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摘要:目的
探讨CENPF在非小细胞肺腺癌(LUAD)中的表达与患者临床预后的关系及其对肺腺癌细胞转移能力的影响。
方法公共数据库分析CENPF在LUAD中的表达及其与患者预后的关系。免疫组织化学染色验证CENPF LUAD在组织芯片中的表达,Kaplan-Meier分析CENPF表达与肺腺癌患者预后的关系;Cox生存风险比例回归模型分析影响患者生存的因素;卡方分析CENPF表达与患者临床病理分期及分级的关系。慢病毒敲除NCI-H2126细胞中CENPF的表达,检测细胞增殖、侵袭及迁移能力的变化。RNA-seq检测CENPF敲除后细胞mRNA表达谱改变,生物信息学分析CENPF下游信号通路及靶基因,Western blot验证下游基因表达水平变化。
结果CENPF在LUAD肿瘤组织中显著上调(P < 0.05),与病理分期显著相关(P=0.013),表达越高患者预后越差(P=0.01, P=0.027)。敲除CENPF表达后,细胞增殖、迁移及侵袭能力均显著降低(P < 0.01);RNA-seq富集分析显示细胞趋化因子通路基因表达改变富集显著(P < 0.001);聚类差异分析则表明,ACKR3/CXCR7及CDH2/N-cadherin显著下调,CDH1/E-cadherin则显著上调;Western blot结果证实,敲除CENPF后,ACKR3/CXCR7及N-cadherin显著下调,E-cadherin则显著上调。
结论CENPF的表达与LUAD患者临床预后呈负相关,其通过ACKR3/CXCR7调控与EMT相关的N-cadherin及E-cadherin的表达促进EMT的发生。
Abstract:ObjectiveTo investigate the relationship between the expression of CENPF in NSCLC adenocarcinoma (LUAD) and the clinical prognosis of patients and its effect on the metastasis of lung adenocarcinoma cells.
MethodsThe expression of CENPF in LUAD and its relationship with patient prognosis were analyzed by online bioinformatics. The expression of CENPF was verified by LUAD tissue microarray immunohistochemical staining. Kaplan-Meier analysis was performed to analyze the relationship between the expression of CENPF and the prognosis of patients with lung adenocarcinoma. Cox survival hazard ratio was used to analyze the factors affecting the survival of patients. Chi-square analysis was adopted to examine the relationship between CENPF expression and clinicopathological stage and grade of patients. The expression of CENPF in NCI-H2126 cells were knocked out by lentivirus, and then the proliferation, invasion, and migration abilities of the cells were detected. Changes in mRNA expression profiles after CENPF knockout were detected by RNA-seq. Bioinformatics analysis of downstream signaling pathways and the target genes of CENPF was also performed. Western blot was used to verify the target gene.
ResultsCENPF was significantly upregulated in LUAD tumor tissue (P < 0.05) and significantly correlated with pathological stage (P=0.013). The higher expression of CENPF, the worse the prognosis of patients (P=0.01, P=0.027). After the expression was CENPF of knocked out, the cell proliferation, migration, and invasion abilities significantly reduced (P < 0.01). The expression of chemokine pathway genes in cells was enriched significantly (P < 0.001). ACKR3/CXCR7 and CDH2/N-cadherin were significantly downregulated, whereas CDH1/E-cadherin was significantly upregulated. After CENPF was knocked out, ACKR3/CXCR7 and N-cadherin were significantly downregulated, whereas E-cadherin significantly increased.
ConclusionThe expression of CENPF is negatively correlated with the clinical prognosis of patients with LUAD, and it promotes the occurrence of EMT by regulating the expression levels of N-cadherin and E-cadherin related to EMT through ACKR3/CXCR7.
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Key words:
- CENPF /
- Lung adenocar-cinoma /
- ACKR3 /
- Epithelial-mesen-chymal transition /
- CXCR7
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0 引言
肺癌是目前全球最常见的恶性肿瘤之一,其发病率和死亡率分别位居世界第二位和第一位[1],尤其在中国,其5年生存率仅有19.7%[2]。NSCLC根据组织结构异质性又分为鳞癌(LUSC)和腺癌(LUAD)[3]。目前,尽管NSCLC的治疗有了非常大的进步,但患者总体预后仍然较差,主要是多数患者确诊时已经是晚期且已发生转移,另一个重要原因则是手术切除后复发[4]。因此寻找新的治疗方法和治疗靶点来改善NSCLC患者预后仍然是目前亟待解决的关键问题。人类着丝粒蛋白F(CENPF)是叉头框蛋白M1(FOXM1)的已知靶点[5],研究表明,CENPF的表达与多种肿瘤的进展及预后显著相关[6-8],且CENPF在LUAD中的功能尚未完全阐明,为此本研究将探讨CENPF在LUAD中表达与患者临床预后及病理分期的关系,同时初步探讨其发挥功能的机制。
1 资料与方法
1.1 临床样本
LUAD组织芯片购自上海芯超生物科技有限公司(HLugA180Su07),样本剔除失访病例后共87例肺腺癌患者组织,其中男47例、女40例,平均年龄(63.8±8.72)岁,T1期患者16例、T2期患者46例、T3期患者20例、T4期患者5例;病理分级为Ⅰ级7例、Ⅱ级50例、Ⅲ级30例。
1.2 材料与设备
肺腺癌NCI-H2126细胞(中国科学院上海细胞库)。DMEM培养基(美国Gibco公司)、胎牛血清(FBS)(美国Hyclone公司)。青霉素、链霉素(美国Hyclone公司)。CENPF慢病毒由吉凯基因公司设计生产。SDS-PAGE试剂(上海生工公司)。ACKR3/CXCR7、E-cadherin、N-cadherin及GAPDH抗体(武汉三鹰)。主要设备有生物安全柜、CO2培养箱、垂直电泳仪、化学发光成像系统、Tecan酶标仪等。
1.3 免疫组织化学染色
SP法检测组织芯片中CENPF的表达。石蜡切片常规脱蜡至水,加0.01 mol/L枸橼酸缓冲液(pH6.0),高压锅进行抗原热修复,加3%H2O2室温避光孵育30 min,PBS洗涤3次。滴加10%的正常非免疫羊血清,37℃孵育60 min,PBS洗涤3次。滴加CENPF一抗工作液(1:100),4℃孵育过夜,PBS洗涤3次。滴加二抗工作液(稀释比例为1:500),37℃孵育60 min,PBS洗涤3次。滴加辣根过氧化物酶(HRP)标记的链霉卵白素,37℃孵育60 min,PBS洗涤3次。DAB显色、苏木精对比染色、脱水、二甲苯透明、中性树胶封片。采用TissueGnostics全景组织扫描仪对染色结果进行扫描,根据染色结果,以所有患者的平均吸光度值的中位表达值为截断值将所有患者分为CENPF高表达组和CENPF低表达组。
1.4 Western blot检测
细胞用RIPA裂解液进行裂解,超声后离心取上清液。BCA试剂盒(上海生工公司)测蛋白浓度。SDS-PAGE电泳用5%的浓缩胶与12%的分离胶进行。电泳后将蛋白转至PVDF膜,5%BSA封闭。分别加CENPF(1:1 000)、ACKR3(1:1 000)、E-cadherin(1:1 000)、N-cadherin(1:1 000)及GAPDH(2:1 000)4℃孵育过夜。洗涤,二抗(1:5 000,武汉三鹰公司)室温孵育,洗涤,曝光显色。
1.5 CENPF稳定敲除细胞株建立
根据人CENPF的mRNA序列设计siRNA慢病毒干扰序列(正义引物: 5’-TAAGAGTCCCATTCTCTTGGG-3’,反义引物: 5’-TAAGAGTCCCATTCTCTTGGG-3’)。参照慢病毒转染说明书感染NCI-H2126细胞,并用嘌呤霉素进行筛选。筛选至第14天时,将筛选细胞进行Western blot鉴定,检测CENPF表达,获得稳定敲除CENPF的单克隆细胞株。
1.6 RNA-seq检测
细胞提取RNA后,按照RNA-seq测序要求制备样品后送第三方测序公司(诺禾致源公司)完成mRNA测序,并对测序结果进行生物信息学分析。
1.7 细胞迁移能力检测
Transwell小室下室加入500 μl含10% FBS的完全培养基,上室中分别接种1×105个无血清培养基重悬的细胞。37℃、5%CO2培养箱中培养24 h后用湿棉签擦去上室面未迁移的肿瘤细胞,固定1 min,然后结晶紫染色,三蒸水洗2次后在普通光学显微镜下随机选取5个视野计总数,取其平均值。
1.8 细胞侵袭能力检测
按照说明将50 mg/L Matrigel(美国Corning公司)1:8稀释液包被Transwell小室底部膜的上室面,4℃风干。纤维粘蛋白(FN)(美国Sigma公司)均匀涂抹在Transwell小室的下室面,24孔板中加入500 μl含10%FBS的完全培养基,然后将Transwell小室置于孔中,上室中接种1×105个无血清培养基重悬的细胞。37℃、CO2培养箱中培养24 h。取出小室,用湿棉签擦去上室面未侵袭的肿瘤细胞。甲醇固定1 min,结晶紫染色,三蒸水洗2次后在显微镜下随机选取5个视野计总数,取其平均值。
1.9 细胞增殖能力检测
取对数期细胞做梯度稀释。6孔板每孔种1 000个细胞,约两周后从培养箱中取出,中间可视细胞生长情况给其换新鲜培养基;PBS小心清洗两遍。每孔加入1 ml甲醇固定20 min后弃甲醇,每孔加入1 ml结晶紫染色20 min,PBS洗两遍,晾干后用核酸凝胶成像仪拍照,观察克隆大小,计数超过50个克隆的数量。取对数期细胞接种于96孔板,每孔2 000个细胞。每孔加入10 μl CCK-8溶液,24、48和72 h时在37℃下孵育2 h。酶标仪测量实验孔在450 nm处的吸光度值。
1.10 统计学方法
SPSS22.0和Graph Pad Prism 8.0软件进行数据分析与作图。计数资料用频数和百分比表示,计量资料采用均数±标准差(x±s)表示。Cox生存风险比例模型分析患者年龄、性别、TNM分期、肿瘤大小、病理分期及CENPF表达与患者死亡风险的关系;Kaplan-Meier分析CENPF表达与肺腺癌患者预后的关系;t检验分析独立样本之间差异,χ2检验或Fisher精确检验进行计数资料分析。P < 0.05为差异有统计学意义。
2 结果
2.1 CENPF在LUAD中表达及与患者预后的关系
GEPIA2数据库(http://gepia2.cancer-pku.cn/#index)分析显示,CENPF在LUAD中表达较正常组织显著上调且与临床病理分期正相关,与患者生存及无疾病进展呈负相关,见图 1A~D。免疫组织化学染色进行验证,结果证实,LUAD中CENPF表达显著升高,见图 1E~F,且其表达与患者预后显著相关,低表达患者预后优于高表达患者,见图 1G。
图 1 CENPF在LUAD中的表达及与患者临床预后的关系Figure 1 Expression of CENPF in LUAD and correlated with clinical prognosis of patients*: P < 0.05, **: P < 0.01; A-D: GEPIA2 database to analyze the expression of CENPF in LUAD and its relationship with the clinical prognosis of patients; E-F: expression of CENPF in normal tissues of LUAD patients and paired tumor tissues was detected by immunohistochemical staining; G: Kaplan-Meier analysis of the relationship between CENPF expression and clinical prognosis of patients with lung adenocarcinoma.2.2 LUAD患者死亡风险因素分析
Cox风险比例回归模型分析结果显示,LUAD患者N分期及CENPF表达与患者死亡风险相关,而T分期、年龄、性别、病理分型及肿瘤大小则与患者死亡风险无关,见表 1。
表 1 Cox生存风险比例分析与患者死亡相关因素Table 1 Cox survival hazard ratio analysis of major factors associated with patient death2.3 CENPF表达与患者临床病理因素的关系
Kaplan-Meier生存分析中CENPF表达的截断值将低表达患者定义为阴性,高表达患者定义为阳性,结果表明,除N分期与CENPF表达有关外,性别、年龄、T分期、病理分级及肿瘤大小均与CENPF表达无关,这表明CENPF的表达与患者淋巴结转移密切相关,见表 2。
表 2 CENPF表达与临床病理因素之间的关联Table 2 Association between CENPF expression and clinicopathological factors2.4 CENPF表达对LUAD细胞表型的影响
体外细胞实验表明,下调NCI-H2126细胞CENPF表达后,细胞增殖能力、迁移能力及侵袭能力显著降低,见图 2。
图 2 下调CENPF表达对LUAD细胞增殖、侵袭及迁移的影响Figure 2 Effect of down-regulation of CENPF on proliferation, invasion and migration of LUAD cellsA: CENPF was knocked out in NCI-H2126 cells; B: cell proliferation was detected by CCK-8; C-D: cell colony formation ability was detected by plate clone formation assay; E-H: cell migration and invasion abilities were detected by Transwell. **: P < 0.01, ***: P < 0.001, compared with sh-con group2.5 CENPF通过下调ACKR3/CXCR7抑制NCI-H2126细胞EMT
NCI-H2126细胞敲除CENPF表达后RNA-seq结果显示,共有132个基因的表达出现显著变化,见图 3A。KEGG富集分析显示细胞趋化因子信号通路富集差异最为显著,见图 3B,其中ACKR3/ CXCR7表达下调最为明显,Western blot验证结果表明,CENPF敲除后,ACKR3/CXCR7表达显著下调。而在聚类差异中分析显示,EMT相关基因CDH1/E-cadherin表达显著上调,而CDH2/N-cadherin表达显著下调,见图 3C,蛋白检测结果显示,敲除CENPF的表达后,E-cadherin显著上调,N-cadherin则显著下调,见图 3D。
图 3 CENPF通过下调ACKR3/ CXCR7抑制NCI-H2126细胞EMTFigure 3 CENPF inhibited EMT in NCI-H2126 cells by downregulating ACKR3/CXCR7A: RNA-seq was used to analyze the changes in mRNA expression profile and the number of differentially-expressed genes after the expression of CENPF was knocked out in NCI-H2126 cells; B: the KEGG signaling pathway enrichment; C: the cluster differential analysis; D: protein expression of ACKR3/CXCR7, E-cadherin, and N-cadherin were verified by Western blot.3 讨论
近年来以表皮生长因子酪氨酸激酶抑制剂为代表的分子靶向及免疫治疗的发展显著延长了NSCLC患者无进展生存期及总生存期[9]。尽管这些治疗方法取得了一定的进展,但由于诊断时多数患者已经是晚期且治疗后易出现复发和耐药性等,导致其治疗前景不甚乐观。因此研究肺癌的发病机制,寻找新的治疗靶点具有重要意义。
CENPF是FOXM1的已知靶点[6],该蛋白于1993年由Yen和Lee鉴定,约367 kDa。CENPF因其与着丝粒-动粒蛋白复合物的结合特征而得名,但这种结合只是短暂的,CENPF还具备其他功能——通过蛋白质相互作用对有丝分裂和细胞增殖进行调节[10-12]。研究表明,CENPF的表达在乳腺癌及膀胱癌中与患者的预后明显相关[13-14];在胃癌中,hnRNPR通过上调CENPF的表达促进胃癌进展[15],但CENPF在胃癌中的作用机制仍然不清楚。研究表明[8, 16],在前列腺癌中,CENPF可通过与FOXM1协同调节靶基因表达和激活与前列腺癌恶性相关的关键信号通路,协同促进前列腺癌生长。此外,FOXM1和CENPF的联合表达还是前列腺癌预后不良和转移的有力指标[17-18]。尽管以上研究都表明CENPF可能在肿瘤的发生发展中扮演着重要的作用,但CENPF在肿瘤中发挥的功能及其作用机制却仍未被完全阐明。本研究发现,在LUAD中,CENPF的表达显著上调,且与患者N分期及临床预后显著相关,这表明,CENPF在LUAD中发挥着非常重要的作用。
肿瘤细胞要入侵周围的组织并最终形成转移需要上皮细胞经历一个去分化的过程,这一过程称为上皮间质转化(EMT)。在EMT期间,上皮细胞失去其极性和细胞间黏附,并获得具有更高运动性的间充质表型[19]。在EMT过程中,上皮细胞黏附连接的蛋白(E-cadherin)被能提供更大连接灵活性的蛋白(N-cadherin)取代,从而导致细胞分离和细胞运动性增强。E-cadherin介导钙依赖性细胞黏附并在正常组织发育中发挥关键作用。在正常组织和细胞中,E-cadherin的表达是可以检测且表达相对较高的,而N-cadherin则只在心肌、平滑肌及睾丸细胞中表达较高,其他的正常组织细胞表达量则较低或基本检测不到[20]。而在大部分的肿瘤细胞中,癌细胞为发动侵袭转移,N-cadherin则经常被上调,E-cadherin则经常被下调。在本研究中,敲除CENPF的表达后,LUAD细胞中被上调的N-cadherin则被逆转消除,而E-cadherin的表达则被恢复,这表明CENPF能够通过影响LUAD的EMT过程促进其发生转移,结果与患者临床样本组织中CENPF的高表达与患者发生淋巴结转移呈正相关相符合。
E-cadherin的表达在多个水平上受到调节,如表观遗传、转录和翻译后修饰等。在转录水平上,E-cadherin受到许多转录因子(E-钙黏蛋白转录抑制因子,EcTRs)的抑制,如ZEB1、ZEB2、SNAI1、SNAI2、E12/E47和Twist1/2[9]。EcTRs能够与E-cadherin启动子区域结合从而抑制其表达,在本研究中,RNA-seq测序未发现LUAD细胞在CENPF表达被改变后EcTRs的表达发生改变,这表明,CENPF可能不是通过调控一系列EcTRs的表达,而可能是通过其他途径抑制了EcTRs与E-cadherin启动子区域的结合。研究表明,ACKR3/ CXCR7能够促进TGF-β1介导的EMT发生[16],而本研究中,RNA-seq结果显示,趋化因子富集信号通路改变显著,其中ACKR3/CXCR7表达变化最大,蛋白质表达验证结果进步一证实,在CENPF敲除后,ACKR3/CXCR7的表达被下调,本研究发现CENPF通过调控ACKR3/CXCR7进而促进肺腺癌细胞的EMT,在后续研究中对CENFP调控EMT标志蛋白E-cadherin及N-cadherin的详细机制仍需要进一步深入研究。
综上所述,CENPF在LUAD中表达上调且与患者预后和淋巴结转移显著相关,它能够通过上调ACKR3/CXCR7进而促进LUAD细胞EMT的进展。本研究也存在一定的局限性,CENPF调控ACKR3/CXCR7的详细机制尚未解释清楚,仍需要进一步的深入研究。
Competing interests: The authors declare that they have no competing interests.作者贡献:顾彤、姜瑜珩:实验实施及数据整理、论文撰写丁姝、陈炜:数据分析罗超:实验设计和文章审核于伟勇、陈小飞:研究设计、数据审核、文稿撰写及修改 -
表 1 Cox生存风险比例分析与患者死亡相关因素
Table 1 Cox survival hazard ratio analysis of major factors associated with patient death
表 2 CENPF表达与临床病理因素之间的关联
Table 2 Association between CENPF expression and clinicopathological factors
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