MMP-2和MMP-9及胃蛋白酶对喉鳞状细胞癌侵袭作用的机制研究

目的探讨基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)及胃蛋白酶(Pepsin)对喉鳞状细胞癌局部侵袭的作用。方法收集2020年10月—2022年3月柳州市人民医院耳鼻咽喉科住院手术并经病理确诊的32例喉鳞状细胞癌患者的癌组织及其癌旁组织新鲜标本,通过实时荧光定量PCR(qRT-PCR)分别检测MMP-2、MMP-9及Pepsin mRNA在上述组织标本中的表达情况,结合临床资料分析。结果喉癌组织中MMP-2、MMP-9及Pepsin mRNA表达高于癌旁组织,差异具有统计学意义(P＜0.05);Ⅲ、Ⅳ期组表达水平高于Ⅰ、Ⅱ期组(P＜0.05),淋巴结转移组高于无转移组(P＜0.05);MMP-2、MMP-9 mRNA在中-低分化鳞状细胞癌组的表达高于高分化组(P＜0.05);Pepsin mRNA在60岁及以上喉癌组中表达高于60岁以下组(P＜0.05)。结论MMP-2、MMP-9和Pepsin在喉鳞状细胞癌的局部侵袭过程中发挥协同促进作用,预防和治疗咽喉反流是防止喉癌局部侵袭的重要环节。     

SHCBP1高表达促进鼻咽癌细胞上皮间质转化过程的分析

目的 探究SHC SH2结构域结合蛋白1(SHCBP1)在鼻咽癌组织及鼻咽癌细胞系中的表达情况,分析其对鼻咽癌细胞系的增殖、侵袭能力及上皮间质转化(EMT)过程的影响。方法 利用TCGA数据库分析SHCBP1在头颈鳞状细胞癌(HNSC)中的表达情况,分析SHCBP1表达程度与肿瘤免疫细胞浸润程度的关系。同时收集2019年9月—2021年9月于贵州医科大学附属医院及贵州医科大学附属肿瘤医院经病理确诊的31例鼻咽癌初诊患者临床组织样本,所有患者就诊前均未接受任何放化疗,并收集同期就诊的31例疑似鼻咽癌但病检示慢性鼻咽炎组织作为对照组。利用逆转录实时荧光定量PCR(RT-qPCR)检测临床样本及鼻咽癌细胞系5-8F中SHCBP1的表达情况。细胞实验以鼻咽癌细胞系5-8F、正常鼻咽部永生化上皮细胞系NP69作为研究对象。采用利用慢病毒载体shRNA-SHCBP1干扰技术沉默5-8F细胞中SHCBP1表达,并分为NC组(空载慢病毒LV-Zs-Green-PURO-NC感染5-8F)、SHCBP1-shRNA组(LV-SHCBP1-shRNA-Zs-Green-PURO沉默5-8F细胞SHCBP1表达)。CCK-8法、克隆形成实验检测SHCBP1对鼻咽癌细胞增殖的影响;划痕实验、Transwell迁移、侵袭实验检测SHCBP1对鼻咽癌细胞的转移、侵袭能力的影响。Western blot检测NC组、SHCBP1-shRNA组中E-cadherin、N-cadherin、基质金属蛋白9(MMP9)、Vimentin的表达情况。结果 在HNSC中SHCBP1高表达,表达量与Th2细胞浸润程度相关。与慢性鼻咽炎组织相比,鼻咽癌组织中SHCBP1明显高表达,差异具有统计学意义(P<0.05)。与NC组相比,SHCBP1-shRNA组鼻咽癌细胞增殖、迁移、侵袭能力均减弱,差异具有统计学意义(P<0.05)。SHCBP1-shRNA组E-cadherin表达较NC组升高,N-cadherin、MMP9、Vimentin表达较NC组降低,差异具有统计学意义(P<0.05)。结论 SHCBP1在鼻咽癌中高表达,并可促进鼻咽癌细胞增殖、侵袭、迁移及激活EMT过程。     

miR 375通过靶向NLK影响鼻咽癌的增殖及迁移

 目的探讨miR 375在鼻咽癌患者中表达的临床意义及其对鼻咽癌细胞的影响。方法收集67例鼻咽癌组织标本和53例慢性鼻咽炎症组织标本,采用qRT PCR检测组织标本和鼻咽癌细胞株CNE1、CNE2、C666 1及人永生化鼻咽上皮细胞株（NP69）中miR 375的表达水平;分别转染miR 375的模拟物或抑制物,CCK8法检测细胞的增殖,Transwell实验检测细胞的迁移;生物信息学靶基因预测miR 375的靶基因,并经双荧光素酶及蛋白质印迹法（Western blotting）验证靶基因。结果鼻咽癌组织中miR 375表达水平与慢性鼻咽炎症组织相比明显下调（P<0.05）;miR 375的表达水平与患者的临床分期、区域淋巴结受累情况以及肿瘤大小有关（P<0.05）,但与患者年龄、性别、吸烟史、分化程度及组织类型无关(P>0.05);鼻咽癌细胞与NP69相比较,miR 375的表达水平均显著低于NP69（P<0.05）;miR 375过表达后C666 1细胞增殖、迁移显著低于慢性鼻咽炎-模拟物组（P<0.05）,Nemo样激酶（Nemo like kinase,NLK）的表达下调。miR 375抑制后CNE1细胞增殖、迁移显著高于慢性鼻咽炎-抑制物组（P<0.05）,miR 375对NLK具有直接靶向调控作用。结论miR 375在鼻咽癌中呈低表达,并与患者的临床分期、区域淋巴结受累情况以及肿瘤大小有关,其可能是通过下调靶基因NLK,从而抑制鼻咽癌的增殖及迁移。     

MiR-98靶向MTDH调控鼻咽癌恶性进展的实验研究

目的探讨miR 98对鼻咽癌细胞体外增殖和侵袭能力的影响及其可能机制。方法利用脂质体介导的miR 98 mimic（及阴性对照）转染鼻咽癌CNE 1和CNE 2细胞,CCK 8检测其体外增殖能力的改变,划痕愈合及Transwell侵袭小室实验检测鼻咽癌细胞的体外迁移及侵袭潜能变化,生物信息学软件预测miR 98可能的靶基因,并经双荧光素酶及Western blotting验证靶基因。结果MiR 98过表达能抑制鼻咽癌CNE 1和CNE 2细胞的体外生长增殖、迁移及侵袭能力;miR 98对MTDH具有直接靶向调控作用。结论本研究表明miR 98能够靶向MTDH调控鼻咽癌细胞株的体外生长增殖及侵袭能力。     

鼻咽癌肿瘤相关成纤维细胞的实验分析

目的 分离、培养和鉴定鼻咽癌肿瘤相关成纤维细胞（CAFs）及正常成纤维细胞（NFs）,并鉴定探讨两种成纤维细胞的生物学差异。方法 临床收集新鲜鼻咽癌组织和鼻咽炎组织经胶原酶和胰酶消化后,利用Matrigel胶混合3D成球培养获得成纤维细胞。通过显微镜观察细胞形态,利用细胞免疫荧光染色、Western bolt和CCK8检测CAFs标准物。结果 分离得到的NFs及CAFs形态和生物学形状存在差异,两者都高表达波形蛋白（Vimentin）,CAFs高表达血管平滑肌肌动蛋白（a-SMA）,且增殖能力增强。结论 利用Matrigel混合3D成球培养的方法能成功分离、培养成纤维细胞;与NFs相比,CAFs高表达a-SMA,且增殖能力增强。     

鼻咽上皮细胞异型性改变与PCNA及MMP-9表达的相关性研究

目的 研究鼻咽上皮细胞癌变过程中不同阶段的增殖细胞核抗原（PCNA）及金属基质蛋白酶9（MMP-9）表达活性.方法 在鼻咽癌高发区,选择慢性鼻咽炎30例、鼻咽上皮细胞不典型增生或化生者42例及鼻咽癌34例作为研究对象,以免疫组化S-P方法检测PCNA及MMP-9表达水平.结果 PCNA在鼻咽炎、不典型增生或化生鼻咽上皮细胞、鼻咽原发灶癌组织表达活性分别为10.0%、73.8%、82.4%,而MMP-9在这些组织的表达水平分别为0.0%、38.1%、67.6%,活性表达差异均具有统计学意义（P＜0.001）.结论 在鼻咽上皮细胞癌变过程中,PCNA及MMP-9依病理分级严重程度而呈现表达增强趋势.     

1,9-二取代β-咔啉在头颈部鳞状细胞癌中抗肿瘤机制的研究

目的 初步探索1，9-二取代β-咔啉（CZY1-4）在头颈部鳞状细胞癌中抗肿瘤机制的研究。方法 CZY1-4是一种新合成的1，9-二取代β-咔啉，具有抗肿瘤潜力。采用CCK-8实验经过CZY1-4浓度梯度（0、2、4、6、10、15、20、25、30、40 μmol/L）处理24 h后检测细胞活力，筛选出两种对药物较为敏感的头颈部鳞状细胞癌细胞系：喉癌细胞系Tu177与舌癌细胞系Cal-27为主要研究对象。以浓度梯度（0、2、4、6、8 μmol/L）进行克隆形成实验检测细胞集落形成能力；随后分别以2 μmol/L与4 μmol/L的CZY1-4处理Tu177与Cal-27 24 h后进行划痕实验和Transwell实验检测细胞侵袭和迁移能力，流式细胞术分析细胞凋亡情况，Western blotting检测细胞相关蛋白表达量变化，免疫荧光实验检测细胞内自噬小体形成情况等。结果 经过上述浓度的CZY1-4处理24 h后，各头颈部鳞状细胞癌细胞系的生长增殖均受到不同程度的抑制，且呈剂量依赖性，其中Tu177与Cal-27抑制效果更为显著，两者的克隆形成能力也被显著抑制（P<0.05）；Tu177与Cal-27的迁移能力被显著抑制（P<0.01）；Tu177与Cal-27的侵袭能力被显著抑制（P<0.001）；Tu177与Cal-27凋亡率变化无显著差异（P>0.05）；自噬相关蛋白ATG5与LC3B，Benclin1蛋白表达量增加，呈剂量依赖性；自噬小体形成显著增加（P<0.01）；药物对被敲除ATG5后的Cal-27生长、增殖的抑制作用显著减弱（P<0.01）。结论 CZY1-4能显著抑制头颈部鳞状细胞癌相关细胞系的生长、增殖作用，并抑制细胞克隆形成、侵袭和迁移，具有一定的抗肿瘤活性，通过实验初步验证该药物主要通过调控ATG5促进细胞自噬的发生进而发挥抗肿瘤效果。     

组织蛋白酶B在鼻咽癌患者组织和血清中的表达及临床意义

目的 通过测定组织蛋白酶B （Capthesin B）在鼻咽癌组织和血清标本中的表达量,探讨Capthesin B作为评估鼻咽癌患者治疗预后的可行性。方法 收集长沙金域医学检验中心2019年8月—2020年2月50例鼻咽癌组织和癌旁正常组织标本,免疫组织化学（IHC）检测组织标本Capthesin B表达,比较分析蛋白表达差异。收集湖南省肿瘤医院2019年7月—2020年3月106例鼻咽癌患者治疗前、治疗后及40例健康体检者的血清标本,ELISA试验检测血清Capthesin B浓度,并比较分析3组间的血清Capthesin B浓度。结果 IHC结果显示,鼻咽癌组织Capthesin B阳性率显著高于癌旁正常组织（P< 0.001）。ELISA结果表明,鼻咽癌患者血清Capthesin B 1.23（0.64,2.27） ng/mL显著高于健康体检组的（0.98 ±0.49） ng/mL （P< 0.05）,鼻咽癌患者治疗后血清Capthesin B 0.69（0.39,1.42） ng/mL显著低于治疗前的1.23（0.64,2.27） ng/mL （P<0.001）。TNM分期III、IV患者血清Capthesin B （2.09 ±1.50） ng/mL显著高于I、II期患者的1.14（0.60,2.12） ng/mL,提示Capthesin B水平与TNM分期显著相关（P<0.05）。鼻咽癌颈淋巴结转移组血清Capthesin B （2.63±1.67） ng/mL显著高于未转移组的1.10（0.59,2.14） ng/mL （P<0.01）,提示Capthesin B与转移相关。工作特征（ROC）曲线下面积（AUC）为0.670（P<0.001）,提示血清Capthesin B可作为鼻咽癌疗效预测的参考指标。结论 鼻咽癌组织Capthesin B高表达和患者血清Capthesin B浓度明显升高,治疗后其浓度降低,提示Capthesin B与鼻咽癌发生发展密切相关。Capthesin B与肿瘤淋巴结转移及TNM分期呈正相关,提示Capthesin B可作为鼻咽癌治疗预后的参考指标。     

环状RNA BCRC-3对鼻咽癌细胞增殖和迁移的影响研究

目的　观察鼻咽癌组织和细胞株中环状RNA（circular RNA,circRNA）BCRC-3的表达,探讨环状RNA BCRC-3对细胞周期依赖性激酶抑制蛋白B（cyclin dependent kinase inhibitors B,DKNIB）基因表达的调控及对鼻咽癌细胞增殖和迁移的影响。方法 荧光实时定量聚合酶链式反应（qPCR）分别检测circRNA BCRC-3在83例鼻咽癌组织及62例慢性鼻咽炎组织、鼻咽癌细胞株及人永生化鼻咽上皮细胞中的表达。以circRNA BCRC-3表达最低的细胞株为转染对象,分别转染阴性对照质粒（对照组） 或载有circRNA BCRC-3序列的质粒（实验组）。MTS法和细胞划痕实验分别检测上调circRNA BCRC-3对细胞增殖和迁移能力的影响。qPCR检测上调环状RNA BCRC-3对DKNIB基因mRNA表达的影响,Western blot检测DKNIB蛋白和PI3K/AKT信号通路蛋白的表达。结果 与慢性鼻咽炎组织相比,circRNA BCRC-3在鼻咽癌组织中表达显著降低（P <0.01）。与人永生化鼻咽上皮细胞相比,环状RNABCRC-3在鼻咽癌细胞株中表达显著降低（P <0.01）,在HONE-1细胞中的表达最少（P <0.01）。与对照组比较,实验组HONE-1细胞增殖能力从第2天开始明显下降（P <0.05）,HONE-1细胞迁移能力明显降低（P <0.01）,HONE-1细胞中DKNIB在mRNA和蛋白水平的表达明显上升（P <0.01）,PI3K/AKT信号通路蛋白PIP3、PDK1、p-AKT和AS160表达明显下降。结论 circRNA BCRC-3在鼻咽癌组织和细胞株中呈低表达,上调circRNA BCRC-3可抑制鼻咽癌HONE-1细胞的增殖和迁移能力,其机制可能是circRNA BCRC-3通过上调DKNIB基因表达,抑制PI3K/AKT信号通路转导。     

SGK1在鼻咽癌中的表达及其对鼻咽癌细胞生物学特性的影响

DOI:10.11798/j.issn.·鼻咽癌专栏·基金项目：第目的探讨SGK1在鼻咽癌（NPC）中的表达及其对肿瘤细胞存活、增殖和迁移的影响。方法采用免疫组织检测NPC组织和慢性鼻咽炎组织中SGK1的表达。使用lipofectamine 2000瞬时转染NPC细胞系HNE 1，Western blot检测siRNA干扰组、阴性对照组及未转染组细胞中的SGK1表达，筛选SGK1沉默效果最佳的NPC细胞。CCK8法检测转染细胞的增殖能力，Transwell细胞迁移实验检测SGK1沉默对细胞迁移能力的影响，流式细胞术检测其凋亡水平。结果NPC组织中SGK1的表达明显高于慢性鼻咽炎组织。Western blot结果显示转染SGK1 siRNA后HNE 1细胞中SGK1蛋白水平明显下降。SGK1基因沉默后，HNE 1细胞表现出较强的细胞增殖和迁移抑制作用。此外，沉默SGK1后，HNE 1细胞的凋亡率增加。结论SGK1在NPC组织中呈高表达。沉默SGK1基因可抑制NPC细胞系HNE 1的增殖、迁移和存活能力。     

基质金属蛋白酶及其抑制剂与鼻咽癌的侵袭性的关系

目的研究基质金属蛋白酶-2,9(matrix metalloproteinase-2,9,MMP-2,9)及组织基质金属蛋白酶抑制剂-1,2(timue inhibitors of the MMP-1,2,TIMP-1,2)与鼻咽癌侵袭的关系。方法用免疫组化S-P法,检测48例鼻咽癌组织及16例鼻咽炎组织中MMP-2,MMP-9,TIMP-1,TIMP-2的表达。结果 鼻咽癌与鼻咽炎两组病人TIMP-1,TIMP-2比较有统计学意义(P<0.01),随着鼻咽癌原发灶T分期的发展,TIMP-1有增加的趋势。结论TIMP-1可以作为监测鼻咽癌侵袭的参数之一。     

In vivo and in vitro study of co-expression of LMP1 and Cripto-1 in nasopharyngeal carcinoma

IntroductionNasopharyngeal carcinoma, an epithelial-derived malignant tumor which because of its anatomical location and atypical early symptoms, when diagnosed invasion and metastasis often have occurred. This requires a better understanding of the development mechanism, identifying diagnostic markers, and developing new treatment strategies.ObjectiveTo study the relationship of LMP1 and Cripto-1 in nasopharyngeal carcinoma.MethodsThe expression of LMP1 and Cripto-1 in specimens obtained from nasopharyngeal carcinoma patients (n = 42) and nasopharyngitis patients (n = 22) were examined. The expression of LMP1 and Cripto-1 in LMP1-negative and LMP1-positive (CNE1-LMP1) cells were also examined.ResultsThe expression of LMP1 and Cripto-1 was significantly higher in nasopharyngeal carcinoma than in nasopharyngitis (p < 0.05). Their expression in nasopharyngeal carcinoma with metastasis were significantly higher than that without metastasis (p < 0.05), which was correlated with TNM staging (p < 0.05). High Cripto-1 expression and high proliferation rate were seen in CNE1-LMP1 cells.ConclusionsThe expression of LMP1 and Cripto-1 in nasopharyngeal carcinoma is positively related. Their co-expression might contribute to the proliferation and metastasis of nasopharyngeal carcinoma.     

Characteristics of Expression of Matrix Metalloproteinases (MMP-2 and MMP-9) in Glottic Squamous Cell Carcinoma and Benign Vocal Fold Lesions

ObjectivesThe aim of this study was to investigate expression profile of matrix metalloproteinases (MMP-2 and MMP-9) in glottic squamous cell carcinoma (SCC) and benign vocal fold lesions (BVFLs) and to correlate it with clinical and pathological features.MethodsThe immunohistochemical expression of MMP-2 and MMP-9 was investigated in specimens taken from 217 patients group, including vocal fold polyps (n=39), recurrent respiratory papillomatosis (n=30), laryngeal keratosis (n=36), glottic SCC (n=112), and the normal tissue of vocal fold (n=12, control group). The expression of MMP-2 and MMP-9, both in epithelium and stroma cells, was graded on a semiquantitative scale, ranging from 0 (no expression) to 18 points (high expression).ResultsExpressions of both, MMP-2 and MMP-9 were significantly higher in the glottic SCC group comparing with BVFL group. Significant higher expression of parenchymal MMP-2 (P<0.001) and stromal MMP-9 (P=0.01) was revealed in the group of moderate/poorly differentiated glottic SCC comparing with well differentiated glottic SCC group. Expression of stroma MMP-2 was found to be correlated with nodal metastasis (P=0.030). Expressions of both, MMP-2 and MMP-9 were not correlated with clinical stage, tumor T value, smoking, alcohol use, age in the glottic SSC patients group. The MMP-2 stroma value of 11.2 points was determined as the optimum point (limiting value) for separating BVFL and glottic SCC patient groups.ConclusionOur results suggest that expressions of both MMP-2 and MMP-9 are up-regulated already in the development of BVFL, the next determinant step is concerned with occurrence of malignization. Limiting value of stroma MMP-2 demonstrates prognostic importance of MMP-2 in glottic SCC carcinogenesis.     

FA-MNP-MMP-9-ASODN纳米复合物对鼻咽癌的体外抑制研究

目的：观察FA-MNP-MMP-9-ASODN纳米复合物对鼻咽癌HNE-1细胞的体外抑制效应。方法：采用RT-PCR、Western-blot方法、MTT方法、流式细胞技术及Matrigel体外侵袭实验观察转染FA-MNP-MMP-9-ASODN 48h后HNE-1细胞MMP-9mRNA及蛋白表达水平和细胞增殖能力、凋亡和侵袭能力的变化。结果：FA-MNP-MMP-9-ASODN组HNE-1细胞MMP-9 mRNA和蛋白的表达水平较对照组及FA-MNP-MMP-9-NSODN无义序列组明显降低。同时FA-MNP-MMP-9-ASODN组HNE-1细胞生长抑制率约为35.66%,增殖活性较对照组和无义序列组明显降低。且HNE-1细胞周期也受到抑制,细胞凋亡率约为12.60%,明显高于对照组和无义序列组。侵袭实验显示FA-MNP-MMP-9-ASODN组穿膜细胞数约为21.00个,明显低于对照组和无义序列组。结论：FA-MNP-MMP-9-ASODN纳米复合物通过抑制MMP-9的表达,可降低鼻咽癌细胞的增殖和侵袭能力,促进凋亡,体外抑制效果良好。     

Role of siRNA silencing of MMP-2 gene on invasion and growth of laryngeal squamous cell carcinoma

The purpose of this study was to explore the effects of MMP-2 silencing on the invasion and growth of laryngeal squamous cell carcinoma (LSCC). Hep-2 cells were transfected with MMP-2-RNAi-Lentivirus, and MMP-2 expression and invasive properties of the cells were evaluated. The experimental tumors in the nude mice were intratumorally injected with the same recombinant lentivirus. The inhibition of tumor growth was observed. The expression of MMP-2 protein in MMP-2 siRNA transfected Hep-2 cells was effectively suppressed. Both the viability and invasive ability of Hep-2 cells were significantly inhibited. The average weight and volume of tumor in MMP-2-RNAi-Lentivirus treated group were significantly lower than those in the control group (P < 0.01). The PCNA index was obviously lower in MMP-2 RNAi treated tumors (P < 0.01). In conclusion, MMP-2 silencing by recombinant lentivirus mediated RNA interference can inhibit invasion and growth of LSCC, and MMP-2 might be a potential target for gene therapy in LSCC.     

Expression of ER stress markers (GRP78/BiP and PERK) in adenoid cystic carcinoma

Conclusion: A high GRP78/BiP expression was proved to be a significant marker for predicting poor outcome after surgery. GRP78/BiP may be a promising molecular target for treatment of ACC. Background: The glucose-regulated protein GRP78/BiP plays a crucial role in the endoplasmic reticulum (ER) stress. The level of GRP78 is highly elevated in various human cancers, but the clinicopathological significance of GRP78/BiP remains controversial in patients with adenoid cystic carcinoma (ACC). Methods: A total of 26 ACC patients were analyzed, and tumor specimens were stained by immunohistochemistry for GRP78/BiP, PERK, Ki-67, and microvessel density (MVD) determined by CD34. Results: GRP78/BiP and PERK were highly expressed in 58% (15/26) and 35% (9/26), respectively. The high expression of GRP78/BiP was significantly associated with PERK, cell proliferation and angiogenesis.     

Expression of matrix metalloproteinases (MMP-2 and MMP-9) in recurrent respiratory papillomas and laryngeal carcinoma: clinical and morphological parallels

The task of the present study was to investigate the expression of MMP-2 and MMP-9 in recurrent respiratory papillomas (RRP) and accomplish a comparative analysis with those in laryngeal squamous cell carcinoma (LSCC). The immunohistochemical expression of MMP-2 and MMP-9 was investigated in specimens taken from RRP (n = 38) and LSCC (n = 39) patient groups, and the normal tissue of vocal fold (n = 12, control group). The expression of MMP-2 and MMP-9, both in epithelium and stroma cells, was graded on a semiquantitative scale, ranging from 0 (no expression) to 18 points (high expression). Statistically significant differences in the expression of MMP-2 and MMP-9, both in epithelium and stroma among the RRP, LSCC patients and control group (epithelium) with the LSCC group having the highest MMPs expression scores were revealed. However, no statistically significant correlations among expression of MMPs and clinical and/or morphological features were found in the group of RRP patients. The MMP-2 stroma value of 10.4 points was determined as the optimum point (limiting value) for separating RRP and LSCC patient groups. Results of the present study indicate that the expression of both MMP-2 and MMP-9 are up-regulated early in development of laryngeal papillomas, when the benign neoplastic lesion begins and the next determinant step is concerned with the occurrence of malignization. These results seem promising, as they may improve our understanding of the molecular events leading to the papilloma formation and development, however, further research is needed.