骨髓分化因子88通过核因子-κB_活化蛋白-1信号通路调控结直肠癌细胞增殖、迁移和侵袭的机制研究.pdf

40实用临床医药杂志Journal of Clinical Medicine in Practice2023,27(14):40-45.骨髓分化因子8 8 通过核因子-kB/活化蛋白-1信号通路调控结直肠癌细胞增殖、迁移和侵袭的机制研究秦蕾，秦鑫（1.北京老年医院普外科，北京，10 0 0 95；2 山西医科大学，山西太原，0 30 0 0 1）摘要：目的探讨骨髓分化因子8 8（MyD88)在结直肠癌发生发展中的功能和潜在机制。方法在SW480细胞中稳定敲低MyD88。采用CCK-8实验检测细胞的增殖能力；采用Transwell和划痕实验检测结直肠癌细胞的迁移能力；采用Transwell实验分析结直肠癌的侵袭能力；采用蛋白质印迹法（Westermn blot)分析MyD88的潜在调控机制。结果实时荧光定量聚合酶链式反应（RT-qPCR）和Westermblot结果显示，本研究在结直肠癌细胞中成功敲低了MyD88的表达。结直肠癌细胞中MyD88的降低可以显著抑制细胞的生长和侵袭。进一步研究发现，在结直肠癌细胞中敲低MyD88可以显著抑制MyD88-NF-kB/AP-1信号通路的表达。结论MyD88基因在结直肠癌的发生发展中起着重要的作用，有望成为结直肠癌的潜在的诊断和预后生物标志物。关键词：结直肠癌；骨髓分化因子8 8；核因子-kB；活化蛋白-1；细胞增殖中图分类号：R735.3；Q 2 7 9文献标志码：A文章编号：16 7 2-2 353(2 0 2 3)14-0 40-0 6 D0I：10.7 6 19/j c mp.2 0 2 2 37 37Myeloid differentiation factor 88 regulates the proliferation,migration and invasion of colorectal cancer cellsthrough the nuclear factor-kB/activatorprotein-1 signaling pathwayQIN Lei,QIN Xin?(1.Department of General Surgery,Beijing Geriatric Hospital,Beijing,100095;2.Shanxi Medical University,Taiyuan,Shanxi,030001)Abstract:Objective To investigate the function and potential mechanism of myeloid differenti-ation factor 88(MyD88)in the occurrence and development of colorectal cancer.Methods Stableknockdown of MyD88 was performed in SW480 cells.The proliferation ability of cells was detected byCCK-8 assay;the migration ability of colorectal cancer cells was examined by Transwell and scratchassay;the invasion ability of colorectal cancer was analyzed by Transwell assay;the potential regula-tory mechanism of MyD88 was analyzed by Western blot.Results Real-time fluorescence quantita-tive polymerase chain reaction(RT-qPCR)and Western blot results showed successful downregula-tion of MyD88 expression in colorectal cancer cells.The decrease of MyD88 in colorectal cancer cellssignificantly inhibited cell growth and invasion.Further studies revealed that knockdown of MyD88 incolorectal cancer cells significantly inhibited the expression of the MyD88-NF-kB/AP-1 signalingpathway.Conclusion MyD88 gene plays an important role in the occurrence and development ofcolorectal cancer and may become a potential diagnostic and prognostic biomarker for colorectal cancer.Key words:colorectal cancer;myeloid differentiation factor 888;nuclear factor-kB;activatorprotein-1;cell proliferation结直肠癌(CRC)是世界上最常见的恶性肿瘤之一,其中晚期结直肠癌患者的预后仍然不收稿日期：2 0 2 2 -12 -14修回日期：2 0 2 3-0 7-0 4基金项目：北京老年医院52 5人才培养项目（RC525-2018-D-06）通信作者：秦鑫，E-mail：q i n a t p 16 3.c o m佳1-2 。研究3 显示包括病毒和细菌感染、酒精、烟草烟雾、衰老、溃疡性结肠炎、久坐的生活方式第14期以及基因突变等因素均可能与结直肠癌有关。CRC的发病与3种类型的遗传畸变有关,即染色体不稳定（CIN）、微卫星不稳定（MSI）和CpG岛甲基化表型（CIMP）4。髓样分化因子8 8（M y D 8 8）是白细胞介素-1（IL-1）和Toll样受体（T LR)信号传导的必要适配分子5。TLRs是一个模式识别受体家族，可识别各种病原体相关分子模式（PAMPs），并激活宿主对病原体人侵的先天免疫防御。MyD88信号通路在激活的CD4+T细胞存活过程中介导全身和心脏细胞因子反应，促进肿瘤细胞增殖、侵袭、转移,并与肝细胞癌(HCC)患者介导的炎症通路损伤和神经退行性组织损伤的预后密切相关6-9研究10-1 证明MyD88表达是卵巢癌的不良预后因素,且在腺病毒角膜炎中发挥作用。另有研究12-13 表明,MyD88 是炎症性肺部疾病的治疗靶点,且在眼表稳态维持中发挥重要作用。既往研究14显示MyD88在结直肠癌患者癌组织及癌旁正常结直肠组织中表达，且癌组织中的表达水平明显高于癌旁组织；MyD88表达水平与结直肠癌患者的临床分期、T分期、M分期及淋巴结转移密切相关,MyD88高表达的结直肠癌患者的生存率明显低于MyD88低表达的患者。本研究探讨MyD88在结直肠癌患者中的作用,通过敲除结直肠癌细胞中MyD88基因以了解其在细胞中的功能作用,并分析MyD88敲低导致相关信号通路改变的机制,现将结果报告如下。1材料与方法1.1主要实验材料SW460细胞（上海中科院细胞库）；MyD88-siRNA重组腺病毒载体及阴性对照质粒购自上海吉玛生物有限公司；DMEM培养基（TOYOBO）；Trizol试剂盒（TaKaRa）；H o e c h s t 332 58 染色试剂；Western印迹试剂盒（美国Sigma公司）；全蛋白抽提试剂盒；Lipofectamine3000转染试剂（In v i t r o g e n，美国）；磷酸盐缓冲液（PBS，美国Amresco公司）；BCA蛋白定量试剂盒（ThermoFisher Scientific);CCK-8（D o j i n d o);M a t r i g e l(BDBioscience），细胞裂解抽提试剂（碧云天），PMSF(Amresco)。1.2主要实验器材苏净Airtech超净工作台（北京六一仪器厂）；SANYOMCO-15AC细胞培养箱（美国强生公司）；实用临床医药杂志Journal of Clinical Medicine in PracticeNikonTi-U/Ti-s倒置荧光显微镜（日本三菱公司）；5810R型高速离心机（日本岛津公司）；微量移液枪（美国Promega公司）；十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PACE，美国Corning公司）；E-Gel Imager凝胶成像仪（美国Beckma公司）。1.3实验方法1.3.1慢病毒感染和稳定细胞系筛选：SW480细胞株培养于含10%胎牛血清（FBS)的DMEM培养基中,置于生化培养箱中培养,条件为37、5%CO，浓度。在HEK293T细胞中将MyD88敲除质粒和空载体与psPAX2和pMD2.G共转染，培养2 d后将上清液过滤。采用上清感染SW480细胞,嘌呤霉素筛选2 周后提取蛋白和mRNA进行验证。将转染空载体和MyD88敲除质粒的SW480细胞分别作为对照组和MyD88-siRNA组。1.3.2细胞总RNA提取和实时荧光定量聚合酶链式反应（RT-qPCR）检测：利用TRIZOL提取2组细胞中总RNA，具体实验步骤参照试剂盒说明书。采用Nanodrop测定的2 6 0 nm与2 8 0 nm下吸光度比值（OD260/280mm比值）计算总RNA的纯度,采用琼脂糖凝胶电泳检测RNA的完整性。取2LRNA按照逆转录试剂盒说明书进行逆转录以构建cDNA文库,以cDNA为模板进行实时定量反应,以 GAPDH 基因作为内参基因。PCR反应条件：9 5下2 min预变性；9 5下15s，6 0 下30 s，设置40 个循环；以2-ACT方法计算基因的相对表达水平。1.3.3细胞总蛋白提取和蛋白质印迹法（West-ernblot）：将细胞采用含1%PMSF的细胞裂解缓冲液进行冰上裂解30 min，随后将裂解液在4、12000g离心10 min，提取上清液，使用BCA蛋白检测试剂盒进行蛋白定量，并加人5SDS煮沸10 min。将50 g蛋白采用12%SDS-PAGE凝胶进行电泳分离,将凝胶转移至0.45mPVDF膜中。采用1%的牛血清白蛋白对PVDF膜进行常温封闭1h，随后采用稀释后的抗体在4摇床上过夜孵育。采用TBST(0.1%Tween-20)将PVDF膜洗涤3次，每次10 min；采用山羊抗兔IgGH&L（H RP）二抗常温孵育1h，随后采用TBST(0.1%Tween-20)将PVDF膜洗涤3次。最后采用增强的化学发光底物检测液对蛋白条带进行检测。本研究所用抗体及稀释浓度为：MyD88（1：10 0 0，AF5195;Affinity)、G A PD H(1:1 0 0 0,a b 18 16 0 2;Abcam)、NF-k B p 6 5（1:1 0 0 0，A F50 0 6;A ffin i-4142ty)、p-NF-k B p 6 5(1:1 0 0 0,#30 33;Ce l l Si g n a-ling)、c-j u n(1:1 0 0 0,b s-0 6 7 0 R;Bi o s s)、p-c-j u n(1:1 000,bs-3172R;Bioss)、g o a t a n t i-r a b b i t Ig GH&L（H RP）（1:2 0 0 0,a b 7 0 90;A b c a m)。1.3.4细胞增殖检测：调整各组细胞浓度至1.0104个/孔并接种于96 孔板中，每组分别设置6 个复孔，置于生化培养箱中培养，条件为37、5%CO 浓度。在培养2 4、48、7 2、96 h后，弃掉上层培养基，添加10 0 L的10%CCK-8无血清培养基,孵育1h后使用微生物板阅读器(Bio-Tek)计算细胞增殖。1.3.5细胞迁移的愈合实验：将各组细胞接种于6 孔板中,每组分别设置6 个复孔,待细胞生长至10 0%时，用2 0 L移液管尖划伤细胞层，将含10%FBS的培养基替换为无血清培养基。拍摄细胞在培养0、48 h后的图像,计算细胞的迁移能力。1.3.6细胞侵袭实验：在培养基上室涂上Matrigel，调整各组细胞浓度至9.0 10 4个/孔接种于上室，下室为含2 0%FBS的DMEM培养基。MyD88实用临床医药杂志Journal of Clinical Medicine in Practice在37、5%CO，条件下孵育48 h后，取出Transwell室,将培养基丢弃并用无钙PBS清洗，而后用甲醇固定细胞30 min，用0.1%结晶紫染色20min，再用棉签轻轻拭去上面未迁移的细胞,并在显微镜下计数。1.4统计学分析采用SPSS19.0统计学软件进行数据分析,计量数据以（xs）表示，组间比较采用独立样本t检验，多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。P0.05为差异有统计学意义。2 结 果2.1siRNA千扰后MyD88蛋白及mRNA表达水平在转染siRNA48 h 后，检测2 组细胞内MyD88的蛋白及mRNA表达水平。RT-qPCR和Westernblot检测结果显示，转染MyD88-siRNA后，MyD88的mRNA和蛋白表达水平均降低,差异有统计学意义(P0.0001)，见图1。1.51.0第2 7 卷*0GAPDH2.2MyD88-siRNA干扰后抑制SW480细胞的增殖CCK-8实验结果显示，与对照组相比，MyD88-siRNA组细胞增殖率、细胞存活率均降低，一对照组2.0MyD88-siRNA组1.51.00.50.5对照组MyD88-siRNA组A：M y D 8 8 蛋白水平；B：M y D 8 8 mRNA 水平（2 组比较，*P0.0001）。图1MyD88敲除后2 组细胞蛋白和mRNA表达水平比较112448时间/h图2 2 组细胞的增殖率和存活率的比较A7296A对照组差异有统计学意义（P0.05），说明MyD88沉默能够显著抑制结直肠癌的细胞增殖。见图2。一对照组1.5MyD88-siRNA组1.00.5MyD88-siRNA组2448时间/hB7296B第14期2.3MyD88-siRNA干扰后抑制SW480细胞的迁移划痕实验结果显示，MyD88-siRNA组的划痕对照组实用临床医药杂志Journal of Clinical Medicine in Practice愈合速度低于对照组,差异有统计学意义（P0.000 1),见图3。150(4 0 J0%)/期443*10050MyD88-siRNA组00h2.4MyD88-siRNA干扰后抑制SW480细胞的侵袭Transwell实验结果显示，MyD88-siRNA组结直肠细胞通过基底膜迁移的细胞数量低于对照48h图32 组细胞划痕试验后细胞愈合情况比较A组,差异有统计学意义（P0.05），说明MyD88敲除可以显著抑制SW480细胞的侵袭。见图4。对照组MyD88-siRNA组B对照组图42 组细胞的侵袭能力比较（放大2 0 0 倍）2.5MyD88对NF-kB和AP-1信号通路的影响Westernblot结果显示，NF-kB（p 6 5）、p-NF-kB(p-p65)、A P-1(c-j u n)和 p-AP-1(p-c-Jun)蛋白在MyD88-siRNA组的表达水平低于对照组，差异有统计学意义（P0.05），说明MyD88沉默可以抑制NF-kB和AP-1信号通路的蛋白表达水平。见图5。3 讨 论MyD88在先天免疫反应中发挥核心作用，其参与调节NF-kB信号通路活性15 和MAPKs-c-jun（A P-1)信号通路116)。MyD88和MyD88相关信号通路已被证明参与内源性和外源性炎症反应,以及癌症相关细胞的进展和癌变过程。此外，MyD88异常表达的检测可用于预测各种人类癌症（如淋巴癌、肝癌）的预后17 。因此，MyD88可作为潜在的致癌标志物进行相关研究，对结直肠癌MyD88-siRNA组AP-1(c-jun)p-AP-1(p-c-jun)NF-B(p65)p-NF-KB(p-p65)GAPDH图52 组细胞中NF-kB和AP-1信号通路相关蛋白的Westernblot结果的治疗有积极的影响。本研究成功敲除了MyD88,并通过RT-qPCR和Westernblot对敲除效率进行了验证分析，并进一步验证MyD88敲除是否会影响结直肠癌细胞的生物学特征,结果表明敲除MyD88可抑制结直肠癌细胞的增殖、侵袭和迁移,证实MyD88在结对照组MyD88-siRNA组44直肠癌细胞增殖、侵袭和迁移中发挥重要的作用。临床研究18 发现MyD88的SNP位点与结直肠癌患者的生存率较差密切相关。此外，MyD88沉默可以降低胰腺癌细胞的侵袭性和迁移能力19。MyD88的高表达与结直肠癌患者肝转移及不良预后相关2 0 然而，MyD88诱导结直肠癌生物学行为的具体机制尚不清楚。有研究16 通过比较 IL-18-、IL-18R1-和MyD88-/-结直肠癌相关小鼠模型以及野生小鼠发现，MyD88可以通过IL-18/MyD88通路保护肠道免受肿瘤发生的影响。相反的，在LPS处理后的MyD88敲除小鼠中，MyD88可通过TLR/MyD88通路促进结肠炎向结直肠癌的恶性转化2 1。目前，MyD88在结直肠癌进展过程中的双重功能尚不清楚,其内在机制需要进一步研究。MyD88信号在调节肠道正常上皮细胞和癌细胞的生长中发挥作用。NF-kB 的上调与很多肿瘤进展过程相关，参与肿瘤细胞的增殖和侵袭；AP-1，特别是c-Jun同样影响肿瘤细胞的增殖、迁移和侵袭2-2 3。多项研究表明，丝裂原活化蛋白激酶（MAPKs）和核因子kB（NF-k B）信号通路是导致脂多糖(LPS)诱导的急性肺损伤(ALI)24、破骨细胞生成2 5、神经炎症2 6 、动脉粥样硬化2 7 、前列腺癌和胶质母细胞瘤的发生2 8 以及人类树突状细胞激活2 9 的关键因素。MyD88敲除可影响结直肠癌细胞的生物学特征，包括细胞的增殖、侵袭和迁移；在MyD88敲除的细胞系中，p65、磷酸化p65、c-j u n 和磷酸化c-jun蛋白表达均有不同程度的下降。敲低MyD88基因可以影响MyD88介导的NF-kB（p 6 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