miR-218靶向Bmi-1抑制肺腺癌细胞增殖的机制探讨.pdf

4301MODERNONCO31.No.232023年12 月现代肿瘤医学第31卷第2 3期miR-218 革靶向Bmi-1抑制肺腺癌细胞增殖的机制探讨焦会珍，金小乐，苑萌，王天伦河北北方学院附属第一医院急诊科，河北张家口0 7 50 0 0【摘要】目的：研究微小RNA-218（m i c r o R NA-2 18,m i R-2 18）靶向B细胞特性莫洛尼鼠白血病病毒插人位点1(B-cell specificmoloneyleukemia virus insertion site1,Bmi-1）抑制肺腺癌细胞增殖的作用及机制。方法：培养肺腺癌细胞株A549,分别转染miR-阴性对照（NC)核苷酸序列、miR-218核苷酸序列、NC质粒、Bmi-1质粒，采用平板克隆形成实验、CCK8实验检测细胞增殖水平，采用荧光定量PCR实验检测miR-218的表达,采用Westernblot实验检测Bmi-1的表达，采用双荧光素酶报告基因实验检测miR-218与Bmi-1的靶向关系。建立移植瘤裸鼠，慢病毒转染miR-NC或miR-218核苷酸序列,检测移植瘤体积、质量及Bmi-1的表达。结果：与转染miR-NC核苷酸序列的miR-NC组比较，转染miR-218核苷酸序列的miR-218组A549细胞miR-218表达水平、增殖抑制率增加,克隆形成率、Bmi-1表达水平及野生型Bmi-1质粒的荧光素酶活性降低（P0.05）；与共转染miR-218核苷酸序列及NC质粒的miR-218+NC质粒组比较，共转染miR-218核苷酸序列及Bmi-1质粒的miR-218+Bmi=1质粒组A549细胞增殖抑制率降低,克隆形成率、Bmi-1表达水平增加（P0.05）;与慢病毒转染miR-NC核苷酸序列的miR-NC组比较，慢病毒转染miR-218核苷酸序列的miR-218组裸鼠移植瘤体积、质量及Bmi-1表达水平均降低（P0.05）。结论：miR-218靶向下调Bmi-1表达并抑制肺腺癌细胞增殖。【关键词】肺腺癌;miR-218;Bmi-1;靶基因;增殖;移植瘤【中图分类号】R734.2【文献标识码】AD0I:10.3969/j.issn.1672-4992.2023.23.002【文章编号】16 7 2-4992-（2 0 2 3)2 3-430 1-0 6Mechanism of miR-218 targeting Bmi-1 to inhibit the proliferation of lung adenocarci-noma cellsJIAO Huizhen,JIN Xiaole,YUAN Meng,WANG TianlunDepartment of Emergency,the First Affiliated Hospital of Hebei North University,Hebei Zhangjiakou 075000,China.AbstractI Objective:To investigate the inhibitory effect and mechanism of B-cell specific moloney leukemia vi-rus insertion site 1(Bmi-1)on the proliferation of lung adenocarcinoma cells by targeting microRNA-218(miR-218).Methods:The lung adenocarcinoma cell line A549 was cultured and transfected with miR-negative control(NC)nucleotide sequence,miR-218 nucleotide sequence,NC plasmid and Bmi-1 plasmid,respectively.The cellproliferation level was detected by plate cloning test and CCK8 test,the expression of miR-218 was detected by fluo-rescence quantitative PCR,the expression of Bmi-1 was detected by Western blot,and the targeting relationship be-tween miR-218 and Bmi-1 was detected by double luciferase reporter gene.Nude mice with transplanted tumor wasestablished,lentivirus was transfected with miR-NC or miR-218 nucleotide sequence and then the volume,qualityand Bmi-1 expression of transplanted tumor was detected.Results:Compared with the miR-NC group transfectedwith miR-NC nucleotide sequence,the miR-218 expression level and proliferation inhibition rate of A549 cells in-creased,and the cloning rate,Bmi-1 expression level and luciferase activity of wild type Bmi-1 plasmid decreasedinthe miR-218 group transfected with miR-218 nucleotide sequence(P0.05).Compared with the miR-218+NCplasmid group co-transfected with miR-218 nucleotide sequence and NC plasmid,the inhibition rate of A549 cellproliferation decreased,while the clone formation rate and Bmi-1 expression level increased in the miR-218+Bmi-1 plasmid group co-transfected with miR-218 nucleotide sequence and Bmi-1 plasmid(P0.05).Com-pared with the miR-NC group transfected with miR-NC nucleotide sequence of lentivirus,the volume,quality andBmi-1 expression level of transplanted tumor in nude mice of miR-218 group transfected with miR-218 nucleotidesequence of lentivirus decreased(P0.05).Conclusion:miR-218 suppresses the proliferation of lung adenocarci-noma cell by targeted downregulation of Bmi-1 expression.【收稿日期】20230507【修回日期】2 0 2 3-0 7-2 1【基金项目】河北省2 0 2 3年度医学科学研究课题（编号：2 0 2 31456）【作者简介】焦会珍（1990 一），女，河北张家口人，主治医师，主要从事急救医学相关工作。Ema i l：j i a o 156 12 30 5396 16 3.c o m4302:miR-218靶向Bmi-1抑制肺腺癌细胞增殖的机制探讨焦会珍，等Key words lung adenocarcinoma,miR-218,Bmi-1,target genes,proliferation,transplant tumorModern Oncology 2023,31(23):43nes,proliferation,transplant tumorModern Oncology 2023,31(23):4301-4306肺癌是全球范围内致死率最高的实体恶性肿瘤，而肺腺癌是最常见的肺癌类型，约占肺癌的40%。晚期肺腺癌的预后差,5年生存率仅10%1-2 ,因此肺腺癌的免疫治疗、靶向治疗成为了近些年的研究热点。癌细胞过度增殖是与肺腺癌复发、转移直接相关的生物学行为，研究肺腺癌细胞增殖的调控机制有助于为肺腺癌的治疗提供合适靶点微小RNA（m i c r o R NA，m i R NA）是通过与靶基因互补位点结合发挥基因表达调控作用的小分子非编码RNA,大量研究表明miRNA在癌细胞增殖、迁移、侵袭中发挥调控作用，与多种恶性肿瘤的发生相关3-4。miR-218 是受到广泛关注的具有抑癌活性的一种miRNA,在多种癌细胞中通过靶向抑制原癌基因B细胞特性莫洛尼鼠白血病病毒插入位点1(B-cell specific moloney leukemia virus insertion site 1,Bmi-1)的方式抑制细胞增殖5-7 。目前已知肺腺癌中miR-218表达下调，而Bmi-1表达上调8 ,并且miR-218对肺腺癌细胞的迁移、侵袭具有调控作用9,但miR-218对肺腺癌细胞增殖的调控作用及相关机制尚不清楚。因此，本研究将首先通过细胞实验分析miR-218靶向Bmi-1抑制肺腺癌细胞增殖的作用及机制,而后通过动物实验对miR-218在肺腺癌中的抑癌作用进行验证，旨在深人全面的认识miR-218在肺腺癌发生发展中的作用。1材料与方法1.1材料1.1.1细胞肺腺癌A549细胞株购自上海通派生物科技有限公司。1.1.2动物20只雄性Balb/c裸鼠购自北京维通利华动物科技有限公司，5周龄、体质量（2 0 2）g（实验分为两组，首批每组6只，第二批每组4只）。1.1.3试剂与仪器miR-阴性对照（NC）核苷酸序列、miR-218核苷酸序列、野生型Bmi-1质粒、突变型Bmi-1质粒购自上海吉玛基因公司,NC质粒、Bmi-1质粒、miR-NC慢病毒、miR-218慢病毒购自广州云舟生物科技股份有限公司，结晶紫购自美国Sigma公司,CCK8检测试剂盒购自武汉伊莱瑞特生物科技股份有限公司，miRcutemiR提取分离试剂盒（货号：DP501）、m i R c u t e 增强型miRcDNA第一链合成试剂盒（货号：KR211）、m iR c u t e 增强型miR荧光定量检测试剂盒（SYBR G r e e n）（货号：FP411）购自北京天根生化公司,Bmi1、-actin特异性一抗购自美国Abcam公司，双荧光素酶报告基因检测试剂盒购自美国Promega公司1.2实验方法1.2.1细胞培养及分组使用含10%胎牛血清的培养基对A549细胞进行培养，定期换液，用0.2 5%胰蛋白酶消化，传代后接种在6 孔细胞培养板内。miR-NC 组转染miR-NC核苷酸序列,浓度为100pmol/L;miR-218组转染miR-218核苷酸序列，浓度为100pmol/L;miR-NC+NC质粒组转染miR-NC核苷酸序列和NC质粒，浓度分别为10 0 pmol/L和1g/mL;miR-218+NC质粒组转染miR-218核苷酸序列和NC质粒，浓度分别为10 0 pmol/L和1g/mL;miR-218+Bmi-1质粒组转染miR-218核苷酸序列和Bmi-1质粒，浓度分别为10 0pmol/L和1 g/mL。转染48 h后用于后续检测1.2.2平板克隆形成实验分组转染的A549细胞用胰蛋白酶消化，重悬后按照1000个/孔接种在6 孔板内，每3天更换1次培养基直至细胞培养板底部出细胞克隆团，用4%多聚甲醛固定细胞30 min，用0.1%结晶紫染液染色30 min，清洗晾干后在显微镜下观察克隆细胞群落数，计算细胞克隆形成率=克隆细胞群落数/接种细胞数10 0%1.2.3CCK8实验分组转染的A549细胞用胰蛋白酶消化，重悬后按照50 0个/孔接种在96 孔细胞培养板内，分别于接种后2 4h、48 h、72h时向每孔内加入2 0 LCCK8检测液，继续培养4h后在酶标仪上检测光密度（optical density,OD)值。计算细胞增殖抑制率=（1-实验组OD值/对照组OD值）10 0%1.2.4荧光定量PCR实验采用miRcutemiR提取分离试剂盒提取各组A549细胞中的miRNA,采用miRcute增强型miRcDNA第一链合成试剂盒将miRNA反转录为cDNA，采用采用miRcute增强型miR荧光定量检测试剂盒进行miR-218表达的检测，首先按照试剂盒说明书配置反应体系：cDNA1uL、试剂盒内反应液10 L、10 mol/LmiR-218或U6的特异性上游引物0.4L、试剂盒中的通用下游引物0.4L,去离子水补足反应体系至2 0.0 uL；而后将反应体系放入荧光定量PCR仪，按照95预变性10 min后95变性5s、6 0 退火/延伸15s的程序循环40 次。完成反应后在软件中生成循环曲线及循环阅值（CT），以U6为内参，按照公式2-ACT计算miR-218的表达水平1.2.5Westernblot实验提取各组A549细胞的蛋白并检测浓度，将含有2 0 g蛋白的样本用于Westernblot实验，在聚丙烯酰胺凝胶中电泳后电转移至硝酸纤维素膜，将膜放人5%脱脂牛奶、室温封闭1h,洗膜后将膜放人1:10 0 0 稀释的Bmi-1特异性一抗或1:50 0 0 稀释的-actin特异性一抗,4孵育过夜。次日洗膜后将膜放人1:2 0 0 0 稀释的辣根过氧化物酶二抗，室温孵育1h。最后，洗膜并在凝胶成像系统中进行化学发光，得到Bmi-1和-actin的条带，在图像录人ImageJ软件并计算条带的OD值,以-actin为内参,计算Bmi-1的表达水平1.2.6双荧光素酶报告基因实验将A549细胞接种在2 4孔细胞培养板内，待细胞融合度达到8 0%后进行转染，共转染miR-NC核苷酸序列+野生型Bmi-1质粒、miR-NC核苷酸序列+突变型Bmi-1质粒、miR-218核苷酸序列+野生型Bmi-1质粒、miR-218核苷酸序列+突变型Bmi-1质粒,转染2 4h后弃去培养基，洗涤后每孔加人2 0 0 uL裂解液，裂解细胞后得到的悬液12000r/min离心10 min,取上清2 0 L并采用双荧光素酶报告基因检测试剂盒Fireflyluciferase值和Renillaluciferase值，得到比值作为质粒的荧光素酶活性。4303MODERNONO.31.No.232023年12 月现代肿瘤医学第31卷第2 3期1.2.7裸鼠成瘤实验NC慢病毒或miR-218慢病毒按照MOI=10感染A549细胞2 4h,更换为含血清的培养基继续培养48 h,胰蛋白酶消化收集细胞,用无血清培养基重悬并调节密度至110/mL,取0.1mL接种在裸鼠皮下建立移植瘤模型。1个月后颈椎脱白处死裸鼠并分离瘤体，测定肿瘤长度a、肿瘤宽度b并计算肿瘤体积=1/2 ab,称量肿瘤质量。1.3统计学方法采用SPSS22.0软件进行统计学处理，实验数据均为计量资料，以（均数标准差）表示，多组间比较采用单因素方差分析，两两比较采用LSD-t法。P0.05为差异有统计学意义。2结果2.1miR-218对A549细胞克隆形成及增殖的影响miR-218组A549细胞中miR-218的表达水平高于miR-NC组（图1A,P0.05），克隆形成率低于miR-NC组（图1B-C,P0.05）,增殖抑制率高于miR-NC组（图1D，P0.05)。ABCD10307-miR-218miR-NC4-groupgroup*miR-NCgroupmiR-218group83206-2-41012000miR-NCgroupmiR-218groupmiR-NCgroupmiR-218group24h48h72h图1miR-NC组与miR-218组A549细胞克隆形成及增殖情况的比较A:miR-218表达水平的比较;B：克隆形成的结晶紫染色；C：克隆形成率的比较;D：增殖抑制率的比较。与miR-NC组比较，*P0.05。Fig.1Comparison of clone formation and proliferation of A549 cell between miR-NC group and miR-218 groupA:Comparison of the expression levels of miR-218.B:Crystal violet staining of clone formation.C:Comparison of clone formation rates.D:Comparisonof proliferation inhibition rates.Compared with miR-NC group,*P0.05.2.2miR-218对A549细胞中Bmi-1表达的靶向调控miR-218组A549细胞中Bmi-1的表达水平低于miR-NC组（图2 A-B,P0.05）,野生型Bmi-1质粒的荧光素酶活性低于miR-NC组（图2 C,P0.05)。ABCD1.51.01.5-miR-NCmiR-2180.8groupgroup1.01.0Bmi-10.60.4-actin0.50.5-0.2-0.00.00.0miR-NCgroupmiR-218groupmiR-NCgroupmiR-218groupmiR-NCgroupmiR-218group图2 miR-NC组与miR-218组A549细胞Bmi-1表达及荧光素酶活性的比较A:Bmi-1的蛋白条带;B:Bmi-1表达水平的比较;C:野生型Bmi-1质粒荧光素酶活性的比较;D：突变型Bmi-1质粒荧光素酶活性的比较。与miR-NC组比较，*P0.05。Fig.2 Comparison of Bmi-1 expression and luciferase activity in A549 cells between miR-NC group and miR-218 groupA:Protein band of Bmi-1.B:Comparison of Bmi-1 expression levels.C:Comparison of luciferase activity of wild type Bmi-1 plasmid.D:Comparisonof luciferase activity of mutant Bmi-1 plasmid.Compared with miR-NC group,*P0.05.2.3Bmi-1对miR-218抑制A549细胞克隆形成、增殖的影响miR-218+NC质粒组A549细胞中miR-218的表达水平高于miR-NC+NC质粒组（图3A,P0.05）;m iR-2 18+NC质粒组A549细胞中Bmi-1的表达水平低于miR-NC+NC质粒组（图3B-C,P0.05）m i R-2 18+Bm i-1质粒组A549细胞中Bmi-1的表达水平高于miR-218+NC质粒组（图3B-C,P0.05）;m i R-2 18+NC质粒组A549细胞的克隆形成率低于miR-NC+NC质粒组（图3D-E,P0.05），miR-218+Bmi-1质粒组A549细胞的克隆形成率高于miR-218+NC质粒组（图3D-E,P0.05）;m iR-2 18+NC质粒组A549细胞的增殖抑制率高于miR-NC+NC质粒组（图3F,P0.05）,m i R-2 18+Bm i-1质粒组A549细胞的增殖抑制率低于miR-218+NC质粒组（图3F,P0.05）2.4miR-218对裸鼠移箱直瘤生长的影响miR-218组移植瘤中miR-218的表达水平高于miR-NC组（图4A,P0.05）;m i R-2 18 组的裸鼠移植瘤体积和质量均低于miR-NC组（图4B-D,P0.05）；m i R-2 18 组移植瘤中Bmi-1的表达水平低于miR-NC组（图4E-F,P0.05)。.4304.miR-218靶向Bmi-1抑制肺腺癌细胞增殖的机制探讨焦会珍，等ABC42.07#miR-NC+miR-218+miR-218+3-NCplasmiddNCplasmidBmi-1plasmid1.5-2-Bmi-11.0-actin0.5-00.0miR-NC+miR-218+miR-218+miR-NC+miR-218+NC plasmid NC plasmid Bmi-1 plasmidmiR-218+NCplasmidNCplasmidBmi-1plasmidDEFmiR-NC+NCplasmidmiR-218+NCplasmidmiR-NC+NCplasmidmiR-218+NCplasmid10-30-miR-218+Bmi-1plasmid8-6-20-4-miR-218+Bmi-1plasmid#2-10-#0miR-NC+miR-218+miR-218+NCplasmidNCplasmidBmi-lplasmid024h48h72h图3三组A549细胞克隆形成及增殖情况的比较A:miR-218表达水平的比较;B:Bmi-1的蛋白条带;C:Bmi-1表达水平的比较;D：克隆形成的结晶紫染色;E：克隆形成率的比较;F:增殖抑制率的比较。与miR-NC+NC质粒组比较，*P0.05;与miR-218+NC质粒组比较，#P0.05。Fig.3Comparison of clone formation and proliferation of A549 cells in three groupsA:Comparison of the expression levels of miR-218.B:Protein band of Bmi-1.C:Comparison of Bmi-1 expression levels.D:Crystal violet staining ofclone formation.E:Comparison of clone formation rates.F:Comparison of proliferation inhibition rates.Compared with miR-NC+NC plasmid group,*P0.05.Compared with miR-218+NC plasmid group,#P0.05.BA3800-800-600-600-2-400-400200-200-00miR-NCgroupmiR-218groupmiR-NCgroup miR-218groupmiR-NCgroup miR-218groupDE1.5FmiR-NCmiR-218groupgroup1.0Bim-1miR-NCgroup0.5-actinmiR-218group0.0miR-NCgroup miR-218group图4miR-NC组和miR-218组裸鼠移植瘤生长的比较A:miR-218表达水平的比较;B：移植瘤体积的比较;C:移植瘤质量的比较;D：移植瘤的图像（n=6);E:Bmi-1表达水平的比较;F:Bmi-1的蛋白条带。与miR-NC组比较，*P0.05。Fig.4Comparison of transplanted tumor growth in nude mice between miR-NC group and miR-218 groupA:Comparison of miR-218 expression levels.B:Comparison of transplanted tumor volume.C:Comparison of transplanted tumor weight.D:Images ofthe transplanted tumor(n=6).E:Comparison of Bmi-1 expression levels.F:Protein bands of Bmi-1.Compared with miR-NC group,*P0.05.MODERNONC.31,No.23.43052023年12 月现代肿瘤医学第31卷第2 3期3讨论肺腺癌是常见的肺癌类型，早期诊断难度大、晚期患者占比高，虽然近些年小分子靶向药物的研发进展迅速，但受限于驱动基因突变、癌细胞耐药等因素，晚期肺腺癌的5年生存率低、预后差。因此，深入挖掘肺腺癌的分子机制有助于发现新的治疗靶点及新的分子标志物。肺腺癌发生发展的相关生物学机制包括癌细胞过度的增殖、迁移、侵袭等，miR-218是具有抑癌作用的miRNA,国内张苑9 的细胞实验证实miR-218抑制肺腺癌细胞的迁移、侵袭及转移,但miR-218在肺腺癌细胞增殖中的调控作用及机制尚不十分清楚。因此，本研究对miR-218对肺腺癌细胞增殖的调控作用及机制进行探索。miRNAs作为一种在转录后水平调节靶基因表达的非编码小分子RNA，虽然本身不编码氨基酸，但能够通过转录降解或翻译抑制的方式导致靶基因表达下调。已有大量研究证明miRNAs调控多种癌基因表达并影响癌细胞的增殖、迁移、侵袭10-1。关于miR-218,研究显示其在结直肠瘤12 、胃癌13、肝癌14、前列腺癌15 等多种癌症组织中表达下调并与肿瘤的病理进展、预后不良相关,且miR-218对癌细胞的增殖、迁移、侵袭具有抑制作用。目前已知miR-218抑制肺腺癌细胞的迁移和侵袭，本研究的细胞功能实验结果表明过表达miR-218抑制肺腺癌细胞株A549的克隆形成及增殖。这与以往研究中miR-218抑制其他癌细胞增殖的结果相似,也从抑制细胞增殖的角度证实了miR-218在肺腺癌中的抑癌作用。miRNAs通过靶向调控癌基因表达影响癌症的发生发展。Bmi-1基因是目前已知受到miR-218调控的靶基因6-7 ,该基因位于人类染色体10 p111.23上，编码产物Bmi-1显著抑制抑瘤基因p53、p 16 INK 4a p 19A R r 的转录、削弱抑癌基因对细胞增殖的抑制作用,进而促进癌细胞增殖、导致癌症发生发展16-18 。本研究中,分子生物学结果及双荧光素酶报告基因实验结果证实miR-218靶向抑制A549细胞中Bmi-1的表达。已有Bmi-1相关的癌细胞功能实验结果表明过表达Bmi-1促进肺腺癌细胞的增殖、迁移、侵袭等19,本研究中回复实验结果显示过表达Bmi-1减弱过表达miR-218对肺腺癌细胞克隆形成及增殖的抑制作用，表明miR-218抑制肺腺癌细胞克隆形成及增殖的作用部分与靶向抑制Bmi-1的表达相关。在癌症病灶的生长和转移过程中,癌细胞的增殖导致病灶体积增大、质量增加。为更加全面地认识miR-218对肺腺癌细胞增殖的抑制作用,本研究进行了裸鼠成瘤的动物实验，过表达miR-218的A549细胞成瘤后的体积缩小、质量减轻、Bmi-1表达降低，与细胞实验中过表达miR-218抑制A549增殖及Bmi-1表达的作用吻合。综上所述，本研究的以上结果表明miR-218作为肺腺癌的抑癌分子，能够靶向抑制原癌基因Bmi-1的表达，miR-218抑制肺腺癌细胞克隆形成及增殖的作用部分与靶向抑制Bmi-1的表达相关。本研究为认识肺腺癌的分子机制、发现新的分子治疗靶点提供了新的理论依据【参考文献】1SUNG H,FERLAY J,SIEGEL RL,et al.Global cancer statistics2020:GLOBOCAN estimates of incidence and mortality worldwidefor 36 cancers in 185 countriesJ.CA Cancer J Clin,2021,71(3):209 249.2LI D,SHI J,DONG X,et al.Epidemiological characteristics andrisk factors of lung adenocarcinoma:A retrospective observationalstudy from North ChinaJ.Frontiers in Oncology,2022,12:892571.3PENG J,LIU J,LIU H,et al.Prognostic value of microRNAs in pa-tients with small cell lung cancer:A Meta-analysis J.WorldJournal of 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