摘要目的 探讨肝癌组织Wnt3a异常表达及其靶向干预的抑制作用. 方法 以自身配对法收集2012年至2013年原发性肝癌患者术后癌和癌旁组织,以组织芯片和免疫组织化学染色法及特异性抗体检测肝癌组织及癌周组织Wnt3a表达情况,结合随访数据评估其预后价值.Crispr/cas9-sgRNA双载体慢病毒构建、感染及筛选,错配酶法在基因水平验证基因敲除效率;蛋白质印迹法在蛋白水平验证基因敲除效率及Wnt通路下游β-catenin表达情况;Cell Counting Kit-8法检测敲除Wnt3a的HepG2细胞增殖活力,并以移植瘤模型证实Wnt3a与肝癌的关系.计数资料采用x2分析,等级资料采用曼惠特尼-U检验,计量资料两个样本均数的比较采用t检验. 结果 肝癌组织Wnt3a主要定位于细胞膜和细胞质中,阳性率95.4％,显著高于自身对照的癌周组(49.4％,x2=47.754,P＜0.001);肝癌组Wnt3a高表达占70.1％ (61/87),而癌旁组仅14.9％ (13/87).Wnt3a高表达与低分化程度、肝硬化、HBV感染、门静脉浸润、TNM分期及5年生存率显著相关.以Crispr/cas9-sgRNA法成功敲除HepG2细胞Wnt3a基因后,Wnt3a蛋白表达水平显著降低,明显下调下游关键蛋白β-catenin,明显抑制癌细胞增殖并呈时间依赖性;与对照组比较,敲除Wnt3a后,肝癌移植瘤生长缓慢,体积显著缩小[(869.4±222.5) mm3与(355.0±99.9) mm3,t=5.168,P＜ 0.001)1、瘤质量显著减少[(0.88±0.20)g与(0.35±0.11)g,t=5.628,P＜0.0011.结论 癌胚型Wnt3a有望成为肝癌基因治疗的分子靶目标.

abstractsObjective To investigate the Wnt3a expression in tissues of HCC and its gene knockout on effects of HepG2 cell proliferation or xenograft tumor growth.Methods Hepatic Wnt3a expressions in 87 HCC and their matched surrounding tissues were observed by tissue microarray and immunohistochemistry for analyzing its clinicopathological characteristics;Wnt3a-knockout HepG2 cell lines were established by Crispr/cas9-sgRNA system and genomic cleavage efficiency was verified at gene level by surveyor assay.The relative proteins were confirmed by Western blotting;Cell Counting Kit-8 assay was used to examine cell proliferation after knocking-out Wnt3a successfully,and the nude mice HepG2 cell xenograft tumors delete that the relationship between Wnt3a and HCC growth.Results The positive Wnt3a with brown staining particles was mainly distributed in cytosol and membrane of hepatocytes.The incidence of hepatic Wnt3a expression in cancerous tissues (95.4％) was significantly higher (x2 =47.754,P ＜ 0.001) than that in their surrounding tissues (49.4％).The high Wnt3a expression was 70.1％ in the HCC and only 14.9％ in the surrounding tissues.High Wnt3a expression was associated with poorly-differentiated grade,liver cirrhosis,HBV infection,portal vein invasion,TNM stage and 5-year survival rate.After knocked-out by Crispr/cas9-sgRNA system successfully,Wnt3a expression was down-regulated significantly at gene or protein level.Key molecule β-catenin in cytoplasma was obviously inhibited.HepG2 cell lines proliferation was suppressed in time-dependent manner.The nude mice HepG2 cell xenograft tumors confirmed that the knock-out of Wnt3a could significantly supressed HCC growth with slower speed (t =6.418,P ＜ 0.001),smaller volume(869.4 ± 222.5 mm3 vs 355.0 ± 99.9 mm3,t=5.168,P ＜ 0.001),and lighter weight (0.88 ± 0.20 g vs 0.35 ± 0.11 g,t =5.628,P ＜ 0.00 l)compared with the control group.Conclusion Abnormal expression of Wnt3a could be expected as a promising target for HCC gene therapy.