PARG基因沉默抑制小鼠结肠癌CT26细胞系体外淋巴管形成

PARG silencing inhibits lymphangiogenesis in mouse colonal carcinoma in vitro

目的初步探讨聚腺苷二磷酸核糖水解酶(PARG)基因沉默对肿瘤淋巴管形成的影响。方法 CT26细胞分为未转染组,空载组和PARG-shRNA慢病毒载体转染组,经嘌呤霉素筛选后获得稳定转染细胞克隆。用Westernblot法检测PARG、PARP-1、NF-κB和VEGF-C蛋白表达。给BALB/c小鼠腹腔注射不完全弗氏佐剂,诱导良性淋巴管瘤形成,分离并培养小鼠淋巴管内皮细胞(LEC)。免疫荧光细胞化学检测VEGFR-3和Podoplanin的表达,共培养实验检测LEC体外淋巴管样结构的形成。结果与对照组相比,PARG-shRNA慢病毒载体转染CT26细胞系后PARP-1、NF-κB及VEGF-C蛋白表达显著减弱,相对灰度值分别为1.0±0.05,0.9±0.02和0.2±0.02(P<0.05)。LEC表达VEGF-C和Podoplanin;PARG沉默组的LEC体外形成淋巴管样结构明显较未转染组和空载组少(P<0.05)。结论 PARG抑制肿瘤淋巴管形成可能与降低VEGF-C的表达有关。

Objective To investigate the influence of PARG silencing on the tumor lymphangiogenesis.MethodsLentiviral-PARG-shRNA was transfected into mouse colon carcinoma CT26 cell line.The CT26 cells expressing PARG gene were established by puromycin selection.The expressions of PARG,PARP-1,NF-κB and VEGF-C protein were detected by Western blot analysis.BALB/c mice benign lymphangiomas in abdominal cavity were induced by injection of 0.2 mL incomplete Freund＇s adjuvant（IFA,1∶ 1 with PBS） and mechanically disrupted to obtain LECs.Expressions of VEGFR-3 and Podoplanin were analyzed by immunofluorescence cytochemistry.The capability of LECs to form lymphatic vessel-like structures was evaluated by a LEC-CT26 cells co-culture method.Results The expression levels of PARP-1,NF-κB,and VEGF-C were reduced in PARG-silencing CT26 cells（P0.05）.Benign lyphangiomas were successfully induced by emulsified IFA.The expressions of VEGFR-3 and Podoplanin were positive in isolated LECs.The lymphatic vessel-like structures in PARG-silencing CT26 cells were significantly less than that in the control group（P 0.05）.Conclusion PARG silencing significantly reduces VEGF-C expression and suppresses the formation of lymphatic vessel-like structures.So PARG silencing may havean antilymphangiogentic effect and therefore prevent the metastatic dissemination through lymph system.