6期
452-458
,共7页
蔚丹丹%姚敏%陈洁%王理%严美娟%顾星%邱历伟%董志珍%姚登福
蔚丹丹%姚敏%陳潔%王理%嚴美娟%顧星%邱歷偉%董誌珍%姚登福
위단단%요민%진길%왕리%엄미연%고성%구력위%동지진%요등복
癌,肝细胞%信号传导%磷脂酰肌醇蛋白聚糖3%短发夹RNA%血管新生
癌,肝細胞%信號傳導%燐脂酰肌醇蛋白聚糖3%短髮夾RNA%血管新生
암,간세포%신호전도%린지선기순단백취당3%단발협RNA%혈관신생
Carcinoma,hepatocellular%Signal transduction%Glypican-3%Short hairpin RNA%Angiogenesis
目的 探讨磷脂酰肌醇蛋白聚糖3 (GPC-3)特异性短发夹RNA (shRNA)对肝癌细胞侵袭和血管新生的抑制作用与机制.方法 将GPC-3特异性shRNA转染肝癌细胞,检测GPC-3 mRNA及蛋白表达的变化；分析其对细胞增殖、凋亡及细胞侵袭能力的影响.样本均数两两比较采用独立样本t检验,多样本均数比较采用单因素方差分析.结果 shRNA转染肝癌细胞的效率大于80％,GPC-3 shRNA1转染HepG2、MHCC-97H和Huh7肝癌细胞后,GPC-3 mRNA水平分别为2.13±1.10、4.94±0.59、3.66±0.32,与空白对照组的19.34±0.90、20.24±1.61、13.97±1.15相比,t值分别为21.786、15.473、15.004,P值均＜0.001,差异均有统计学意义,且GPC-3 shRNA1可明显诱导细胞凋亡,抑制肝癌细胞增殖、运动及侵袭能力.HepG2、MHCC-97H和Huh7肝癌细胞干扰组β-连环蛋白mRNA表达分别为6.82±0.21、4.01±0.52和5.47±0.90,与阴性对照组的12.52±0.30、12.03±0.98、12.45±0.26比较,t值分别为26.903、12.578和12.870,P值均＜0.001,差异均有统计学意义;Glil mRNA水平分别为9.78±1.35、10.78±1.15和10.93±0.97,与阴性对照组的6.49±0.72、6.80±0.79、10.03±0.97比较,t值分别为-3.730、-4.918和-3.083,P值均＜0.05,差异均有统计学意义.GPC-3 shRNA1还可下调HepG2和MHCC-97H细胞胰岛素样生长因子Ⅱ和血管内皮生长因子表达.结论 GPC-3shRNA1可下调GPC-3 mRNA水平,通过Wnt/β-连环蛋白和Hedgehogs信号通路抑制癌细胞迁移、侵袭和血管新生,提示GPC-3为肝癌基因治疗潜在的分子靶目标.
目的 探討燐脂酰肌醇蛋白聚糖3 (GPC-3)特異性短髮夾RNA (shRNA)對肝癌細胞侵襲和血管新生的抑製作用與機製.方法 將GPC-3特異性shRNA轉染肝癌細胞,檢測GPC-3 mRNA及蛋白錶達的變化；分析其對細胞增殖、凋亡及細胞侵襲能力的影響.樣本均數兩兩比較採用獨立樣本t檢驗,多樣本均數比較採用單因素方差分析.結果 shRNA轉染肝癌細胞的效率大于80％,GPC-3 shRNA1轉染HepG2、MHCC-97H和Huh7肝癌細胞後,GPC-3 mRNA水平分彆為2.13±1.10、4.94±0.59、3.66±0.32,與空白對照組的19.34±0.90、20.24±1.61、13.97±1.15相比,t值分彆為21.786、15.473、15.004,P值均＜0.001,差異均有統計學意義,且GPC-3 shRNA1可明顯誘導細胞凋亡,抑製肝癌細胞增殖、運動及侵襲能力.HepG2、MHCC-97H和Huh7肝癌細胞榦擾組β-連環蛋白mRNA錶達分彆為6.82±0.21、4.01±0.52和5.47±0.90,與陰性對照組的12.52±0.30、12.03±0.98、12.45±0.26比較,t值分彆為26.903、12.578和12.870,P值均＜0.001,差異均有統計學意義;Glil mRNA水平分彆為9.78±1.35、10.78±1.15和10.93±0.97,與陰性對照組的6.49±0.72、6.80±0.79、10.03±0.97比較,t值分彆為-3.730、-4.918和-3.083,P值均＜0.05,差異均有統計學意義.GPC-3 shRNA1還可下調HepG2和MHCC-97H細胞胰島素樣生長因子Ⅱ和血管內皮生長因子錶達.結論 GPC-3shRNA1可下調GPC-3 mRNA水平,通過Wnt/β-連環蛋白和Hedgehogs信號通路抑製癌細胞遷移、侵襲和血管新生,提示GPC-3為肝癌基因治療潛在的分子靶目標.
목적 탐토린지선기순단백취당3 (GPC-3)특이성단발협RNA (shRNA)대간암세포침습화혈관신생적억제작용여궤제.방법 장GPC-3특이성shRNA전염간암세포,검측GPC-3 mRNA급단백표체적변화；분석기대세포증식、조망급세포침습능력적영향.양본균수량량비교채용독립양본t검험,다양본균수비교채용단인소방차분석.결과 shRNA전염간암세포적효솔대우80％,GPC-3 shRNA1전염HepG2、MHCC-97H화Huh7간암세포후,GPC-3 mRNA수평분별위2.13±1.10、4.94±0.59、3.66±0.32,여공백대조조적19.34±0.90、20.24±1.61、13.97±1.15상비,t치분별위21.786、15.473、15.004,P치균＜0.001,차이균유통계학의의,차GPC-3 shRNA1가명현유도세포조망,억제간암세포증식、운동급침습능력.HepG2、MHCC-97H화Huh7간암세포간우조β-련배단백mRNA표체분별위6.82±0.21、4.01±0.52화5.47±0.90,여음성대조조적12.52±0.30、12.03±0.98、12.45±0.26비교,t치분별위26.903、12.578화12.870,P치균＜0.001,차이균유통계학의의;Glil mRNA수평분별위9.78±1.35、10.78±1.15화10.93±0.97,여음성대조조적6.49±0.72、6.80±0.79、10.03±0.97비교,t치분별위-3.730、-4.918화-3.083,P치균＜0.05,차이균유통계학의의.GPC-3 shRNA1환가하조HepG2화MHCC-97H세포이도소양생장인자Ⅱ화혈관내피생장인자표체.결론 GPC-3shRNA1가하조GPC-3 mRNA수평,통과Wnt/β-련배단백화Hedgehogs신호통로억제암세포천이、침습화혈관신생,제시GPC-3위간암기인치료잠재적분자파목표.
Objective To construct glypican-3 (GPC-3) short hairpin RNA (shRNA) and investigate the effects of GPC-3 transcription silencing on hepatoma cell invasion and angiogenesis mechanisms.Methods GPC-3-specific shRNA and non-target control shRNA were constructed and transfected into the human hepatoma cell lines HepG2,MHCC-97H,and Huh7.shRNA-mediated silencing of GPC-3 expression was confirmed at the mRNA and protein levels by fluorescence quantitative reverse transcription (FQRT)-PCR and western blotting,respectively.The effect of silenced GPC-3 expression on cell proliferation was detected by EdU and sulforhodamine B assays,on migration by wound healing (scratch) assay,on invasion by transwell chamber assay,and on apoptosis by luminescence assay of caspase-3/7 activity.The effect of silenced GPC-3 expression on angiogenesis-related signaling factors was detected by FQRT-PCR (for the glioma-associated oncogene homolog-1 hedgehog signaling factor,GLI1,and the beta-catenin Wnt signaling factor,β-catenin),immunofluorescent staining (for the insulin-like growth factor-Ⅱ,IGF-Ⅱ),and ELISA (for the vascular endothelial growth factor,VEGF).Pairwise comparisons were made by the independent sample t-test,and multiple comparisons were made by one-way ANOVA.Results In all cell lines,transfection with the GPC-3-specific shRNA significantly reduced GPC-3 mRNA levels (％ reduction as compared to the non-target control shRNA:HepG2,89.2 + 6.0％,t =-25.753,P ＜ 0.001; MHCC-97H,75.3 + 4.9％,t =-26.487,P ＜ 0.001; Huh7,73.6 ± 4.6％,t =-27.607,P ＜ 0.001); the GPC-3 protein levels were similarly reduced.The GPC-3 shRNA-silenced cells showed significantly reduced proliferative,migratory and invasive capacities,as well as significantly increased apoptosis.The shRNA-mediated GPC-3 silencing was accompanied by significant down-regulation of β-catenin mRNA (HepG2,46.9 + 0.6％; MHCC-97H,67.5 + 2.7％; Huh7,56.3 + 8.4％) and significant up-regulation ofGLI1 mRNA (HepG2,49.2 ± 28.6％; MHCC-97H,54.6 ± 24.4％; Huh7,31.6 ±15.7％).At 72 h after transfection,the HepG2 cells showed significant down-regulation of VEGF protein (54.3 + 1.5％,t =46.746,P ＜ 0.001).Conclusion GPC-3 contributes to migration,invasion,angiogenesis,and apoptosis of hepatoma cells,possibly through its interactions with the Wnt/β-catenin and Hedgehog signaling pathways.GPC-3 may represent a useful target for gene silencing by molecular-based therapies to treat hepatocellular carcinoma.
