Progress on Multi-disciplinary Combined Therapy for Locally Advanced Hypopharyngeal Carcinoma
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摘要:
由于下咽独特的位置与功能，器官功能的保全与生存的提高同样重要，对于下咽癌的最佳保喉策略在持续探索中。手术、放疗、化疗、分子靶向治疗以及免疫治疗等手段的不断进步，新的保喉治疗策略不断涌现。本文主要介绍下咽癌保喉治疗策略的确立、发展以及对未来的展望。
Abstract:Given the important position and function of the hypopharynx, the preservation of organ function and survival improvement are equally important. The optimal role of multi-disciplinary combined treatment modality becoming increasingly important. The optimal laryngeal preservation strategy for hypopharyngeal cancer is under continuous exploration. With the constant research of surgery, radiotherapy, chemotherapy, and molecular targeted therapy, new laryngeal preservation strategies continue to emerge. Herein, we primarily summarize the advances in multi-disciplinary combined treatment and the future direction in the treatment of hypopharyngeal carcinoma.
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0 引言
结肠癌早期特异性症状不明显，而术后复发和转移影响了治疗效果。当前，阐明结肠癌发生发展的分子机制对寻找新的分子靶点具有重要意义。miRNA是一类非编码小分子RNA。通过与靶基因mRNA 3’-UTR完全或不完全互补配对结合，导致靶miRNA降解或翻译抑制来调控靶基因的表达。研究证实，在结肠癌细胞中存在多个miRNA表达异常，且与结肠癌患者的分化程度、远处转移等病理特征之间存在相关性，提示miRNA可能参与结肠癌的发生及发展[1-2]。SMURF1是HECT型E3泛素连接酶，参与多个生物学信号通路，近来研究发现，其在结肠癌组织中呈现高表达，并协同泛素蛋白酶系统降解抑癌蛋白，表现出促癌功能[3]。miRNA-125a在多种肿瘤的形成和转移中发挥重要作用[4]。本研究旨在分析miRNA-125a与结肠癌相关临床指标的相关性，并探寻其通过靶向调控SMURF1在结肠癌的病理过程中所发挥的作用，为结肠癌的诊断及治疗寻找新靶点。
1 资料与方法
1.1 研究对象
收集2017年6月至2018年1月在我院收治的50例结肠癌患者，其中男21例、女29例，平均年龄（59.72±5.36）岁。所有患者无化疗或放疗史。
1.2 外周血采集
纳入研究的结肠癌患者均于清晨空腹状况下进行外周静脉血抽取，具体操作如下：采用一次性血清分离胶管，抽取外周静脉血约3 ml；待血清析出后以1 000 g离心10 min，取上层血清保存至-80℃冰箱待检测。
1.3 qRT-PCR检测外周血miRNA-125a和SMURF1的表达
取0.5 ml血清标本，采用TRIzol法提取血清总RNA，实验步骤按照TRIzol试剂盒说明书进行。将提取到的总RNA按照反转录试剂盒说明书进行RNA反转录，合成cDNA。按照qRT-PCR试剂盒说明书进行PCR反应。miRNA-125a的测序PCR引物为：上游5’-TGTGTCTCTTTCACAGTGGATC-3’和下游5’-CCATCG TGTGGGTCTCAAG-3’。
1.4 SW480细胞培养
使用含10%胎牛血清、100 u/ml青霉素、100 μg/ml链霉素的DMEM培养基在37℃、5%CO2饱和湿度培养条件下培养SW480细胞。
1.5 细胞转染
根据转染SW480细胞的转染物进行分组：miR-NC组（转染negative control miRNA mimics），miRNA-125a模拟物转染组（转染miRNA-125a模拟物），miRNA-125a模拟物+SMURF1转染组（转染miRNA-125a模拟物和SMURF1）。
1.6 荧光素酶报告基因法检测miRNA-125a与靶基因SMURF1的结合
利用microRNA靶基因数据库预测miRNA-125a与SMURF1可能的作用位点。构建SMURF1野生型3’-UTR荧光素酶质粒pMIR-WT和突变型质粒pMIR-Mut：根据GenBank中登录的human SMURF1基因序列，上游：5’-ACTCCTGGTACAGCGACTCCGAAATCCT-3’，下游：5’-GTCCCTGCACTGTTGTCCTTTGCTCATA-3’，采用Primer Premier5.0软件设计PCR引物，PCR扩增目的基因片段，并连接入野生型载体，转化及菌液PCR鉴定，送南京金斯瑞科技有限公司测序，测序鉴定正确的重组载体命名为pMIR-WT。突变载体的构建步骤与双荧光素酶载体的构建基本一致，利用PCR突变法在野生型载体基础上设计突变引物，引物由南京金斯瑞生物科技有限公司合成。以野生型载体为模板进行扩增，鉴定正确的重组载体命名为pMIR-Mut。将pMIR-WT、pMIR-Mut与miR-125a模拟物和negative control miRNA mimics共转染进SW480细胞，荧光素酶检测实验检测各组荧光素酶活性。
1.7 Western blot检测SMURF1蛋白表达
将miRNA-125a模拟物和negative control miRNA mimics分别转染SW480细胞中，36 h后用IP裂解液收集细胞。按照Western blot操作流程检测SMURF1蛋白的表达水平。
1.8 HE染色
将肿瘤组织经固定液固定，石蜡包埋，3~4 μm厚切片，45℃恒温箱中烘干后进行HE染色。
1.9 CCK-8检测细胞增殖
参照CCK-8试剂盒说明书步骤进行，各组转染24 h后，接种于96孔培养板，每孔加10 μl CCK-8，37℃培养2 h，酶标仪上480 nm波长处测定A值。
1.10 细胞划痕实验检测细胞迁移
各组转染48 h后，细胞经胰酶消化吹打为单细胞悬液，接种于12孔细胞培养板，恒温培养24 h。待细胞长成单层（铺满培养板底部）弃去培养液，在每孔中央划出一个划痕，洗去死亡细胞，显微镜下拍照。利用软件测量并计算24 h细胞迁移距离。
1.11 流式细胞法检测细胞周期
悬浮并收集不同时间点细胞，0.9%氯化钠溶液冲洗细胞两遍，70%乙醇固定。加入碘化丙啶DNA荧光染色（PI:50 mg/L, 1% Triton X-100），4℃冰箱避光染色30 min，铜网过滤，取合格的单细胞悬液，流式细胞仪检测细胞周期。
1.12 构建结肠癌肿瘤异种移植小鼠模型
取分别转染miRNA-125a模拟物和negative control miRNA mimics的结肠癌SW480细胞，调整细胞密度至2×108个/毫升。取0.1 ml细胞悬液注射于每只裸鼠左前肢腋窝皮下，将裸鼠随机分为两组，分别注射转染了miRNA-125a模拟物和转染了negative control miRNA mimics的SW480细胞。每日观察裸鼠皮下肿瘤生长情况，21天后将肿瘤从裸鼠身上分离，PCNA染色分析细胞增殖情况。
1.13 统计学方法
采用SPSS 22.0统计软件进行数据分析，计量资料用（x±s）表示，两组样本均数比较采用t检验，P < 0.05为差异有统计学意义。
2 结果
2.1 miRNA-125a在结肠癌和癌旁正常组织的表达
qRT-PCR结果显示，相比癌旁正常组织，结肠癌组织中miRNA-125a的表达水平显著降低，差异有统计学意义（P < 0.01），见图 1A。同时，miRNA-125a在发生远端转移肿瘤组织中的表达水平显著低于未转移肿瘤组织，差异有统计学意义（P < 0.01），见图 1B。提示miRNA-125a可能与结肠癌的发生及发展有关。
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图 1 miRNA-125a在结肠癌和癌旁正常组织中的表达Figure 1 miRNA-125a expression in colon cancer and adjacent normal tissuesA: miRNA-125a expression in non-tumor tissues and colon cancer tissues; B: miRNA-125a expression in distal metastatic and non-metastatic tumor tissues2.2 miRNA-125a与结肠癌患者临床指标的相关性
分析结果表明，miRNA-125a的表达水平与肿瘤分化程度、淋巴结转移、远端转移、肿瘤浸润深度和肿瘤直径相关（均P < 0.01），与性别和年龄无关，见表 1。
表 1 miRNA-125a与结肠癌患者临床指标的关系Table 1 Relationship between miRNA-125a and clinical indicators of patients with colon cancer
2.3 miRNA-125a通过结合SMURF1的3' -UTR来抑制SMURF1的表达
根据microRNA靶基因数据库预测潜在的miRNA-125a靶基因为SMURF1，见图 2A。将miRNA-125a模拟物、negative control miRNA mimics（miR-NC）以及构建的pMIR-WT和pMIR-Mut质粒转染入SW480细胞，36 h后检测荧光素酶活性。结果显示，相比miR-NC组，miRNA-125a模拟物转染组pMIR-WT质粒荧光素酶活性显著降低（P < 0.01），而pMIR-Mut质粒荧光素酶活性无明显变化，见图 2B。结果显示miRNA-125a可以通过结合SMURF1的3’-UTR进而抑制SMURF1的表达。
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图 2 miRNA-125a结合SMURF1的3’-UTR抑制荧光素酶活性Figure 2 miRNA-125a binded 3' -UTR of SMURF1 to inhibit luciferase activityA: gene sequences of miRNA-125a and SMURF1; B: luciferase activity of pMIR-WT and pMIR-Mut in miR-NC group and miRNA-125a mimics transfection group2.4 miRNA-125a抑制SMURF1 mRNA翻译及蛋白水平
qRT-PCR检测结果显示miRNA-125a模拟物转染组中SMURF1 mRNA表达水平明显低于miR-NC组，差异有统计学意义（P < 0.01），见图 3A。Western blot检测结果显示，相比miR-NC组，miRNA-125a模拟物转染组中SMURF1蛋白表达量明显降低，差异有统计学意义（P < 0.01），见图 3B。
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图 3 miRNA-125a抑制SMURF1 mRNA和蛋白表达Figure 3 miRNA-125a inhibited mRNA and protein expression of SMURF1A: SMURF1 mRNA expression in miR-NC group and miRNA-125a mimics transfection group; B: SMURF1 protein expression in miR-NC group and miRNA-125a mimics transfection group2.5 SMURF1与结肠癌患者临床指标的相关性
统计分析发现，SMURF1的表达水平与肿瘤分化程度、淋巴转移、远端转移、肿瘤浸润深度和肿瘤直径呈正相关（P < 0.01），与性别和年龄无关，见表 2。
表 2 SMURF1与结肠癌患者临床指标的关系Table 2 Relationship between SMURF1 and clinical indicators of patients with colon cancer
2.6 SMURF1在结肠癌组织中的表达
HE染色结果显示，相比癌旁正常组织，结肠癌组织中SMURF1表达水平明显升高，见图 4A。而结肠癌患者血清中miRNA-125a表达水平与SMURF1表达水平呈负相关性，见图 4B。
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图 4 SMURF1在肿瘤组织中的表达Figure 4 Expression of SMURF1 in colon cancer tissuesA: HE staining and immunohistochemical staining of SMURF1 in miR-NC group and miRNA-125a mimics transfection group; B: Correlation analysis between serum miRNA-125a and SMURF1 expression level in patients with colon cancer2.7 miRNA-125a和SMURF1与细胞增殖、迁移及周期的相关性
CCK-8检测结果显示，miRNA-125a模拟物转染组A值明显低于miR-NC组，差异有统计学意义（P=0.029）。而miRNA-125a模拟物+SMURF1转染组A值明显高于miRNA-125a模拟物转染组，差异有统计学意义（P=0.006），见图 5A。流式细胞术检测结果显示，miRNA-125a模拟物转染组的S期细胞比值明显低于miR-NC组，差异有统计学意义（P=0.048），而miRNA-125a模拟物+SMURF1转染组的S期细胞比值明显高于miRNA-125a模拟物转染组，差异有统计学意义（P=0.004），见图 5B。细胞划痕实验检测结果显示，miRNA-125a模拟物转染组的细胞迁移距离明显低于miR-NC组，差异有统计学意义（P=0.031），而miRNA-125a模拟物+SMURF1转染组的细胞迁移距离明显高于miRNA-125a模拟物转染组，差异有统计学意义（P=0.002），见图 5C。
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图 5 miRNA-125a和SMURF1对细胞增殖、迁移及周期的影响Figure 5 Effects of miRNA-125a and SMURF1 on cell proliferation, migration and cell cycle of colon cancer**: P < 0.01, compared with miRNA-125a+SMURF1 group2.8 miRNA-125a抑制小鼠体内肿瘤生长
采用SW480细胞构建结肠癌肿瘤异种移植小鼠模型，将negative control miRNA mimics（miR-NC），miRNA-125a模拟物转染SW480细胞并注射到裸鼠皮下21 d后，切除肿瘤并称重，miRNA-125a模拟物转染组肿瘤重量明显低于miR-NC对照组，差异有统计学意义（P=0.003），见图 6A。PCNA染色结果显示, 相比miR-NC对照组，miRNA-125a模拟物转染组的SW480细胞中SMURF1阳性信号明显减少，且细胞增殖明显降低，见图 6B。
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图 6 miRNA-125a抑制小鼠体内肿瘤生长Figure 6 miRNA-125a inhibited tumor growth in miceA: Tumor weight in miR-NC group and miRNA-125a mimics transfection group; B: immunohistochemical staining of SMURF1 and PCNA in miR-NC group and miRNA-125a mimics transfection group A: Tumor weight in miR-NC group and miRNA-125a mimics transfection group; B: immunohistochemical staining of SMURF1 and PCNA in miR-NC group and miRNA-125a mimics transfection group3 讨论
结肠癌是常见的消化系统恶性肿瘤，具有疾病隐匿、症状不典型且易转移的生物学行为特征。目前虽然大量研究证实TGF-β/Smad[5]、RhoA[6]、PI3K/AKT[7]、Wnt[8]等信号通路的异常活化或失活与结肠癌的转移密切相关，但对结肠癌的临床治疗仍未有较为突出的贡献。因此，从新的生物学层面挖掘其发展机制具有重要意义。miRNA是一类长度为22~28个核苷酸的内源性非编码单链小分子RNA，与许多恶性肿瘤的发生发展存在密切关系，它参与调控细胞凋亡、增殖、侵袭和转移相关的生物学事件，起着癌基因或抑癌基因的作用。有报道证实miR-143、miR-145及miR-101在结肠癌中低表达[9-10]，可能是潜在的抑癌因子。miR-106a、miR-181b、miR-203[11-13]等在结肠癌组织中高表达，提示miRNA在结肠癌的发生及发展过程中发挥重要作用。
Lyu等[14]发现miRNA-125a可通过抑制ERBB2和ERBB3的表达来抑制乳腺癌细胞的增殖和转移；miRNA-125a表达受EGFR调节，抑制肺癌细胞的转移；此外，miRNA-125a可以抑制ARID3B来负性调节卵巢癌EMT，从而抑制卵巢癌细胞的转移。然而，miRNA-125a在结肠癌中的作用尚未有相关报道。本研究发现miRNA-125a表达量随结肠癌发展程度的加深呈降低趋势，提示miRNA-125a与结肠癌的发生发展密切相关。
SMURF1属于HECT型E3泛素连接酶，参与TGF-β/Smad、Wnt、BMP、MAPK等多种生物学信号通路的调控。SMURF1高表达可以影响肿瘤组织的侵袭、转移，与肿瘤预后关系密切。研究发现SMURF1促进抑癌蛋白的快速降解，是促进结直肠癌发生发展的一个重要因子[15]。本研究检测到结肠癌患者肿瘤组织中miRNA-125a的表达水平显著降低，且结肠癌患者血清中SMURF1表达水平随结肠癌发展程度（肿瘤分化程度、淋巴转移、远端转移、肿瘤浸润深度和肿瘤直径）加深而呈升高趋势，而miRNA-125a表达水平与SMURF1表达水平呈负相关性。进一步HE染色发现，SMURF1在结肠癌组织中表达水平明显升高，提示SMURF1在结肠癌中发挥促癌作用，与以往研究结果相似[16]。本研究采用microRNA靶基因数据库预测获得SMURF1为miRNA-125a潜在靶基因，并验证了miRNA-125a可以通过结合SMURF1的3’-UTR进而抑制SMURF1的表达，同样miRNA-125a能够抑制SMURF1的mRNA翻译和SMURF1蛋白水平表达。这些结果提示miRNA-125a可能通过对SMURF1的靶向调控参与到结肠癌的病理过程中。本研究还发现miRNA-125a可通过抑制SMURF1的表达水平从而抑制结肠癌的发展。在本研究中，我们将miRNA-125a模拟物转染的SW480细胞注射到裸鼠皮下，观察到miRNA-125a能够抑制肿瘤生长及SMURF1的表达。这进一步验证了miRNA-125a对结肠癌发挥抑癌作用。
综上所述，miRNA-125a通过结合靶基因SMURF1的3’-UTR，抑制结肠癌促癌因子SMURF1的表达，进而抑制癌细胞的增殖、迁移和S期细胞的聚集，在结肠癌的发展过程中发挥抑制作用。因此，miRNA-125a可能为结肠癌的诊断及治疗提供新的研究方向。
Competing interests: The authors declare that they have no competing interests.利益冲突声明：所有作者均声明不存在利益冲突。作者贡献：罗 希：文章撰写易俊林：文章修改 -
表 1 下咽癌的保喉策略探索的相关研究
Table 1 Key trials of laryngeal preservation strategies for hypopharyngeal carcinoma
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