基于LAMP2A介导的自噬探讨仙鹤草-黄连含药血清对结直肠癌细胞的作用及机制研究  

Exploring effects and mechanisms of Agrimoniae Herba-Coptidis Rhizoma containing serum on colorectal cancer cells via LAMP2A-mediated autophagy

作　　者：何亚萍 侯敏艳 沈鑫玲 李智育 徐敏 陈轩 张淑娟 熊晗 彭海燕[1,2] HE Ya-ping;HOU Min-yan;SHEN Xin-ling;LI Zhi-yu;XU Min;CHEN Xuan;ZHANG Shu-juan;XIONG Han;PENG Hai-yan(the First Clinical College,Nanjing University of Chinese Medicine,Nanjing 210029,China;the Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China)

机构地区：[1]南京中医药大学第一临床医学院,江苏南京210029 [2]南京中医药大学附属医院,江苏南京210029

出　　处：《中国中药杂志》2024年第21期5730-5742,共13页China Journal of Chinese Materia Medica

基　　金：江苏省自然科学基金项目(BK20201400);国家中医药管理局全国名中医刘沈林工作室项目(国中医药办人教函[2018]119号);江苏高校优势学科建设工程项目(苏政办发[2018]87号);研究生《中医学导论》微课程建设的探索与实践项目(YJS-YB-2023-49)。

摘　　要：探讨仙鹤草-黄连(Agrimoniae Herba-Coptidis Rhizoma,XHC-HL)含药血清通过溶酶体相关膜蛋白2A型(lysosome-associated membrane protein type 2A,LAMP2A)介导的自噬对人结直肠癌HT29和HCT116细胞增殖、迁移、侵袭、凋亡的影响。通过生物信息学分析LAMP2A在结直肠癌发生发展中的作用机制;蛋白免疫印迹(Westem blot,WB)检测LAMP2A蛋白在结直肠癌细胞株中的表达;利用慢病毒转染技术构建LAMP2A干扰HT29及过表达HCT116结直肠癌细胞模型;实时荧光定量聚合酶链式反应(real-time qPCR)实验检测转染效率;结直肠癌HT29和HCT116细胞分别给予不同浓度的仙鹤草-黄连含药血清,采用cell counting kit-8(CCK-8)法检测细胞增殖,筛选含药血清最佳干预浓度及时间。将HT29细胞分为空白对照(NC)组、XHC-HL(仙鹤草-黄连药对处理)组、XHC-HL+shLAMP2A(仙鹤草-黄连药对处理+干扰LAMP2A)组;将HCT116细胞分为空白对照(NC)组、XHC-HL(仙鹤草-黄连药对处理)组、XHC-HL+LAMP2A(仙鹤草-黄连药对处理+过表达LAMP2A)组,CCK-8检测细胞活力;平板克隆实验检测细胞增殖能力;划痕实验及Transwell迁移实验检测细胞迁移能力;Transwell侵袭实验检测细胞侵袭能力;流式细胞术检测细胞凋亡率;WB技术和real-time qPCR技术检测仙鹤草-黄连药对对结直肠癌细胞LAMP2A、热休克同源蛋白70(heat shock cognate protein 70,HSC70)、热休克蛋白90(heat shock protein 90,HSP90)、甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)的蛋白表达水平及mRNA的影响。通过差异表达分析发现结直肠癌患者相对于正常对照样本的LAMP2A表达水平有显著升高,生存分析提示伴侣介导的自噬(chaperone-mediated autophagy,CMA)关键分子LAMP2A与结直肠癌的进展密切关联,通过基因集功能富集分析发现LAMP2A高表达患者显著上调了血管生成、免疫抑制等肿瘤进展相关信号通路,且免疫浸润分析发现LAMP2A高表达患者的肿瘤微�This study investigated the effects of Agrimoniae Herba-Coptidis Rhizoma(XHC-HL)-medicated serum on the proliferation,migration,invasion,and apoptosis of human colorectal cancer HT29 and HCT116 cells via the autophagy mediated by lysosome-associated membrane protein type 2A(LAMP2A).Bioinformatics analysis was conducted to explore the role of LAMP2A in the development and progression of colorectal cancer.Western blot(WB)was used to detect the expression of LAMP2A protein in colorectal cancer cell lines.Lentiviral transfection was utilized to construct LAMP2A knockdown in HT29 and overexpression in HCT116 colorectal cancer cell models.Real-time fluorescence quantitative polymerase chain reaction(real-time qPCR)was performed to assess transfection efficiency.HT29 and HCT116 cells were treated with different concentrations of XHC-HL-medicated serum.The cell counting kit-8(CCK-8)assay was used to detect cell proliferation and determine the optimal concentration and duration of medicated serum intervention.HT29 cells were divided into a normal control(NC)group,an XHC-HL(medicated serum treatment)group,and an XHC-HL+shLAMP2A(medicated serum treatment+LAMP2A knockdown)group.HCT116 cells were divided into a NC group,an XHC-HL group,and an XHC-HL+LAMP2A(medicated serum treatment+LAMP2A overexpression)group.CCK-8 was used to measure cell viability.Colony formation assay was employed to assess cell proliferation ability.Scratch and Transwell migration assays were conducted to evaluate cell migration ability,and Transwell invasion assay was used to detect cell invasion ability.Flow cytometry was adopted to determine apoptosis rates.WB and real-time qPCR were employed to detect the effect of XHC-HL on the protein and mRNA expression of LAMP2A,heat shock cognate protein 70(HSC70),heat shock protein 90(HSP90),and glyceraldehyde-3-phosphate dehydrogenase(GAPDH)in colorectal cancer cells.Differential expression analysis revealed that LAMP2A expression was significantly higher in colorectal cancer patients compared to that in norma

关 键 词：仙鹤草-黄连 药对 结直肠癌 LAMP2A 分子伴侣介导自噬 

分 类 号：R285[医药卫生—中药学]