CLCA4对结直肠癌细胞增殖、侵袭、迁移及上皮-间质转化的影响及机制.pdf

第一作者简介:李亮,副主任医师,研究方向:胃肠道肿瘤。E-mail:37056064 通讯作者:李建刚,硕士,副主任医师,研究方向:胃肠道肿瘤。E-mail:15199142320 doi:10.3969/j.issn.1006-5709.2023.08.014CLCA4 对结直肠癌细胞增殖、侵袭、迁移及上皮-间质转化的影响及机制李亮,李建刚,王俊新疆医科大学第二附属医院普外科,新疆 乌鲁木齐 830063【摘要】目的探讨 CLCA4 对结直肠癌细胞增殖、侵袭、迁移及上皮-间质转化的影响及可能机制。方法收集 100 例患者的结直肠癌组织及癌旁正常组织。培养 SW620 细胞,根据转染质粒不同分为 Vector 组(转染 pcDNA3.1)、CLCA4 组(转染 pcDNA3.1-CLCA4)、sh-NC 组(转 染 sh-NC)、sh-CLCA4 组(转 染 sh-CLCA4)及 Control 组(无 处 理)。sh-NC 组 及 sh-CLCA4 组 细 胞 用GSK2126458 分处理并定义为 sh-NC+GSK2126458 组、sh-CLCA4+GSK2126458 组。分析 CLCA4 与结直肠癌临床病理特征、预后的关系,CCK-8 检测细胞增殖能力,Tanswell 实验检测细胞侵袭能力,划痕实验检测细胞迁移能力,qRT-PCR 检测细胞、组织中 CLCA4 mRNA 表达水平,Western blotting 检测细胞或组织中 CLCA4、E-cadherin、N-cadherin、Vimentin、PI3K、p-PI3K、AKT、p-AKT、mTOR、p-mTOR 蛋白表达水平。结果结直肠癌组织 CLCA4 mRNA、蛋白表达水平低于癌旁正常组织(P0.05)。CLCA4 表达水平与临床分期、淋巴结转移、脉管侵犯、神经侵犯及血清癌胚抗原水平有关(P0.05)。CLCA4 高表达组患者总体生存率高于 CLCA4 低表达组(P=0.006)。CLCA4 组细胞 48 h、72 h OD 值、侵袭细胞数,Vimentin、N-cadherin 表达水平及 p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR 值低于 Vector 组,迁移率及 CLCA4、E-cadherin 表达水平高于 Vector 组(P0.05);sh-CLCA4 组细胞 48 h、72 h OD 值、侵袭细胞数,Vimentin、N-cadherin 表达水平及 p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR 值高于 sh-NC 组,迁移率及 CLCA4、E-cadherin 表达水平低于 sh-NC 组(P0.05);sh-NC+GSK2126458 组细胞 48 h、72 h OD 值、侵袭细胞数低于 sh-NC 组,迁移率高于 sh-NC 组(P0.05),sh-CLCA4+GSK2126458 组细胞 48 h、72 h OD 值、侵袭细胞数低于 sh-CLCA4 组,迁移率高于 sh-CLCA4 组(P0.05)。结论CLCA4在结直肠癌组织中低表达,与肿瘤临床病理特征、预后有关,CLCA4 通过 PI3K/AKT/mTOR 信号通路抑制结直肠癌细胞的增殖、侵袭、迁移及上皮-间质转化。【关键词】CLCA4;PI3K/AKT/mTOR;结直肠癌;上皮-间质转化中图分类号:R735.3文献标识码:A文章编号:1006-5709(2023)08-0908-07收稿日期:2022-08-10Effect and mechanism of CLCA4 on proliferation,invasion,migration and epithelial mesenchymal transformation in colorectal cancer cellsLI Liang,LI Jiangang,WANG JunDepartment of General Surgery,the Second Affiliated Hospital of Xinjiang Medical University,Urumqi 830063,China【Abstract】ObjectiveTo investigate the effect and possible mechanism of calcium activated chloride channel A4(CLCA4)on proliferation,invasion,migration and epithelial mesenchymal transformation(EMT)in colorectal cancer(CRC)cells.MethodsA total of 100 cases of CRC tissues and adjacent normal tissues were collected.SW620 cells were cultured and divided into Vector group(transfected with pcDNA3.1),CLCA4 group(transfected with pcDNA3.1-CLCA4),sh-NC group(transfected with sh-NC),sh-CLCA4 group(transfected with sh-CLCA4)and Control group(no treatment).sh-NC group and sh-CLCA4 group were treated with GSK2126458,which were defined as sh-NC+GSK2126458 group and sh-CLCA4+GSK2126458 group.The relationship between CLCA4 and clinicopathological fea-tures and prognosis of CRC were analyzed.CCK-8 was used to detect cell proliferation.Tanswell test was used to detect cell invasion.Scratch test was used to detect cell migration.qRT-PCR was used to detect the expression level of CLCA4 mRNA in cells and tissues.Western blotting was used to detect CLCA4,E-cadherin,N-cadherin,Vimentin,PI3K,p-PI3K,AKT,p-AKT,mTOR,p-mTOR protein expression.ResultsThe expression levels of CLCA4 mRNA and protein in CRC were lower than those in adjacent normal tissues(P0.05).The expression level of CLCA4 was related to clinical stage,lymph node metastasis,vascular invasion,nerve invasion and serum carcinoembryonic antigen level(P0.05).The overall survival rate of patients with high expression of CLCA4 was higher than that of patients with low expression of CLCA4(P=0.006).OD values at 48 hours and 72 hours,the number of invasive cells,expression levels of Vimentin and N-cadherin,p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR values in CLCA4 group were lower than 809胃肠病学和肝病学杂志2023 年 8 月第 32 卷第 8 期Chin J Gastroenterol Hepatol,Aug 2023,Vol.32,No.8those in Vector group.The mobility and expression of CLCA4 and E-cadherin in Vector group were higher than those in Vector group(P0.05).OD values at 48 hours and 72 hours,number of invasive cells,expression levels of Vimentin and N-cadherin,p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR values in sh-CLCA4 group were higher than those in sh-NC group.The mobility and expression levels of CLCA4 and E-cadherin in sh-NC group were lower than those in sh-NC group(P0.05).The 48 hours and 72 hours OD values of cells in sh-NC+GSK2126458 group were lower than those in sh-NC group.The mobility was higher than that in sh-NC group(P0.05).The 48 hours and 72 hours OD values of cells in sh-CLCA4+GSK2126458 group were lower than those in sh-CLCA4 group,but the mobility was higher than that in sh-CLCA4 group(P5 cm)。本研究通过我院伦理委员会批准,批号:2019(069)号。1.2 随访采用电话或门诊复诊等方式,截止时间为2021 年 12 月 1 日,根据 CLCA4 mRNA 表达截断值分为CLCA4 低表达组、高表达组,使用 R 包“Survival”分析两组患者总体生存率:确诊之日至患者死亡或最后1 次随访日期。1.3细胞、试剂及仪器人结直肠癌 SW620 细胞株(本实验室保存)。DMEM/F12 培养基、胰蛋白酶(05-200-1A,03-045-1B,BI,以色列),CCK-8(E606335,上海生工生物有限公司),RIPA 蛋白裂解液、BCA 试剂盒(20-188,71285-M,Sigma,美 国),兔 抗 人 CLCA4、E-cadherin、N-cadherin、Vimentin、PI3K、p-PI3K、AKT、p-AKT、mTOR、p-mTOR及GAPDH(10494-1-AP、20874-1-AP、22018-1-AP、10366-1-AP、20584-1-AP、67121-1-Ig、60203-2-Ig、80455-1-RR、66888-1-Ig、67778-1-Ig、60004-1-Ig,Proteintech,美国),山羊抗兔 IgG-辣根过氧化物酶(30000-0-AP,北京博尔西科技有限公司),TRIzol(15596026,Invitrogen,美 国),Matrigel(354234,BD Bioscienc