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miR-2110通过调节CDT1影响肺腺癌的生物学行为

米源, 李旭哲, 王聪, 蔺晨曦, 赵志超, 颜晓菲, 史平, 王雷

米源, 李旭哲, 王聪, 等. miR-2110通过调节CDT1影响肺腺癌的生物学行为[J]. 四川大学学报(医学版), 2024, 55(5): 1202-1209. DOI: 10.12182/20240960505
引用本文: 米源, 李旭哲, 王聪, 等. miR-2110通过调节CDT1影响肺腺癌的生物学行为[J]. 四川大学学报(医学版), 2024, 55(5): 1202-1209. DOI: 10.12182/20240960505
MI Yuan, LI Xuzhe, WANG Cong, et al. miR-2110 Affects the Biological Behaviors of Lung Adenocarcinoma by Regulating CDT1[J]. Journal of Sichuan University (Medical Sciences), 2024, 55(5): 1202-1209. DOI: 10.12182/20240960505
Citation: MI Yuan, LI Xuzhe, WANG Cong, et al. miR-2110 Affects the Biological Behaviors of Lung Adenocarcinoma by Regulating CDT1[J]. Journal of Sichuan University (Medical Sciences), 2024, 55(5): 1202-1209. DOI: 10.12182/20240960505

miR-2110通过调节CDT1影响肺腺癌的生物学行为

基金项目: 河北省卫生厅课题(No. 20221353)、河北省科技厅(No. 22377769D)、河北省省级科技计划基础研究专项(自然科学基金)联合基金项目(No. H202206391)和河北医科大学第四医院科研创新团队项目(No. 2023B02)资助
详细信息
    通讯作者:

    王雷: E-mail:Yuankundu@163.com

miR-2110 Affects the Biological Behaviors of Lung Adenocarcinoma by Regulating CDT1

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  • 摘要:
    目的 

    通过细胞实验和动物实验结果,探讨miR-2110对肺腺癌(lung adenocarcinoma, LUAD)细胞增殖、凋亡及转移等生物学行为的影响。

    方法 

    通过生物信息学网站(ENCORI、TargetScan、miRTarBase和Tarbase)分析在LUAD组织中miR-2110的表达变化并进行miR-2110靶基因的预测。收集LUAD组织样本及肺腺癌细胞通过PCR技术验证miR-2110的表达变化。采用CCK-8、克隆形成实验、Transwell和流式细胞术分析LUAD细胞的功能变化。将10只6~8周龄BALB/c雌性裸鼠随机分为2组,通过体内实验验证miR-2110对LUAD的影响。

    结果 

    miR-2110在LUAD组织及细胞中相比于正常肺组织显著降低,其过表达可抑制LUAD细胞的增殖、转移并促进了肿瘤细胞的凋亡(P<0.05)。通过生物信息学预测和双荧光素酶报告基因测定,验证了miR-2110可靶向结合CDT1,并通过过表达CDT1基因发现相比于过表达miR-2110组,逆转了其增殖、转移和凋亡的作用(P<0.05)。裸鼠体内实验发现相比于对照组,过表达miR-2110可以明显降低肿瘤的增殖指标Ki67和转移指标vimentin、MMP9的表达。

    结论 

    miR-2110可以通过靶向CDT1抑制LUAD的增殖、转移,为LUAD的治疗提供新的依据。

     

    Abstract:
    Objective 

    To investigate the effect of miR-2110 on the biological behaviors, such as cell proliferation, apoptosis, and metastasis, of lung adenocarcinoma (LUAD) cells by means of cell and animal experiments.

    Methods 

    Bioinformatics websites, including ENCORI, TargetScan, miRTarBase, and Tarbase, were used to analyze the changes in the expression of miR-2110 in LUAD samples and to predict miR-2110 target. LUAD tissue samples and cells were collected and the changes in the expression of miR-2110 were verified through PCR technology. CCK-8 assay, clonogenic assay, Transwell assay, and flow cytometry were conducted to analyze alterations in the functions of LUAD cells. In addition, 10 BALB/c female nude mice aged 6 to 8 weeks were randomly divided into 2 groups, and the effect of miR-2110 on LUAD was investigated by in vivo experiments.

    Results 

    miR-2110 was significantly decreased in LUAD tissues and cells compared with the normal lung tissues. miR-2110 overexpression inhibited the proliferation and metastasis of LUAD cells and promoted the apoptosis of tumor cells (P<0.05). Bioinformatics prediction and dual luciferase reporter gene assay results confirmed that miR-2110 could target and bind to CDT1. In addition, overexpression of CDT1 gene reversed the proliferation, metastasis, and apoptosis of miR-2110 compared with the miR-2110 overexpression group (P<0.05). Nude mice in vivo experiments showed that miR-2110 overexpression significantly decreased the expression of Ki67, a tumor proliferation index, and vimentin and MMP9, two metastasis indices, compared with the control group.

    Conclusion 

    miR-2110 can inhibit proliferation and metastasis of LUAD by targeting CDT1, providing a new rationale for the treatment of LUAD.

     

  • 肺癌是全球癌症死亡的主要原因,占全球癌症相关死亡病例的18.0%[1]。2020年,肺癌是全球第二大常见癌症,约有221万新发病例[2]。肺腺癌(lung adenocarcinoma, LUAD)是肺癌的主要组织学亚型,占非小细胞肺癌(non-small cell lung cancer, NSCLC)50%以上[3]。近年来LUAD病例数呈上升趋势,LUAD的侵袭和转移是导致死亡的主要因素,对人民健康构成了严重威胁[4]。目前,对LUAD的治疗即使采取包括手术切除的综合治疗,其5年生存率仍低于20%[5]。因此,深入挖掘LUAD的分子机理,有利于全面了解LUAD的发生与发展过程,从而为LUAD的临床治疗奠定坚实的理论基础。

    微小RNA(miRNA)是一类小的非编码 RNA,由19~25个核苷酸组成,通过与靶基因mRNA的3'-UTR的互补性结合进行负向调节靶基因表达[6-7]。miRNA与细胞上皮-间质转化等多种细胞生理过程有关,其异常表达与多种人类肿瘤的进展和转移密切有关[8]。有研究发现,miR-2110与肿瘤的发生有关。ZHU等[9]在研究鼻咽癌和正常组织中miRNA表达时首次发现了人miR-2110。miR-2110位于染色体10q25.3中,后续研究表明,miR-2110是一种肿瘤抑制因子,可通过靶向SP1、TSKU来抑制乳腺癌和神经母细胞瘤的发病机制[10-11]。同样,FERRACIN等[12]发现miR-2110在结直肠癌患者中低表达并与肿瘤的进展相关。然而目前尚不清楚miR-2110是否参与LUAD的发病机制,其具体作用机制未见明确报道。

    因此,本研究通过数据库分析在LUAD中miR-2110的表达,并利用生物信息学技术进行靶点的预测。此外,通过基础细胞实验及裸鼠体内实验验证miR-2110对LUAD迁移、增殖的影响,以期为LUAD的临床治疗提供理论基础。

    人肺腺癌细胞系A549由河北医科大学药学院提供,30例临床LUAD及30例正常肺组织标本由河北医科大学第四医院标本库提供,已通过河北医科大学第四医院伦理委员会审批(2022KY129)。miR-2110 mimics和NC-mimics(苏州,吉玛生物科技有限公司,B01001),CDT1过表达载体pEGFP-N1-CDT1和对照载体pEGFP-N1(上海,生工生物工程股份有限公司),si-miR-2110和si-NC(苏州,吉玛生物科技有限公司,A09001),miR-2110和U6引物(上海,生工生物工程股份有限公司),miRNA反转录试剂盒(南京,诺唯赞生物科技有限公司,MR101-01),miRNA荧光定量PCR试剂盒(南京,诺唯赞生物科技有限公司,MQ101-01),CDT1兔抗人单克隆抗体(武汉,博士德生物工程有限公司,BM5203),GAPDH兔抗人单克隆抗体(武汉,武汉三鹰生物技术有限公司,10494-1-AP)。CCK-8试剂盒(美国,MCE公司,HY-K0301),Transwell膜嵌套(美国,Corning公司,CLS3396),凋亡试剂盒(美国,BD公司,556547)。

    在ENCORI(https://rnasysu.com/encori/)数据库中下载LUAD组织及正常组织中miR-2110表达数据,并进行miR-2110与CDT1的相关性分析;通过TargetScan(https://www.targetscan.org/vert_80/)、miRTarBase(https://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase_2022/php/index.php)和Tarbase(https://dianalab.e-ce.uth.gr/tarbasev9)数据库进行miR-2110的靶基因的预测。

    A549细胞系用含10%胎牛血清、1%青链霉素的DMEM的高糖培养基,在体积分数为5%的CO2、37 ℃恒温培养箱培养,将长到90%左右的细胞用含0.25%胰蛋白酶的EDTA消化液消化细胞进行1∶3传代培养。

    将呈对数生长期的A549细胞平均接种于6孔板上,待细胞生长到50%~60%时,更换无血清无抗体培养基,根据实验分组将细胞分为miR-2110过表达组(miR-2110-mimics组)、miR-2110对照组(NC-mimics组);miR-2110敲低对照组(si-NC组)和miR-2110敲低组(miR-2110-inhibitor组);以及miR-2110过表达+CDT1对照组(miR-2110-mimics+pcDNA-NC组)、miR-2110过表达+CDT1过表达组(miR-2110-mimics+pcDNA-CDT1组),按照LipofectamineTM 3000说明书分别转染相应质粒,24 h后提取mRNA,48 h后收集蛋白。

    LUAD及正常肺组织样本破碎后加入Trizol裂解液裂解并提取RNA,同样各组A549细胞用Trizol裂解液进行裂解并提取总RNA进行定量,然后按照miRNA反转录试剂盒说明书进行cDNA的反转录。最后按照miRNA PCR试剂盒说明书进行基因扩增。以U6为miRNA的内参,采用2-∆∆Ct法计算miR-2110表达量。

    收集各组A549细胞,用含有蛋白酶抑制剂的RIPA裂解液裂解细胞提取总蛋白并应用BCA试剂盒进行定量。通过10%SDS-聚丙烯酰胺凝胶电泳分离蛋白质样品。然后转移到PVDF膜。室温用含5%脱脂奶粉液进行膜的封闭2 h,然后加入CDT1(1∶2000)一抗放置于4 ℃冰箱孵育过夜。第2天用TBST洗膜3次、每次10 min,洗膜后加入对应二抗孵育1.5 h,最后用ECL试剂显影条带,用Image软件分析蛋白的表达。内参蛋白为GAPDH。

    将转染的miR-2110-mimics组、NC-mimics组、miR-2110-mimics+pcDNA-NC、miR-2110-mimics+pcDNA-CDT1组的A549细胞用含0.25%胰蛋白酶的EDTA消化液进行消化,PBS洗细胞2次,用Binding Buffer稀释细胞;取适量各组细胞加入5 μL FITC(BD公司,美国)和5 μL PI试剂(BD公司,美国),室温避光孵育15 min,设立单FITC和单PI对照组,最后每管加入400 μL Binding Buffer稀释细胞,于1 h内上机进行检测。

    将miR-2110-mimics组、NC-mimics组、miR-2110-mimics+pcDNA-NC组、miR-2110-mimics+pcDNA-CDT1组的A549细胞消化重悬于无血清培养基中。将1×105个细胞溶解在300 μL无血清培养基中加到Transwell上室中。将含有10%胎牛血清的完全培养基600 μL加入到下室中,在 37 ℃培养箱中孵育24 h,取出Transwell小室进行常规固定,并用结晶紫染色,然后弃去多余的结晶紫。干燥后,观察小室底膜上的细胞,并在显微镜(×100)下计数。

    将miR-2110-mimics组、NC-mimics组、miR-2110-mimics+pcDNA-NC组、miR-2110-mimics+pcDNA-CDT1组的A549细胞接种在6孔板中(每孔3000个细胞)。并在37 ℃、体积分数为5%CO2的培养箱中培养5 d左右。在显微镜下可见明显克隆后先用PBS清洗细胞2次,然后用多聚甲醛固定细胞15 min,再用PBS清洗细胞2次后,结晶紫染色20 min。最后PBS清洗多余的结晶紫,待干燥后进行拍照计数,按(克隆数/3000)×100%计算克隆形成率。

    将miR-2110-mimics组、NC-mimics组、miR-2110-mimics+pcDNA-NC、miR-2110-mimics+pcDNA-CDT1组的A549细胞接种到96孔培养板中(1×103细胞/孔),然后置于37 ℃、体积分数为5%CO2培养箱中进行常规培养。培养0、24、48、72、96 h时,加入10 μL CCK-8检测试剂,将细胞置于培养箱中孵育2 h。最后使用酶标仪测量450 nm的吸光度。

    分别将CDT1 3'UTR区包含miR-2110结合位点的基因片段进行PCR扩增,并克隆到pmirGLO载体上,命名为pmirGLO-CDT1 3'UTR WT。应用Q5® Site Directed Mutagenesis Kit试剂盒进行结合位点的点突变,突变质粒命名为pmirGLO-CDT1 3'UTR MUT。将构建的WT质粒和MUT质粒与miR-2110 (NC/mimic) 混合,共转染到A549细胞中,48 h后用含0.25%EDTA的胰蛋白酶消化每组细胞,收集并加入500 μL裂解物,然后在冰上孵育5 min。将细胞完全裂解后,将20 μL细胞裂解液加入黑色微孔板中,摇动与100 μL萤火虫荧光素酶反应液混合,以检测萤火虫荧光素酶的活性。同时,将孔板与100 μL肾荧光素酶反应液混合,摇动检测肾荧光素酶的活性。

    动物研究符合《实验动物护理和使用指南》(伦理号:2022114),裸鼠购自(斯贝福(北京)生物技术有限公司),饲养于IVC系统动物室。选取6~8周龄BALB/c雌性裸鼠10只,随机分为NC-mimics组(n=5)和miR-2110-mimics组(n=5),每只裸鼠前肢腋窝皮下接种含有5×106个稳定转染NC-mimics或miR-2110-mimics的A549细胞PBS试剂。3周后取肿瘤组织制作石蜡切片进行免疫组化染色。

    载玻片在56 ℃下过夜,然后在分级醇中脱水。石蜡切片用EDTA修复液在100 ℃下水化15 min,提取抗原。室温下冷却30 min后,用3%过氧化氢暗箱培养20 min,以消除内源性过氧化物酶活性。然后用10%正常山羊血清阻断非特异性结合位点,室温作用45 min。抗CDT1(1∶200)、抗Ki67(1∶400)、抗波形蛋白(vimentin)(1∶400)、抗基质金属蛋白酶9(matrix metalloproteinase-9, MMP9)(1∶100)与载玻片在4 ℃孵育过夜。PBS洗涤后,用辣根过氧化物酶(HRP)偶联二抗与载玻片孵育1 h, PBS洗涤载玻片,室温下加入3,3-二氨基联苯胺溶液。最后苏木精染色,酒精脱水,裱片。根据染色程度和染色强度评价CDT1、Ki67、vimentin、MMP9的表达。

    所有数据均应用GraphPad Prism 8.0软件进行统计分析,并采用$ \overline{x}\pm s $表示。两组之间用独立样本t检验进行分析,3组数据比较采用单因素方差分析(ANOVA),两两比较通过Tukey检测进行分析,CCK-8组间分析采用重复测量数据方差分析, α=0.05。

    通过ENCORI网站数据分析发现在LUAD组织中miR-2110表达明显降低(图1A),在收集的临床LUAD组织及正常肺组织样本中也验证了这一结果(P<0.001,图1B)。将正常组织样本进行混合后作为对照组与肺腺癌A549进行比较,同样发现miR-2110在A549中表达相对较低(P<0.001,图1C)。

    图  1  miR-2110在LUAD中低表达
    Figure  1.  miR-2110 is low expression in LUAD
    RPM: reads per million. A, The expression changes of miR-2110 in the ENCORI database; B, expression changes of miR-2110 in lung adenocarcinoma tissues and normal lung tissues (n=30); C, expression changes of miR-2110 in normal lung cells and A549 cells (n=3). *** P<0.001.

    miR-2110-mimics组A549细胞中miR-2110表达高于NC-mimics组(11.98±2.84 vs. 1.08±0.553,P=0.0028)(图2A);通过CCK-8实验及克隆形成实验发现在miR-2110-mimics组肿瘤细胞增殖能力降低〔克隆形成率(11.33±4.16)% vs. (32.00±5.29)%,P<0.0001〕,差异有统计学意义(图2B2C);通过Transwell实验同样观察到miR-2110-mimics组肿瘤细胞转移能力降低〔(18.00±2.000)% vs. (34.00±5.292)%,P=0.008〕,差异有统计学意义(图2D);通过流式细胞术检测细胞凋亡可见miR-2110-mimics组肿瘤细胞凋亡增加〔(68.08±1.843)% vs. (3.710±0.175)%,P<0.0001〕,差异有统计学意义(图2E)。

    图  2  过表达miR-2110可以抑制A549的增殖、转移并促进细胞凋亡
    Figure  2.  Overexpression of miR-2110 can inhibit the proliferation and metastasis of A549 and promote apoptosis
    A, The expression of miR-2110 was determined by PCR; B, CCK-8 assay was performed to determine the change in cell proliferation; C, clone formation assay was performed to determine the change in cell proliferation ability; D, Transwell assay was performed to determine the changes in cell metastasis ability (original magnification ×100); E, the changes in apoptosis were determined by flow cytometry. * P<0.05, ** P<0.01 (n=3).

    通过Tarbase、miRTarBase和TargetScan三个网站发现了32个可能的miR-2110靶基因(图3A);通过ENCORI数据库信息发现CDT1与miR-2110表达呈负相关(r=-0.166,P=1.68e-04)(图3B);通过应用miR-2110-inhibitor可以明显降低miR-2110在A549细胞中的表达(图3C);敲低miR-2110后CDT1表达升高,过表达miR-2110后CDT1表达降低(图3D、3E);最后通过双荧光素酶实验发现CDT1 WT-3'UTR随着miR-2110的过表达荧光强度明显下降,而对CDT1 MUT-3'UTR的荧光强度无明显影响,证明了miR-2110与CDT1存在靶向关系(图3F、3G)。

    图  3  LUAD中miR-2110与CDT1靶向关系
    Figure  3.  The targeting relationship between miR-2110 and CDT1 in LUAD
    A, Prediction of miR-2110 target genes by Tarbase, miRTarBase, and TargetScan databases; B, correlation analysis of miR-2110 and CDT1 in ENCORI database; C, the expression changes of miR-2110 were determined by PCR; D, expression changes of CDT1 after the knockdown and overexpression of miR-2110; E, histogram analysis of CDT1 protein expression; F, bioinformatics sites for predicting potential binding sequences of miR-2110 and CDT1; G, double luciferase reporter gene assay was performed to confirm that CDT1 was the target of miR-2110 in lung adenocarcinoma cells. * P<0.05, *** P<0.001 (n=3).

    Western blot实验示(图4A4B),相比于miR-2110-mimics+pcDNA-NC组,miR-2110-mimics+pcDNA-CDT1组中CDT1表达增高( 0.26±0.111vs. 1.37±0.341,P<0.0001);CCK-8实验表明与NC-mimics相比,miR-2110-mimics+pcDNA-NC组增殖能力明显降低,而miR-2110-mimics+pcDNA-CDT1组相比于miR-2110-mimics+pcDNA-NC组增殖能力得到恢复(图4C);克隆形成实验同样表明与NC-mimics相比,miR-2110-mimics+pcDNA-NC组增殖能力降低〔克隆形成率(27.33±2.082)% vs. (15.67±2.517)%,P=0.0011〕,而miR-2110-mimics+pcDNA-CDT1组相比于miR-2110-mimics+pcDNA-NC组增殖能力得到恢复〔(21.67±1.528)% vs. (27.33±2.082)%,P=0.0287〕(图4D);Transwell实验表明与NC-mimics相比,miR-2110-mimics+pcDNA-NC组转移能力降低〔(58.00±3.000)% vs. (16.33±1.528)%,P<0.0001〕,而miR-2110-mimics+pcDNA-CDT1组相比于miR-2110-mimics+pcDNA-NC组增殖能力得到恢复〔(25.00±3.000)% vs. (16.33±1.528)%,P=0.0154〕(图4E);流式细胞术检测细胞凋亡表明与NC-mimics相比,miR-2110-mimics+pcDNA-NC组细胞凋亡升高〔(6.96±0.770)% vs. (71.71±3.123)%,P<0.0001〕,而miR-2110-mimics+pcDNA-CDT1组相比于miR-2110-mimics+pcDNA-NC组细胞凋亡得到部分逆转〔(37.88±2.549)% vs. (71.71±3.123)%,P<0.0001〕(图4F)。

    图  4  过表达CDT1后部分逆转了过表达miR-2110对LUAD的生物学行为
    Figure  4.  Overexpression of CDT1 partially reversed the biological behaviors of the overexpression of miR-2110 on LUAD
    A, The changes in CDT1 expression were determined by Western blot (n=6). B, The changes in CDT1 expression were analyzed by histogram (n=3). C, CCK-8 assay was performed to determine the changes in cell proliferation ability, # P<0.05, vs. miR-2110-mimics+pcDNA-NC, P<0.05, vs. NC-mimics (n=3). D, Clone formation assay was performed to determine the changes in cell proliferation ability (n=3). E, Transwell assay was performed to determine the changes in cell metastasis ability (original magnification ×100) (n=3). F, The changes in apoptosis were determined by flow cytometry (n=3). * P<0.05, ** P<0.01, *** P<0.001.

    将各组细胞注射到裸鼠皮下,3周后处死裸鼠,可见miR-2110-mimics组肿瘤体积小于NC-mimics组(图5A);剥离瘤体并称量发现(图5B),与NC-mimics组相比,miR-2110-mimics组裸鼠肿瘤质量降低〔(1725.0±236.7) mg vs. (1058.0±172.5) mg,P=0.0009〕;免疫组化检测结果提示,与NC-mimics组相比,miR-2110-mimics组肿瘤组织中CDT1、Ki67、 vimentin、MMP9表达降低(图5C)。

    图  5  miR-2110在体内影响LUAD的增殖与转移
    Figure  5.  miR-2110 affects the proliferation and metastasis of LUAD in vivo
    A, Subcutaneous tumor transplantation was performed on nude mice in each group; B, the changes in subcutaneous grafts in each group were analyzed with histogram (n=5), *** P<0.001; C, the expression changes in CDT1, Ki67, vimentin, and MMP9 in each group were analyzed by immunohistochemistry staining.

    大量研究表明,miRNA主要参与转录后调控。miRNA表达异常可导致肿瘤的发生发展。miRNA主要通过与靶基因mRNA部分互补配对,在转录后水平抑制靶基因表达。研究表明,miRNA可参与多种生物过程,包括细胞增殖、凋亡、分化、代谢发育和肿瘤转移[13]。免疫系统的失调也是癌症发展的重要原因,多种miRNA与肿瘤免疫检测和肿瘤免疫逃逸过程有关。miRNA也可以被外泌体包裹并释放到细胞外,参与细胞间信息传递调控肿瘤的进展[14]。但目前对miR-2110的研究很少,仅有少量研究发现miR-2110在乳腺癌、神经母细胞瘤、直肠癌等肿瘤中起到抑制肿瘤的作用[10-11]。有生物信息学分析表明miR-2110在LUAD中可能通过作用微染色体维持家族2的表达影响肿瘤的侵袭[15]。但其在LUAD中的具体作用及相关机制目前尚未明确报道。因此,本研究首先通过生物信息学方法观察到miRNA-2110在LUAD组织中显著下调。有研究通过生物信息学分析也发现在LUAD中miR-2110为差异表达的miRNA,有望成为新的治疗标点[15]。为进一步验证数据库信息的准确性,本课题组又通过临床LUAD样本及LUAD细胞系进行了测定,所得结果与数据库信息是一致的。表明miR-2110在LUAD中可能起到抑制肿瘤的作用。这与先前研究在其他肿瘤中miR-2110作为抑制因子的作用是一致的[11]

    增殖转移是肿瘤的主要功能表现[16]。为进一步明确miR-2110在LUAD中抗肿瘤作用,本研究通过过表达miR-2110的方法发现,随着miR-2110表达的增加肿瘤增殖转移能力明显降低。先前的研究表明,miR-2110被长链非编码RNA AFAP1-AS1吸收,降低 SP1的靶向性,从而促进乳腺侵袭[10]。然而,这些研究缺乏动物模型来验证miR-2110的功能。本研究建立了miR-2110在LUAD细胞中的稳定过表达体系。体外细胞实验研究表明,miR-2110显著抑制了LUAD细胞的增殖和转移,并且促进了LUAD细胞的凋亡。并且通过裸鼠体内实验发现过表达miR-2110后肿瘤增殖标志物Ki67明显降低;上皮间质转化是肿瘤转移的重要机制,本研究同样发现在过表达miR-2110后上皮间质转化间质标志蛋白vimentin和MMP9的表达明显降低,表明肿瘤的转移能力明显下降[17],这些数据支持了miR-2110 在LUAD中起着抑制因子的作用。

    miR-2110需要调控下游靶基因才能发挥特定功能,本研究对miR-2110可能作用的靶基因进行了验证,通过生物信息技术进行了预测并根据本课题先前的研究发现,在LUAD中抑制CDT1可以抑制肿瘤的增殖转移并与患者预后相关[18],因此本课题将CDT1作为miR-2110的靶点,并通过数据库信息及双荧光素酶报告实验表明miR-2110可以靶向抑制CDT1的表达。在多种肿瘤中都发现CDT1可以通过抑制上皮间质转化等机制抑制肿瘤的增殖转移[19]。为了进一步证实miR-2110靶向抑制CDT1可以起到抗肿瘤作用,本研究通过共转染的方法,发现过表达CDT1可以明显恢复过表达miR-2110后的抑制肿瘤增殖转移及促凋亡作用。这些结果表明在LUAD中miR-2110可以通过作用于CDT1起到调控肿瘤的作用。在鼻咽癌中也发现miR-2110发挥了重要作用,并通过直接靶向FGFR1提高了PTEN蛋白的稳定性,抑制鼻咽癌转移[20]。同时有研究发现miR-2110可能在宫颈癌中影响免疫疗效[21]。因此miR-2110也可能在调节免疫等方面发挥着重要作用。

    综上所述,本研究结果表明miRNA-2110在LUAD中表达下调,促进了LUAD细胞的增殖和转移并抑制了细胞凋亡。此外,本研究通过体外实验揭示了miRNA-2110通过靶向CDT1影响LUAD的增殖转移和凋亡,并通过体内实验验证了在LUAD中miR-2110起到了明显的抗肿瘤作用,但该结论尚未得到临床多中心数据的验证,以及miR-2110通过CDT1具体的调控通路也有待进一步地明确。因此,进一步明确miR-2110在LUAD中作用将为开发miRNA疗法候选药物奠定基础,为LUAD预防及与化疗、放疗、免疫治疗互补治疗提供新的方向,希望通过利用miR-2110强大的抑制肿瘤的作用,使广大LUAD患者从中受益。

    *    *    *

    作者贡献声明 米源负责论文构思和初稿写作,李旭哲负责调查研究和研究方法,王聪负责数据审编,蔺晨曦负责正式分析,赵志超负责软件,颜晓菲负责验证,史平负责可视化,王雷负责经费获取、研究项目管理、提供资源、监督指导和审读与编辑写作。所有作者已经同意将文章提交给本刊,且对将要发表的版本进行最终定稿,并同意对工作的所有方面负责。

    Author Contribution  MI Yuan is responsible for conceptualization and writing--original draft. LI Xuzhe is responsible for investigation and methodology. WANG Cong is responsible for data curation. LIN Chenxi is responsible for formal analysis. ZHAO Zhichao is responsible for software. YAN Xiaofei is responsible for validation. SHI Ping is responsible for visualization. WANG Lei is responsible for funding acquisition, project administration, resources, supervision, and writing--review and editing. All authors consented to the submission of the article to the Journal. All authors approved the final version to be published and agreed to take responsibility for all aspects of the work.

    利益冲突 所有作者均声明不存在利益冲突

    Declaration of Conflicting Interests All authors declare no competing interests.

  • 图  1   miR-2110在LUAD中低表达

    Figure  1.   miR-2110 is low expression in LUAD

    RPM: reads per million. A, The expression changes of miR-2110 in the ENCORI database; B, expression changes of miR-2110 in lung adenocarcinoma tissues and normal lung tissues (n=30); C, expression changes of miR-2110 in normal lung cells and A549 cells (n=3). *** P<0.001.

    图  2   过表达miR-2110可以抑制A549的增殖、转移并促进细胞凋亡

    Figure  2.   Overexpression of miR-2110 can inhibit the proliferation and metastasis of A549 and promote apoptosis

    A, The expression of miR-2110 was determined by PCR; B, CCK-8 assay was performed to determine the change in cell proliferation; C, clone formation assay was performed to determine the change in cell proliferation ability; D, Transwell assay was performed to determine the changes in cell metastasis ability (original magnification ×100); E, the changes in apoptosis were determined by flow cytometry. * P<0.05, ** P<0.01 (n=3).

    图  3   LUAD中miR-2110与CDT1靶向关系

    Figure  3.   The targeting relationship between miR-2110 and CDT1 in LUAD

    A, Prediction of miR-2110 target genes by Tarbase, miRTarBase, and TargetScan databases; B, correlation analysis of miR-2110 and CDT1 in ENCORI database; C, the expression changes of miR-2110 were determined by PCR; D, expression changes of CDT1 after the knockdown and overexpression of miR-2110; E, histogram analysis of CDT1 protein expression; F, bioinformatics sites for predicting potential binding sequences of miR-2110 and CDT1; G, double luciferase reporter gene assay was performed to confirm that CDT1 was the target of miR-2110 in lung adenocarcinoma cells. * P<0.05, *** P<0.001 (n=3).

    图  4   过表达CDT1后部分逆转了过表达miR-2110对LUAD的生物学行为

    Figure  4.   Overexpression of CDT1 partially reversed the biological behaviors of the overexpression of miR-2110 on LUAD

    A, The changes in CDT1 expression were determined by Western blot (n=6). B, The changes in CDT1 expression were analyzed by histogram (n=3). C, CCK-8 assay was performed to determine the changes in cell proliferation ability, # P<0.05, vs. miR-2110-mimics+pcDNA-NC, P<0.05, vs. NC-mimics (n=3). D, Clone formation assay was performed to determine the changes in cell proliferation ability (n=3). E, Transwell assay was performed to determine the changes in cell metastasis ability (original magnification ×100) (n=3). F, The changes in apoptosis were determined by flow cytometry (n=3). * P<0.05, ** P<0.01, *** P<0.001.

    图  5   miR-2110在体内影响LUAD的增殖与转移

    Figure  5.   miR-2110 affects the proliferation and metastasis of LUAD in vivo

    A, Subcutaneous tumor transplantation was performed on nude mice in each group; B, the changes in subcutaneous grafts in each group were analyzed with histogram (n=5), *** P<0.001; C, the expression changes in CDT1, Ki67, vimentin, and MMP9 in each group were analyzed by immunohistochemistry staining.

  • [1]

    HUIHUI L, ZEXIN C, NING C, et al. Applications of lung cancer organoids in precision medicine: from bench to bedside. Cell Commun Signal, 2023, 21(1): 350. doi: 10.1186/s12964-023-01332-9.

    [2]

    MAHMOOD Y, HABIB J, SOMAYEH H, et al. Cost of lung cancer: a systematic review. Value Health Reg Issues, 2022, 33: 17–26. doi: 10.1016/j.vhri.2022.07.007.

    [3]

    ROSAMARIA P, DANIELA P, ROSANNA L, et al. KRAS-driven lung adenocarcinoma and b cell infiltration: novel insights for immunotherapy. Cancers (Basel), 2019, 11(8): 1145. doi: 10.3390/cancers11081145.

    [4]

    SHAN J, AN Y, BUES M, et al. Robust optimization in IMPT using quadratic objective functions to account for the minimum MU constraint. Med Phys, 2018, 45(1): 460–469. doi: 10.1002/mp.12677.

    [5]

    JIANG X, LI Y, ZHANG N, et al. RRM2 silencing suppresses malignant phenotype and enhances radiosensitivity via activating cGAS/STING signaling pathway in lung adenocarcinoma. Cell Biosci, 2021, 11(1): 74. doi: 10.1186/s13578-021-00586-5.

    [6]

    NAKANISHI T, YONESHIMA Y, OKAMURA K, et al. MicroRNA-326 negatively regulates CD155 expression in lung adenocarcinoma. Cancer Sci, 2023, 114(10): 4101–4113. doi: 10.1111/cas.15921.

    [7] 张丽萍, 白俊, 胡雅琼 等. MiR-204通过靶向调控HNRNPA2B1抑制乳腺癌的侵袭和转移. 南方医科大学学报, 2020, 40(6): 869–875. doi: 10.12122/j.issn.1673-4254.2020.06.15.

    ZHANG L P, BAI J, HU Y Q, et al. MiR-204 inhibits invasion and metastasis of breast cancer cells by targeted regulation of HNRNPA2B1. J South Med Univ, 2020, 40(6): 869–875. doi: 10.12122/j.issn.1673-4254.2020.06.15.

    [8]

    SON S M, YUN J, KIM D W, et al. MicroRNA 29a therapy for CEACAM6-expressing lung adenocarcinoma. BMC Cancer, 2023, 23(1): 843. doi: 10.1186/s12885-023-11352-w.

    [9]

    ZHU J Y, PFUHL T, MOTSCH N, et al. Identification of novel Epstein-Barr virus microRNA genes from nasopharyngeal carcinomas. J Virol, 2009, 83(7): 3333–3341. doi: 10.1128/JVI.01689-08.

    [10]

    ZHANG X, LI F, ZHOU Y, et al. Long noncoding RNA AFAP1-AS1 promotes tumor progression and invasion by regulating the miR-2110/Sp1 axis in triple-negative breast cancer. Cell Death Dis, 2021, 12(7): 627. doi: 10.1038/s41419-021-03917-z.

    [11]

    ZHAO Z, PARTRIDGE V, SOUSARES M, et al. microRNA-2110 functions as an onco-suppressor in neuroblastoma by directly targeting Tsukushi. PLoS One, 2018, 13(12): e0208777. doi: 10.1371/journal.pone.0208777.

    [12]

    FERRACIN M, LUPINI L, SALAMON I, et al. Absolute quantification of cell-free microRNAs in cancer patients. Oncotarget, 2015, 6(16): 14545–14555. doi: 10.18632/oncotarget.3859.

    [13]

    ZHANG B, WANG D, WANG Y, et al. miRNA-338-3p inhibits the migration, invasion and proliferation of human lung adenocarcinoma cells by targeting MAP3K2. Aging (Albany NY), 2022, 14(15): 6094–6110. doi: 10.18632/aging.204198.

    [14]

    HE B, ZHAO Z, CAI Q, et al. miRNA-based biomarkers, therapies, and resistance in Cancer. Int J Biol Sci, 2020, 16(14): 2628–2647. doi: 10.7150/ijbs.47203.

    [15]

    TANIGAWA K, TOMIOKA Y, MISONO S, et al. Minichromosome maintenance proteins in lung adenocarcinoma: clinical significance and therapeutic targets. FEBS Open Bio, 2023, 13(9): 1737–1755. doi: 10.1002/2211-5463.13681.

    [16]

    MAJIDPOOR J, MORTEZAEE K. Steps in metastasis: an updated review. Med Oncol, 2021, 38(1): 3. doi: 10.1007/s12032-020-01447-w.

    [17]

    KIM J, YOU H J, YOUN C. SCARA3 inhibits cell proliferation and EMT through AKT signaling pathway in lung cancer. BMC Cancer, 2022, 22(1): 552. doi: 10.1186/s12885-022-09631-z.

    [18] 米源, 梁宇翔, 王聪 等. 肺腺癌中通过靶向CDT1抑制肿瘤生长及其机制研究. 安徽医科大学学报, 2024, 59(2): 260–266. doi: 10.19405/ j.cnki. issn1000-1492.2024.02.013.

    MI Y, LIANG Y X, WANG C, et al. Targeting CDT1 inhibit tumor growth and its mechanism in lung adenocarcinoma. Acta Univer Med Anhui, 2024, 59(2): 260–266. doi: 10.19405/ j.cnki. issn1000-1492.2024.02.013.

    [19]

    SHI H X, HUANG S W, LUO W J, et al. Elevated expression of CDT1 in childhood acute lymphoblastic leukemia promotes cell proliferation, invasion and migration through activation of EMT. J Biol Regul Homeost Agents, 2021, 35 (Special Issue on Internal Medicine n.1). doi: 10.23812/21-SI1-6.

    [20]

    FANG S, PENG L, ZHANG M, et al. MiR-2110 induced by chemically synthesized cinobufagin functions as a tumor-metastatic suppressor via targeting FGFR1 to reduce PTEN ubiquitination degradation in nasopharyngeal carcinoma. Environ Toxicol, 2024, 39(6): 3548–3562. doi: 10.1002/tox.24197.

    [21]

    HAN Y H, MA D Y, LEE S J, et al. Bioinformatics analysis of novel targets for treating cervical cancer by immunotherapy based on immune escape. Cancer Genomics Proteomics, 2023, 20(4): 383–397. doi: 10.21873/cgp.20390.

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  • 收稿日期:  2024-05-09
  • 修回日期:  2024-08-14
  • 发布日期:  2024-09-19
  • 刊出日期:  2024-09-19

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