鉴定头颈部鳞癌中异常甲基化的差异表达基因及其通路

目的 基于头颈部鳞癌(HNSCC)的生物标记物及其通路尚不明确的现状,研究旨在分析和鉴定HNSCC中异常甲基化的差异表达基因,探讨其关键基因和潜在通路。 方法 从GEO数据库下载基因表达的数据集GSE107591和甲基化数据集GSE33202。通过R软件筛选异常甲基化基因和差异表达基因,两者取交集后获得低甲基化高表达基因(Hypo-HGs)和高甲基化低表达基因(Hyper-LGs)。利用Enrichr对两组基因进行功能富集分析。蛋白互作(PPI)网络由STRING构建并在Cytoscape中可视化,最后利用生存分析来鉴定出关键基因。此外,还进行了免疫组化分析,利用CMap寻找可能逆转HNSCC基因表达的候选小分子。 结果 共鉴定出28个低甲基化高表达基因,GO富集分析显示其主要参与T细胞趋化性的正调节,表皮发育、细胞-基底连接组件及调节T细胞趋化性等方面。Wiki通路分析的结果表明,其主要参与典型和非典型TGF-β信号传导、血液凝结级联反应、α6β4信号通路、补体和凝血级联反应及癌症中的衰老和自噬途径。同时,发现了24个高甲基化低表达基因,主要富集于血管生成及发育的调节,对干扰素-γ反应的负调节,对干扰素-γ介导的信号通路的负调节和上皮发育的生物学过程。Wiki通路分析显示其主要参与哺乳动物含黄素单加氧酶(FMOs)的催化循环,HIF1A和PPARG调节糖酵解以及苯和黄曲霉毒素B1的代谢。此外,鉴定出与HNSCC预后相关的关键基因,分别是SERPINE1、PLAU、MMRN1、LAMB3、LAMC2、PDPN和CXCL13。 结论 通过生物信息学分析并鉴定出HNSCC中异常甲基化差异表达的基因和作用途径,为揭示HNSCC发病机制提供了重要的分子学基础。包括SERPINE1、PLAU、MMRN1、LAMB3、LAMC2、PDPN和CXCL13在内的关键基因可能作为基于甲基化的异常生物标志物,为未来寻找HNSCC诊断和治疗靶点提供了新的思路。     

3MA抑制自噬降低头颈鳞癌细胞放疗抵抗性的实验研究

目的探讨6-氨基-3-甲基嘌呤(3MA)对头颈鳞癌细胞放疗抵抗性的影响。方法蛋白印迹法（Western blotting）检测CNE2、6 10B及其放疗抵抗细胞CNE2 Rs、6 10BRs中LC3B蛋白的表达;3MA作用于CNE2 Rs及TU686细胞后LC3B蛋白的表达;克隆集落形成实验检测3MA对头颈鳞癌细胞的放疗抵抗能力及生存分数的改变;细胞免疫荧光法检测3MA对放疗致DNA双链损伤相关指标γ H2AX焦点数量的改变。结果LC3B蛋白在放疗抵抗细胞较亲本细胞中表达增加;3MA能有效抑制自噬膜蛋白LC3B的表达;3MA抑制自噬后CNE2 Rs及TU686细胞克隆集落形成能力减弱,生存分数降低;3MA抑制自噬后CNE2 Rs及TU686细胞放疗组中γ H2AX焦点数量较对照组显著增加。结论3MA抑制自噬后可能影响放疗后DNA双链损伤修复,进而降低头颈鳞癌细胞的放疗抵抗性。     

喉癌组织中CHFR基因mRNA表达水平及启动子区甲基化的研究

目的:探讨CHFR基因表达水平及启动子区CpG岛过甲基化与喉癌发生发展的关系.方法:采用荧光定量PCR技术和甲基化特异性PCR技术检测50例喉癌组织(喉癌组)和15例正常喉组织(对照组)CHFR基因mRNA表达情况及启动子区CpG岛过甲基化情况.结果:①CHFR基因在对照组mRNA全部表达,在喉癌组mRNA有2例(4%)表达缺失,48例表达量明显下调(相对表达量为0.50±0.12),其中Ⅰ和Ⅱ期相对表达量(0.30±0.04),Ⅲ期和Ⅳ期相对表达量(0.70±0.21),与对照组比较,差异有统计学意义(P<0.01).②在对照组中未发现CHFR基因启动子区甲基化,在喉癌组中CHFR基因启动子区甲基化率为22%(11/50),其中Ⅰ期和Ⅱ期患者共10例,Ⅲ期1例,Ⅳ期未发现甲基化,与对照组比较,差异均有统计学意义(P<0.01).③甲基化的喉癌标本mRNA相对表达量为0.11±0.05,2例mRNA表达缺失.未甲基化的喉癌标本mRNA相对表达量为0.75±0.13.甲基化和mRNA表达相关,γ=0.387(P<0.05).结论:喉癌组织中CHFR基因mRNA表达缺失或下调,CHFR基因启动子区CpG岛过甲基化在喉癌组织中是频发事件,二者密切相关,可能与喉癌的发生发展有关,有望能成为喉癌的早期诊断及治疗靶点基因之一.     

人乳头状瘤病毒阳性的头颈鳞状细胞癌特异性基因生物信息学研究

目的分析人乳头状瘤病毒(HPV)阳性的头颈鳞状细胞癌(鳞癌)特异表达基因及关键信号通路,为HPV相关头颈鳞癌筛选有价值的基因标记物,并为进一步的肿瘤机制研究提供参考。方法从GEO高通量基因芯片数据库中筛选出头颈鳞癌具有HPV感染信息的芯片,从中筛出差异基因进行基因本体分析及京都基因和基因组(KEGG)信号通路富集分析,并筛出头颈鳞癌的特征基因簇和通路,以及关键基因并进行蛋白质相互作用网络可视化分析。通过Cbioportal信息门户以及癌症基因组图谱(TCGA)数据库验证这些特异基因在HPV(+)与HPV(-)头颈鳞癌中的表达差异并分析特异基因与头颈鳞癌患者生存预后的相关性。结果从数据集GSE52088与GSE39366中筛选出42个共同差异基因,其中上调基因25个,下调基因17个,经Cytoscape两轮筛选确定白介素-6(IL-6)、细胞表面标记物CD44、基质金属蛋白酶1(MMP1)、CXC趋化因子配体基序1(CXCL1) 4个特异基因。信号通路富集分析显示共同差异基因参与细胞周期、NOD样受体信号通路、肿瘤坏死因子(TNF)信号通路途径等信号通路(P < 0.01)。经TCGA数据库以及Cbioportal检验证实特异基因在HPV(+)与HPV(-)头颈鳞癌中的表达差异,且IL-6、CD44表达水平与头颈鳞癌生存预后呈负相关(P < 0.01)。结论HPV(+)头颈鳞癌具有特异性基因表达,并可能参与关键信号通路调控肿瘤的发生发展。IL-6、CD44、MMP1、CXCL1 4个特异基因可能参与HPV(+)头颈鳞癌发展及侵袭过程,其中MMP1、CXCL1有望作为诊断及预后的标志物,IL-6、CD44与头颈鳞癌预后存在相关性,有望成为治疗HPV(+)头颈鳞癌的潜在靶点。     

头颈鳞癌和颈清扫淋巴结D9S171位点杂合性缺失的研究

目的探讨头颈鳞状细胞癌在D9S171微卫星多态性位点的杂合性缺失(Loss of heterozygosity,LOH)的发生率、临床意义及对检测颈淋巴结转移的应用价值.方法标准酚氯仿法提取肿瘤组织及颈清扫淋巴结组织的基因组DNA,采用D9S171微卫星多态性位点进行聚合酶链反应(Polymerase cham reaction,PCR)扩增、变性聚丙烯酰胺凝胶电泳、硝酸银染色.分析D9S171微卫星多态性位点的LOH.结果头颈鳞癌原发癌组织的D9S171多态性位点的LOH发生率为62.96%;LOH发生与患者的临床分期有相关关系(P＜0.01).颈清扫淋巴结组织D9S171多态性位点的LOH发生率(35.60%),高于常规病检的阳性率(17.0%),(P＜0.01).结论D9S171多态性位点LOH与头颈鳞癌的发生有密切关系,LOH分析可能作为检测头颈鳞癌颈淋巴结转移的手段之一.     

MicroRNA-24对人喉鳞癌细胞生物学行为的影响

目的 探讨MicroRNA-24(miR-24)对喉鳞状细胞癌(LSCC)Hep-2、AMC-HN-8细胞增殖、侵袭及凋亡等生物学行为的影响。 方法 应用Lipofectamine^TM 2000转染上调Hep-2、AMC-HN-8细胞中miR-24的表达,通过荧光定量PCR验证转染效率;然后分别采用MTT法、克隆形成实验、细胞划痕实验、Transwell侵袭实验流式细胞分析技术分析外源上调miR-24后Hep-2、AMC-HN-8细胞增殖、迁移、侵袭及凋亡的影响。 结果 miR-24质粒稳定转染Hep-2、AMC-HN-8细胞后miR-24表达明显上调。与转染miR-NC组和空白对照组相比,转染miR-24能明显降低Hep-2细胞的增殖、迁移和侵袭能力;同时,转染miR-24能明显增加Hep-2、AMC-HN-8细胞的凋亡能力。 结论 miR-24异常表达与LSCC Hep-2、AMC-HN-8细胞的生物学行为密切相关,可能发挥抑癌作用。     

喉癌组织中IGFBP-rP1基因的甲基化及表达

目的：探讨喉癌中胰岛素样生长因子结合蛋白一相关蛋白1（IGFBP—rP1）基因启动子区甲基化与蛋白表达之间的关系。方法：采用甲基化特异性PCR方法和免疫组织化学的方法分别检测45例喉癌组织及18例癌旁组织中IGFBP-rP1基因的甲基化状态和蛋白表达情况。结果：喉癌组织IGFBP—rP1启动子区甲基化率为33．3％（15／45）,相应癌旁组织甲基化率为5．6％（1／18）,喉癌组织中IGFBP—rP1基因甲基化率明显高于癌旁组织（P〈0．05）。喉癌组织中IGFBP—rP1蛋白表达显著低于癌旁正常组织（P％0．05）,且与其启动子区甲基化状态呈负相关。结论：IGFBP—rP1基因启动子区甲基化导致基因沉默可能是喉癌发生的机制之一。     

CK13基因5′旁侧活性与其组织特异性表达的相关性研究

目的 检测CK13基因′旁侧在不同细胞中的报告基因活性，探讨CK13基因组织特异性表达及上皮性肿瘤中异常表达与CK13基因′旁侧活性的关系。方法 采用报告基因分析的方法，构建CK13基因′旁侧帝侧413bp与氯霉素乙酰转移酶报告基因载体pCAT－Enhancer的重组体，并通过脂质体介导的转染技术导入不同细胞，检测该重组体报告基因的瞬间表达，探讨CK13基因′旁侧报告基因枯不同细胞中的活性。同时采用Northern-blot的方法检测这些细胞中CK13基因的表达情况，探讨CK13基因组织特异性表达与其5′旁侧活性的关系。结果 不同类型细胞中的CK13基因′旁侧报告基因活性不一，报告基因活性很低的细胞不表达CK13基因，报告基因活性很强的细胞明显表达CK13基因。结论 CK13基因组织特异性表达及上皮性肿瘤中异常表达与CK13基因′旁侧活性明显相关。     

喉癌细胞系中抑癌基因MGMT表达与DNA甲基化及组蛋白H3-K9甲基化的关系

目的:探讨喉癌Hep-2细胞中组蛋白H3-K9甲基化与DNA甲基化及抑癌基因MGMT表达的关系。方法:应用去甲基化制剂5-氮杂-2′-脱氧胞苷(5-Aza-dC)处理体外培养的Hep-2细胞。应用染色质免疫沉淀技术、甲基化特异性聚合酶链反应和实时定量逆转录聚合酶链反应分析药物作用前后MGMT基因启动子区组蛋白H3-K9甲基化、DNA甲基化和MGMT表达情况。结果:①在Hep-2细胞未经药物干预前,MGMT表现为DNA甲基化,组蛋白H3-K9高甲基化,MGMT低表达。②在5-Aza-dC的作用下,Hep-2细胞中MGMT的组蛋白H3-K9甲基化状态被降低;MGMT的DNA甲基化状态得到了逆转;原来低表达的MGMT表达上调。结论:喉癌细胞系中MGMT启动子区甲基化可能是导致其基因失活的主要原因。DNA甲基化可能导致组蛋白H3-K9高甲基化。应用5-Aza-dC能够通过逆转MGMT的DNA甲基化水平从而降低MGMT组蛋白H3-K9甲基化水平来使抑癌基因表达上调,从而抑制肿瘤的发生和发展。     

头颈部鳞状细胞癌的靶向治疗研究进展

头颈部肿瘤是常见肿瘤之一,超过95%的病理类型是鳞状细胞癌,手术与放化疗结合的综合治疗方案是头颈部鳞状细胞癌(HNSCC)的主要治疗方案,但是总体生存率并不高,主要原因是肿瘤复发和/或转移;同时复发性或转移性HNSCC常无法进行手术治疗,放化疗效果也差。靶向治疗的发现为HNSCC、特别是复发性或转移性HNSCC的治疗提供了新的方法。为了进一步认识靶向治疗的临床治疗作用,就HNSCC的靶向治疗研究进展做一综述。     

Cancer stem cells in head and neck squamous cell carcinoma

Recent studies have demonstrated that cancer stem cells (CSC) play an important role in the pathobiology of head and neck squamous cell carcinomas (HNSCC). This subpopulation of undifferentiated, self-renewing cells is responsible for resistance to conventional anti-cancer therapy, cancer recurrence, metastasis and ability to form a heterogeneous tumor. CSC are identified on the basis of specific markers, including membrane proteins or cell enzymes, or by using their self-renewal properties. As their resistance to standard HNSCC treatment may eventually lead to the lack of treatment success, there is an urgent need to better understanding CSC biology and identify them as potential target new treatment modality.     

Patterns of bone metastases from head and neck squamous cell carcinoma

ObjectiveTo report clinical features of bone metastases (BM) from head and neck squamous cell carcinoma (HNSCC).MethodsAmong 772 patients with HNSCC diagnosed at our hospital over 9 years, 30 patients (3.9%) had clinical evidence of BM (24 men and 6 women; mean age: 63 years). We assessed the time interval from the primary diagnosis to BM development, symptoms attributable to BM, presence of distant metastases to other organs, number of BM, sites of BM, morphologic changes on computed tomography (CT) images, treatment for BM, and overall survival (OS).ResultsBM at the initial stage were found in 9 patients with HNSCC (30%), and in 21 patients (70%) with HNSCC during the course of the disease. In the later patients, the median time interval from the primary diagnosis was 11.5 months. Nineteen patients (63%) did not have BM-related symptoms, 6 (20%) had pain, 3 (10%) had neurologic symptoms resulting from vertebral or skull metastases, and 2 (7%) had hypercalcemia. Seventeen patients (57%) showed bone-exclusive metastases, and 13 (43%) had distant metastases in other organs. Eleven patients (37%) had monostotic metastases (solitary BM), and 19 patients (63%) had polyostotic metastases (multiple BM). When combined, 9 patients (30%) showed bone-exclusive and monostotic metastases. The most commonly affected site was the thoracolumbar spine, accounting for 34% of total BM, followed by the pelvis (24%), shoulder and thorax (21%), and the extremities (17%). Notably, metastases to bones above the clavicle (craniofacial bones and cervical spine) accounted for only 3% of all bone lesions. CT images showed variable morphologic patterns with osteolytic type in 17 patients (57%), intertrabecular in 7 (23%), osteoblastic in 4 (13%), and mixed in 2 (7%). Systematic chemotherapy for BM was performed in 19 patients and radiotherapy in 18. The median survival time for patients with bone-exclusive and monostotic metastases was significantly longer than that for patients with multi-organ metastases or polyostotic metastases at 18.2 months vs. 5.7 months (p = 0.02). Neither chemotherapy nor radiotherapy extended OS.ConclusionThirty percent of BM cases from HNSCC showed bone-exclusive and monostotic metastases. These patients tended to show a more favorable prognosis than patients with multi-organ metastases or polyostotic metastases.     

新辅助免疫治疗在头颈部鳞状细胞癌中的研究进展

头颈部鳞状细胞癌(HNSCC)是全球第八大常见癌症,超过一半的HNSCC患者可出现局部复发或远处转移。随着医学的发展,免疫治疗药物的陆续问世为复发/转移性HNSCC患者带来了新的希望。目前多项研究已经证实,以程序性死亡受体1(PD-1)检查点抑制剂为代表的新辅助免疫治疗具有较好的疗效,且安全性良好,而以新辅助免疫治疗为基础的联合治疗也成为研究热点,包括新辅助免疫治疗联合化疗、放疗、放化疗、靶向治疗以及新辅助双免疫联合治疗等新型治疗模式。本文将对新辅助免疫治疗在HNSCC中的研究进展作一综述。     

Reovirus oncolysis in human head and neck squamous carcinoma cells

OBJECTIVE: To determine whether reovirus, a double-standed RNA virus is effective on the growth of a human head and neck squamous cell carcinoma cell line. DESIGNS: In vitro cell proliferation assay, KB cells, a human oral floor squamous cell carcinoma cell line, were treated with reovirus and the number of cells was quantitated by an assay, using trypan blue staining. In vivo tumor growth assay, KB cells were injected subcutaneously into athymic nude mice, which were given an intratumoral injection of reovirus to a maximum four times in every week. The tumor size was measured once a week. Simultaneously, apoptosis and necrosis of KB cells were investigated, using technique of immunohistochemistry. RESULTS: In vitro, the multiplication of the KB cell was inhibited depending on the concentration of reovirus. In vivo, athymic nude mice bearing KB tumors were injected with the virus intratumorally, and the tumor growth was suppressed proportionally depending on the injection time of reovirus. Necrosis was recognized extensively in the pathological specimen. On the other hand, apoptosis-inducing effect was not obvious in these specimen. CONCLUSIONS: Reovirus suppressed tumor growth of KB cells in vivo as well as in vitro. The possibility that reovirus could become the means of treatment for head and neck carcinoma, was suggested with further work.     

人乳头状瘤病毒及pRb在头颈鳞状细胞癌中的表达

目的：探讨人乳头状瘤病毒(HPV)和视网膜母细胞瘤蛋白(pRb)在头颈鳞状细胞癌中的表达及其临床意义。方法：对首选手术治疗的73例头颈鳞状细胞癌患者，用GP5( )bioGP6( )介导的酶联吸附免疫PCR和type-specific PCR检测HPV；免疫组织化学法检测pRb在肿瘤组织中的表达。结果：HPV DNA在73例肿瘤组织中的阳性率为12．3％，均为HPV 16 DNA；口咽癌患者HPV阳性率为18．0％，口腔癌患者HPV阳性率为7．5％。pRb在73例肿瘤组织中的阴性率为12．3％。结论：尽管HPV阳性肿瘤临床多为进展期，常伴有颈淋巴结转移，但HPV阳性患者预后较阴性患者为好．提示HPV阳性、pRb阴性的头颈鳞状细胞癌对放疗反应敏感．     

Occult node metastases in head and neck squamous carcinoma

Summary The present study examined the Liverpool database in an attempt to determine what proportion of N0 necks for various head and neck primary sites harbored subclinical squamous cell carcinoma and whether empiric treatment of occult disease improved survival over and above a wait-and-watch policy (treatment when metastasis becomes manifest). One hundred seventeen neck dissections were carried out for N0 necks, with 32% of specimens found to contain squamous cell carcinoma. The risk of carcinoma was highest in the hypopharynx, with 50% of specimens associated with a pyriform fossa primary cancer. Twenty-nine percent of neck dissection specimens for oral cavity cancer contained carcinoma and this was commonly associated with lateral border of tongue or anterior floor of mouth carcinomas. Twenty-five percent of specimens when primary tumor was in the oropharynx contained carcinoma and were due to tonsillar carcinoma. Twenty-one percent of laryngeal cancers produced histologically positive nodes and were mostly associated with posterior epiglottic tumors. Two hundred forty-six patients had a pyriform fossa cancer and of these only 37 had N0 disease and surgical treatment. Of these, 23 patients had radical neck dissections, whereas in 14 the necks were not treated. There was no difference in survival between the two groups (₁ ² = 0.787, P = NS). The Liverpool database also contained 1631 previously untreated patients with no clinical evidence of neck node metastases. Of these only 107 had a neck dissection. There was no difference in survival (₁ ² = 2.79, P = NS). When these data were analyzed by multivariate methods (Cox's proportional hazards model) prophylactic neck dissection was found to have no significant effect.Based on a presentation at the International Symposium on the N0 neck: Göttingen, September 1992 Correspondence to A. S. Jones     

头颈部鳞癌及癌旁组织端粒酶活性检测

目的：研究原发头颈部鳞癌及相关癌旁组织中端粒酶活性表达,探讨春作为头颈部鳞癌分子生物学标志物的可能性。方法：采用ＴＲＡＰ－ＰＣＲ－ＥＬＩＳＡ,对３２例原发头颈部鳞癌及１５例癌旁组织进行端粒酶活性检测。结果：３２例原发头颈部鳞癌中,２７例端粒酶活化,阳性率为８４．４％;１５例癌旁组织中５例端粒酶活化,阳性率为３３．３％。有淋巴结累及者端粒酶阳性率（８６．７％）高于无淋巴结累及者（８２．４％）,低分化