摘要
目的:研究片仔癀(PZH)体外对长链非编码RNA-ANRIL(LncRNA-ANRIL)介导大肠癌淋巴管新生的抑制作用及其机制。方法:将体外培养人大肠癌细胞HCT-116用PZH进行干预,分为对照组和PZH干预组(0.25、0.50、0.75 mg/mL),采用CCK-8实验检测细胞活力,用RT-qPCR检测LncRNA-ANRIL的表达,用Western blot法检测促淋巴管新生因子VEGF-C蛋白表达;用Lipofectamine RNAiMAX转染HCT-116细胞,分为Si-NC组和Si-ANRIL组,采用RT-qPCR法检测LncRNA-ANRIL的表达,在倒置显微镜下观察细胞形态变化,用CCK-8实验检测细胞活力,Western blot检测促淋巴管新生因子VEGF-C蛋白表达;分别以Si-NC、Si-ANRIL和Si-NC+PZH(0.25 mg/mL)干预HCT-116细胞并收集细胞培养上清液即肿瘤培养上清(TSNs)用于培养人淋巴内皮细胞(HLEC),采用CCK-8实验检测细胞活力,Transwell实验观察细胞迁移能力和细胞侵袭能力,管腔形成实验观察细胞管腔形成能力。结果:不同浓度PZH干预HCT-116细胞均可显著抑制其活力(P<0.01),下调LncRNA-ANRIL表达(P<0.01)和VEGF-C蛋白表达,呈浓度依赖性;在HCT-116细胞中沉默LncRNA-ANRIL的表达,与Si-NC组比较,Si-ANRIL组LncRNA-ANRIL表达显著降低(P<0.05),且可降低细胞密度、细胞活力(P<0.01)和VEGF-C蛋白表达;收集的TSNs上清干预HLEC后,与Si-NC组比较,Si-ANRIL和Si-NC+PZH(0.25 mg/mL)组可显著降低HLEC活力、迁移能力、侵袭能力和管腔形成能力(P<0.01)。结论:PZH可抑制大肠癌细胞LncRNA-ANRIL表达及其介导的大肠癌淋巴管新生,LncRNA-ANRIL是PZH抑制大肠癌淋巴管新生的作用靶点之一。
Objective:To evaluate the inhibitory effect and mechanism of Pien Tze Huang(PZH)on long non-coding RNA-ANRIL(LncRNA-ANRIL)mediated lymphangiogenesis in colorectal cancer(CRC)in vitro.Methods:HCT-116 cells were cultured in vitro,and divided into control group and PZH group(treated with 0.25,0.50,0.75 mg/mL).CCK-8 assay was used to evaluate cell ability.The expression of LncRNA-ANRIL was detected by RT-qPCR,and the expression of VEGF-C was examined by Western blot.HCT-116 cells were transfected with Lipofectamine RNAiMAX and divided into Si-NC and Si-ANRIL groups.RT-qPCR was used to detect the expression of LncRNA-ANRIL.Phase-contrast microscope was used to observe the cell morphology.CCK-8 assay was used to detect cell viability.Western blot was used to detect the expression of VEGF-C protein.Treat HCT-116 cells with Si-NC,Si-ANRIL and Si-NC+PZH(0.25 mg/mL)respectively and collect cell culture supernatants(Tumor culture supernatants,TSNs)for theculture of human lymphatic endothelium cell(HLEC).CCK-8 assay was used to detect cell proliferation,cell migration and invasion were evaluated by Transwell assay.Tube Formation assay was used to observe lymphatic vessel formation ability.Results:Treatment of HCT-116 cells with different doses of PZH significantly inhibited the viability(P<0.01).At the same time,PZH treatment in HCT-116 cells also reduced the expression of LncRNA-ANRIL(P<0.01)and the expression of VEGF-C protein in a dose-dependent.Silencing LncRNA-ANRIL in HCT-116 cells,compared with Si-NC group,the expression of LncRNA-ANRIL in Si-ANRIL group was significantly reduced(P<0.05),and the expression of cell density,cell viability(P<0.01)and VEGF-C protein was also decreased.After the collected TSNs supernatant was used to treat HLEC cells,compared with the Si-NC group,Si-ANRIL and Si-NC+PZH(0.25 mg/mL)groups could remarkably decrease HLEC cell viability,migration and invasion ability and tube formation ability(P<0.01).Conclusion:PZH can inhibit the expression of LncRNA-ANRIL in colorectal cancer cells and its mediated lymphangiogenesis in colorectal cancer.LncRNA-ANRIL is one of the targets of PZH in inhibiting lymphangiogenesis in colorectal cancer.
作者
逯遥
张敏
刘洁
毛倩倩
曹治云
林久茂
LU Yao;ZHANG Min;LIU Jie;MAO Qianqian;CAO Zhiyun;LIN Jiumao(Fujian Academy of Integrative Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou,Fujian 350122,China;Fujian Key Laboratory of Integrative Medicine on Geriatrics,Fuzhou,Fujian 350122,China;Key Laboratory of Integrative Medicine of Fujian Province University(Fujian University of Traditional Chinese Medicine),Fuzhou,Fujian 350122,China)
出处
《康复学报》
CSCD
2020年第5期387-394,共8页
Rehabilitation Medicine
基金
国家自然科学基金项目(81774121)。
