原花青素介导的自噬流对喉癌细胞增殖影响及其机制探讨  

Investigate the effect of proanthocyanidin-mediated autophagic flow on the proliferation of laryngeal carcinoma cells and its mechanism

作　　者：刘炜 于锋[1,2] 周毅波 龚辉成[1,2] 张群慧 艾毛毛 LIU Wei;YU Feng;ZHOU Yi-bo;GONG Hui-cheng;ZHANG Qun-hui;AI Mao-mao(Institute of Otolaryngology Head and Neck Surgery Affiliated to Guangzhou Medical University,Guangzhou 510620,P.R.China;Department of Otolaryngology Head and Neck Surgery,Guangzhou Hospital of Otolaryngology Head and Neck Surgery,Guangzhou 510620,P.R.China)

机构地区：[1]广州医科大学耳鼻咽喉头颈外科研究所,广东广州510620 [2]广州市耳鼻咽喉头颈外科医院耳鼻咽喉头颈外科,广东广州510620

出　　处：《中华肿瘤防治杂志》2018年第20期1413-1421,共9页Chinese Journal of Cancer Prevention and Treatment

基　　金：广州市科技计划(201604020005;201804010201);广东省医学科研基金(B2018004);广州市医药卫生科技项目(20181A011047)

摘　　要：目的 原花青素(procyanidins,OPC)通过抑制细胞周期、促进凋亡等机制发挥抗肿瘤作用。本研究旨在探讨OPC介导的自噬流对喉癌TU686细胞增殖活力的影响,并进一步探究OPC促进喉癌TU686细胞自噬流可能的分子机制。方法 单丹磺酰尸胺(dansylcadaverine,MDC)染色法及流式细胞术检测不同浓度OPC对TU686细胞自噬的诱导作用;蛋白质印迹法检测TU686细胞LC3-Ⅱ和p62蛋白表达变化;并从翻译水平检测相关Atg蛋白表达,使用SiRNA干扰Atg5基因,并采用q-PCR和蛋白质印迹法检测干扰后Atg5转录和翻译水平变化,进一步检测干扰Atg5对LC3-Ⅱ和p62影响;检测PI3K/AKT途径中活化PI3Kp85α和磷酸化P-AKT1蛋白表达情况,采取PI3K/AKT途径激动剂胰岛素样生长因子-1(insulin-like growth factors-1,IGF-1)与OPC共培养细胞的方式以阐明PI3K/AKT途径在OPC诱导的自噬流中具体作用。CCK-8法和台盼蓝染色法检测OPC诱导的自噬流对TU686细胞增殖活力影响。结果 OPC处理组(20~40μg/mL)LC3-Ⅱ相对蛋白表达量高于0加药组,F=1 688.710,P<0.001;p62相对蛋白表达量低于0加药组,F=73.875,P<0.001。40μg/mL OPC作用后Atg5和Atg12蛋白相对表达量分别为3.20±0.08和7.52±0.13,高于0加药组,F 值均为-3.079,P 值均为0.012;40μg/mL OPC作用后PI3Kp85α和P-AKT1蛋白相对表达量分别为0.18±0.01和0.48±0.06,低于0加药组,F 值均为3.079,P 值均为0.012。IGF-1可以逆转OPC引起的LC3-Ⅱ升高和p62降低,与CON组相比,OPC组、IGF-1组和OPC+IGF-1组LC3-Ⅱ相对表达量分别为10.32±0.31、1.02±0.20和5.73±0.11,均P<0.001;p62相对表达量分别为0.64±0.02、1.03±0.02和0.87±0.01,均P<0.001。3-MA预处理后可以逆转OPC引起的LC3-Ⅱ和p62变化,与CON组相比,OPC组、3-MA组和OPC+3-MA组LC3-Ⅱ蛋白相对表达量分别为4.74±0.10、1.05±0.13和1.81±0.05,均P<0.001;p62蛋白相对表达量分别为0.59±0.02、0.94±0.03和0.78±0.03,P 值分别为<0.001、0.002和<0.001。SiRNA干扰Atg5后,�OBJECTIVE OPC exerts anti-tumor effects by inhibiting cell cycle and promoting apoptosis.The aim of this study was to investigate the effect of OPC-mediated autophagic flow on the proliferation of laryngeal TU686 cells,and to explore the molecular mechanism of OPC promoting the autophagic flow of laryngeal TU686 cells.METHODS MDC staining method was used to detect the induction of TU686 cells autophagy by different concentrations of OPC by fluorescence microscopy and flow cytometry.The expression of LC3-Ⅱ and p62 protein in TU686 cells was detected by western blot assay.The expression level of Atg protein was detected from the translation level,and Atg5 gene was interfered by siRNA.The changes of Atg5 transcription and translation levels after interference were detected by Q-PCR and western blotting,and the effect of interference with Atg5 on LC3-Ⅱ and p62 was further detected.Detection of the expression of activated PI3 Kp85αand phosphorylated P-AKT1 protein in the PI3 K/AKT pathway,by using the PI3 K/AKT pathway agonist IGF-1 and OPC the manner in which cells were cultured to elucidate the specific role of the PI3 K/AKT pathway in OPC-induced autophagic flow;the effects of OPC-induced autophagic flow on the proliferation of TU686 cells were detected by CCK-8 method and trypan blue staining.RESULTS The relative protein expression levels of LC3-Ⅱin the OPC treatment group(20-40μg/ml)were higher than that of 0 medicated group(F=1 688.710,P<0.001),p62 were lower than that of 0 medicated group(F=73.875,P<0.001).After 40μg/ml OPC treatment,the expression of Atg5(3.20±0.08)and Atg12(7.52±0.13)were higher than those of the 0 medicated group(Fvalues were-3.079,Pvalues were0.012);the expression of PI3 Kp85α(0.18±0.01)and P-AKT1(0.48±0.06)were lower than those of the 0 medicated group(Fvalues were-3.079,P values were 0.012).IGF-1 could reverse the increase of LC3-Ⅱ and decrease of p62 caused by OPC,compared with the CON group,the relative expression of LC3-Ⅱin OPC group,IGF-1 group and OPC+IGF-1 group we

关 键 词：原花青素 PI3K/AKT通路 喉癌 自噬流 

分 类 号：R739.65[医药卫生—肿瘤]