miR-124抑制口腔鳞状细胞癌CAL27细胞的增殖活性及其对EZH2的靶向调控

目的:探讨miR-124对口腔鳞状细胞癌CAL27细胞增殖活性的影响以及其与EZH2的靶向关系。方法:构建pre-miR-124、野生型EZH2(EZH2-WT)和突变型EZH2(EZH2-MT)真核表达载体,采用荧光实时定量PCR(qRT-PCR)检测pre-miR-124的转染效率,MTT法检测miR-124过表达对CAL27细胞增殖活性的影响;另外,利用双荧光素酶报告基因系统、qRT-PCR和Western blot验证miR-124与EZH2的靶向关系。结果:采用NCBI BLAST软件比对载体测序,结果显示pcDNATM6.2-GW-pre-miR-124、pmirGLO- EZH2-WT和pmirGLO- EZH2-MT表达载体构建成功,qRT-PCR显示转染pre-miR-124后CAL27细胞内miR-124的表达水平显著升高(P＜0.001)。MTT分析结果显示miR-124过表达能够明显抑制CAL27细胞的增殖活性(P＜0.05),双光素酶报告基因显示共转染pre-miR-124和EZH2-WT的CAL27细胞荧光活性显著下降(P＜0.001),且qRT-PCR的结果也显示转染pre-miR-124的CAL27细胞内EZH2的表达水平明显低于对照组(P＜0.001),Western blot显示miR-124的过表达能够显著抑制EZH2蛋白水平的表达。结论:miR-124的过表达能够显著抑制口腔鳞状细胞癌CAL27细胞的增殖,并且与EZH2之间存在具有良好的靶向关系。     

下调miR-21表达对口腔鳞状细胞癌细胞增殖的影响

目的 :探讨下调miR-21表达对口腔鳞状细胞癌细胞增殖的影响及可能机制。方法 :体外培养SCC15和CAL27口腔鳞癌细胞,根据转染不同,分别将细胞分为miR-21抑制物组、miR-阴性对照物组和空白对照组。MTT法检测不同转染组细胞增殖情况,检测不同转染组细胞凋亡情况,实时荧光定量PCR检测细胞中miR-21、β-catenin、C-myc和Dkk1表达,Western blot法检测细胞中β-catenin、C-myc和Dkk1蛋白表达。结果 :SCC15和CAL27细胞中,miR-21抑制物组细胞转染48 h和72 h时,细胞增殖活性均低于miR-阴性对照组和空白对照组,差异均有统计学意义(P<0.05);SCC15和CAL27细胞中,miR-21抑制物组细胞凋亡率均显著高于miR-阴性对照组和空白对照组,差异均有统计学意义(P<0.05);SCC15和CAL27细胞中,miR-21抑制物组细胞中miR-21、β-catenin基因和蛋白、C-myc基因和蛋白表达量均低于miR-阴性对照组和空白对照组,而Dkk1基因和蛋白表达量均高于miR-阴性对照组和空白对照组,差异均有统计学意义(P<0.05)。结论:下调miR-21可显著抑制口腔鳞状细胞癌细胞增殖,促使细胞发生凋亡,可能与促进Dkk1基因表达而抑制Wnt/β-catenin信号通路有关。     

MicroRNA-27b通过靶向调控FZD7抑制口腔鳞状细胞癌细胞的增殖

目的探讨microRNA-27b(miR-27b)对口腔鳞状细胞癌细胞增殖的影响及其作用的分子生物学机制。方法 MTT方法检测细胞增殖水平;实时定量PCR方法检测miR-27b表达水平以及卷曲蛋白7(Frizzled7,FZD7)、cyclin D1和c-myc的信使RNA(mRNA)表达水平;Western blot方法检测FZD7的蛋白表达水平;荧光素酶报告基因方法检测miR-27b与FZD7的靶向关系以及Wnt信号通路的活性。结果 miR-27b在口腔鳞状细胞癌细胞株中表达降低;过表达miR-27b可以显著抑制口腔鳞状细胞癌细胞的增殖;miR-27b可以靶向FZD7并抑制FZD7的表达及其下游的Wnt信号通路。结论 miR-27b通过靶向抑制FZD7的表达来抑制口腔鳞状细胞癌细胞的增殖,其机制可能与抑制FZD7介导的Wnt信号通路的激活有关。     

血浆miR-1290表达水平对口腔鳞状细胞癌患者预后预测的价值

目的探讨血浆miR-1290表达水平对口腔鳞状细胞癌(OSCC)患者预后预测的价值。方法选取2014年1月—2018年12月儋州市人民医院收治的OSCC患者70例,采用实时荧光定量聚合酶链反应检测OSCC患者血浆miR-1290表达水平。通过ROC曲线确定血浆miR-1290表达水平的最佳界值,并以此为标准将OSCC患者分为高miR-1290组(n=31)和低miR-1290组(n=39),对2组临床病理特征进行比较。应用Kaplan-Meier法绘制生存曲线,采用单因素及多因素COX回归模型分析影响OSCC患者预后不良的危险因素。结果 OSCC组血浆miR-1290表达水平(0.65±0.14)明显低于对照组(2.06±0.90),二者间差异有统计学意义(t=13.912,P<0.001)。血浆miR-1290低表达与OSCC患者的临床分期、分化程度、肿瘤直径及淋巴结转移有关(P<0.05)。生存分析显示,低miR-1290组患者总生存率和无进展生存率明显低于高miR-1290组(P<0.01)。多因素COX回归模型分析显示,临床分期、淋巴结转移及血浆miR-129...     

KLF4对口腔鳞癌细胞增殖的抑制作用

目的 探讨KLF4对人口腔鳞癌细胞CAL27的增殖抑制作用.方法 构建KLF4慢病毒表达载体,并转染人口腔鳞癌细胞系CAL27,转染不含KLF4开放读码框的慢病毒载体LV105作为对照.采用RT-PCR、免疫细胞化学法对KLF4表达进行鉴定.MTT实验、流式细胞术检测KLF4对口腔鳞癌细胞系CAL27的增殖抑制作用.结果 RT-PCR、免疫细胞化学实验均表明实验组细胞KLF4的表达明显高于对照组(P＜0.01).MTT实验结果表明与对照组CAL27/LV105组相比,CAL27/KLF4能够抑制细胞的增殖能力(P＜0.01).CAL27/KLF4主要通过使细胞周期中的G2/M期阻滞来抑制CAL27细胞的增殖(P＜0.01).结论 KLF4转染能够抑制口腔鳞癌细胞系CAL27的增殖能力,可能在口腔鳞状细胞癌中作为抑癌基因发挥作用.     

血浆miR-1290在口腔鳞状细胞癌中的表达及临床意义

目的:探讨血浆miR-1290表达水平对口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)患者预后预测的价值。方法:选取2015年1月～2018年10月儋州市人民医院收治的OSCC患者82例,采用实时荧光定量PCR技术检测OSCC患者血浆miR-1290表达水平。通过受试者工作特征(receiver operating characteristic,ROC)曲线确定血浆miR-1290表达水平的最佳截断值,并以此为标准将OSCC患者分为高miR-1290组(n=48)和低miR-1290组(n=34),对两组临床病理特征进行比较。应用Kaplan-Meier法绘制生存曲线,采用单因素及多因素COX回归模型分析影响OSCC患者预后不良的危险因素。结果:OSCC组血浆miR-1290表达水平明显低于对照组(P<0. 01)。血浆miR-1290低表达与OSCC患者的临床分期、分化程度、肿瘤直径及淋巴结转移有关(P<0. 05)。生存分析显示,低miR-1290组患者总生存率明显低于高miR-1290组(P<0. 01),低miR-1290...     

基于生物信息学分析miR-655在口腔鳞状细胞癌中的表达及功能探讨

目的:基于生物信息学分析miR-655在口腔鳞状细胞癌中的表达及功能探讨.方法:下载TCGA数据库中的OSCC(n=342)及癌旁组织(n=44),同时收取湖北省恩施土家族苗族自治州中心医院口腔科手术切除的40例OSCC患者标本;比较miR-655的表达,分析miR-655的表达与OSCC预后的关系,体外实验初步探讨m...     

术前诱导化疗对口腔鳞癌细胞凋亡和增殖的影响

细胞过度增殖是恶性肿瘤的重要特征之一,长期以来,抗癌药物的研究多集中在抑制肿瘤细胞增殖活性方面.近年发现,细胞凋亡(apoptosis, APO)与肿瘤的发生、发展、治疗及预后关系密切,APO在肿瘤化疗中的作用成为研究热点[1,2].本文比较24例口腔鳞癌(oral squamous cell carcinoma,OSCC)患者经VM(vincristine methotrexate)方案化疗前后APO和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的变化,以探讨VM方案的抗癌机理.     

口腔鳞癌组织超氧化物歧化酶免疫组化定位观察

作者对７０例口腔鳞状细胞癌组织标本进行超氧化物歧化酶（ＳＯＤ）免疫组化研究（ＡＢＣ法）。结果发现，鳞癌组织中ＳＯＤ标记阳性率和阳性程度均显著高于癌旁和正常上皮组织（Ｐ＜０．００１）；癌组织分化程度越差，其ＳＯＤ标记阳性率和阳性程度亦越高（Ｐ＜０．０５）；高分化舌癌的ＳＯＤ阳性率和阳性程度较高分化唇癌为高（Ｐ＜０．０５）。结果表明：①本研究方法具有定位及半定量特点，特异性及灵敏度高，组织对比性好，检测误差少，结果判断可靠等优点。②口腔鳞癌组织的生物学特性，分化程度及预后与ＳＯＤ含量增高有关。     

口腔鳞癌间质微血管的特征及意义

目的观察口腔鳞癌间质微血管的空间分布和形态学特征，分析它们与临床病理学参数间的关系，探讨抗血管生成治疗在口腔鳞癌治疗中的作用。方法应用双标免疫组织化学及计算机图像分析技术，检测和分析62例口腔鳞癌及30例癌旁正常组织和10例正常口腔黏膜的微血管分布及形态。结果口腔鳞癌间质微血管与癌旁和正常口腔黏膜微血管相比，前者具有血管密度高、不成熟、形态不规则、血管周长较小的特点（P〈0．01）；口腔鳞癌癌灶边缘的血管与癌灶内血管相比，血管密度更高（P〈0．01）、更不成熟（P〈0．05）；微血管密度与肿瘤的淋巴结转移有关（P〈0．01）。结论口腔鳞癌间质微血管与正常口腔组织血管比较，差异有统计学意义，该差异有利于抗血管生成治疗的应用。癌灶边缘可能是抗血管生成治疗的重要靶区。微血管密度可望成为判断肿瘤恶性程度的一项指标。     

MicroRNA-27b inhibits cell proliferation in oral squamous cell carcinoma by targeting FZD7 and Wnt signaling pathway

This study intended to investigate the role of microRNA-27b (miR-27b) in proliferation of oral squamous cell carcinoma (OSCC) cells and to explore the potential molecular mechanism. Cell proliferation was detected by MTT assay. The expression levels of miR-27b, Frizzled7 (FZD7), cyclin D1 and c-myc were detected by quantitative real time polymerase chain reaction (qRT-PCR). The protein expression level of FZD7 was detected by western blot analysis. The relationship between miR-27b and FZD7, and the activity of Wnt signaling pathway were determined using luciferase reporter assay. The miR-27b expression in OSCC cell lines was significantly decreased compared with control. Overexpression of miR-27b remarkably inhibited OSCC cell proliferation. Additionally, miR-27b could target and inhibit FZD7 expression and decrease the activity of Wnt signaling pathway.miR-27b could inhibit OSCC cell proliferation through inhibiting FZD7 and FZD7-mediated Wnt signaling pathway.     

MiR-200c inhibited the proliferation of oral squamous cell carcinoma cells by targeting Akt pathway and its downstream Glut1

ObjectiveOur study aimed to investigate the functionality of miR-200c in oral squamous cell carcinoma.MethodsTumor tissues and adjacent tissues were obtained from oral squamous cell carcinoma patients, and blood samples were extracted from both oral squamous cell carcinoma patients and healthy controls. Expression of miR-200c in those tissues was detected by qRT-PCR. All patients were followed-up for 5 years and ROC curves as well as survival analyses were performed to evaluate the diagnostic as well as prognostic values of serum miR-200c for oral squamous cell carcinoma. miR-200c and Glut1 overexpression oral squamous cell carcinoma cell lines were constructed and cell proliferation was detected by CCK-8 assay. Glucose uptake was determined by glucose uptake assay. Interactions between miR-200c, Akt and Glut1 were explored by western blot.ResultsExpression of miR-200c was significantly downregulated in tumor tissues comparing with adjacent tissues in most oral squamous cell carcinoma patients. Serum level of miR-200c was lower in oral squamous cell carcinoma patients than that in healthy controls, and it was decreased with increased primary tumor stages. Serum levels of miR-200c can been used to effectively distinguish oral squamous cell carcinoma patients from healthy control, and patients with lower serum level of miR-200c showed shorter survival time. miR-200c overexpression inactivated Akt and Glut1 expression, while Akt activator and Glut1 overexpression showed no significant effects on miR-200c. Akt activator promoted Glut1 expression, but Glut1 overexpression showed no significant effects on Akt. MiR-200c inhibited cell proliferation and glucose uptake, while Akt activator and Glut1 overexpression reduced the inhibitory effect of miR-200c overexpression on cell proliferation.ConclusionmiR-200c can inhibit the proliferation of oral squamous cell carcinoma cells by targeting Akt pathway and its downstream Glut1.     

mTOR调控口腔鳞状细胞癌增殖转移的机制研究

目的：研究Toll样受体4（TOLL-like receptor 4,TLR4）分子高表达与口腔鳞状细胞癌预后的关系,探讨哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin,mTOR）通过TLR4信号通路间接调控口腔鳞状细胞癌增殖转移的潜在机制。方法：采用免疫组织化学法检测50例口腔鳞状细胞癌患者病理切片中TLR4的表达;使用mTOR抑制剂(雷帕霉素)作用于口腔鳞状细胞癌细胞系CAL27后,再用LPS激活TLR4信号通路,通过MTT法检测CAL27细胞的增殖能力,Transwell法检测CAL27细胞的迁移能力, Western 免疫印迹检测其对肿瘤细胞中NF-κB 及MAPK信号通路的影响。采用SPSS 16.0软件包对数据进行统计学分析。结果：TLR4分子高表达与口腔鳞状细胞癌预后差显著相关（P<0.05）;雷帕霉素能够显著抑制TLR4激活后的CAL27细胞的增殖和迁移能力（P<0.01）,显著抑制TLR4下游的NF-κB 及MAPK信号通路（P<0.01）。结论： mTOR通过TLR4信号通路调控口腔鳞状细胞癌的增殖与转移,有望成为治疗口腔鳞状细胞癌的新靶点。     

B7-H3在口腔鳞癌细胞中的表达及其意义

目的：研究B7-H3在口腔鳞癌组织和正常口腔黏膜组织中的表达差异,以及B7-H3对口腔鳞癌细胞生物学的影响。方法：RT-qPCR、免疫组化检测B7-H3在口腔鳞癌组织和正常口腔黏膜组织的表达差异;构建B7-H3腺病毒表达载体,感染人舌鳞癌细胞Tca8113,CCK-8、PI染色流式细胞仪检测B7-H3高表达对Tca8113细胞增殖和细胞周期的影响。采用SPSS11.0软件包对数据进行统计学处理。结果：RT-qPCR结果显示,口腔鳞癌组织B7-H3 mRNA表达为3.021±0.2310,显著高于正常口腔黏膜组织（0.6002±0.1010）;与对照组比较,10、50、100感染复数组在作用24h呈现促进细胞增殖作用,S期细胞比例显著增加,72h时增殖作用最强（P〈0.05）;与感染复数1组比较,同时间段50、100组促细胞增殖作用和S期细胞比例显著增加（P〈0.05）,50与100组比较无显著差异（P〉0.05）。结论：口腔鳞癌组织B7-H3mRNA和蛋白表达显著高于正常口腔黏膜组织;B7-H3高表达,可促进口腔鳞癌细胞增殖。     

LLY-283靶向PRMT5抑制头颈部鳞癌增殖和转移的实验研究

目的： 探讨LLY-283通过蛋白质精氨酸甲基转移酶（protein arginine methyltransferase 5,PRMT5）对头颈部鳞状细胞癌（head and neck squamous cell carcinoma,HNSCC）增殖和转移生物学行为的影响。方法： 通过TCGA数据库、实时荧光定量PCR和免疫印迹实验检测PRMT5在HNSCC组织、细胞系中表达水平;利用慢病毒技术构建PRMT5敲低稳转细胞株,分析PRMT5对HNSCC细胞生物学行为影响,药物杀伤实验观察LLY-283在细胞系中IC50变化;细胞实验和裸鼠移植瘤实验进一步检测LLY-283通过PRMT5对HNSCC生物学作用。结果： PRMT5在HNSCC组织和细胞系中高表达,促进HNSCC增殖和转移能力,降低药物LLY-283的IC₅₀值。LLY-283可通过PRMT5显著降低HNSCC增殖和转移能力、体内裸鼠移植瘤体积和Ki-67表达量。结论： LLY-283可能通过抑制PRMT5和Ki-67表达,降低HNSCC增殖、转移和裸鼠移植瘤形成能力,发挥抗肿瘤作用。     

Acetylshikonin inhibits growth of oral squamous cell carcinoma by inducing apoptosis

ObjectivesRecently, shikonin derivatives from Lithospermum erythrorhizon have been suggested as potential chemotherapeutic agents against numerous types of cancers in addition to their traditional uses, e.g., as anti-inflammatory agents. Acetylshikonin, one of shikonin derivatives, has also been reported to possess anticancer activity. However, few studies of the effectiveness of acetylshikonin against cancer cells have been conducted, and there are no studies of oral cancers. In this study, we investigated the usefulness of acetylshikonin as a treatment regimen for oral cancers by observing the growth inhibitory function of acetylshikonin and the involved mechanisms.DesignsThe viability, cell cycle, and ratio of apoptotic cells of oral squamous cell carcinoma (OSCC) cells were observed after treatment with acetylshikonin using MTT assay, flow cytometric analysis, and Annexin V/PI staining, respectively. In addition, molecular changes of apoptosis-related pathways and the role of reactive oxygen species (ROS) were analyzed in acetylshikonin-treated cells.ResultsWe observed that acetylshikonin significantly suppressed the growth of OSCC cells by inducing apoptotic cell death, and acetylshikonin affected the viability of a normal keratinocyte cell line HaCaT to a lesser degree, suggesting that acetylshikonin may be a good chemotherapeutic reagent with less toxicity to normal tissues. In addition, we found that acetylshikonin-induced apoptosis of OSCC cells is mediated by ROS as well as G2 cell cycle arrest. ROS production in response to acetylshikonin treatment enhanced the phosphorylation of JNK and p38 MAPK, which are in the major pathways of apoptotic cell death mechanisms.ConclusionsIn summary, our data suggest that acetylshikonin is a strong candidate for use as a selective chemotherapeutic agent for the treatment of OSCC.     

Spindly和Bub3在口腔鳞状细胞癌中的表达及意义

目的：探讨Spindly和Bub3在口腔鳞状细胞癌（oral squamous cell carcinoma,OSCC）中的表达水平及意义。方法：选取河北医科大学第四医院2017年3月—2019年3月收治的65例OSCC患者的口腔鳞癌组织及癌旁正常组织,采用RT-PCR检测口腔鳞癌组织及癌旁正常组织中Spindly、Bub3 mRNA的表达水平;对OSCC细胞株进行培养,siRNA转染干扰Spindly、Bub3基因表达后,采用MTT法检测细胞增殖能力,划痕实验检测细胞迁移能力。采用SPSS 22.0软件包对数据进行统计学分析。结果：Spindly和Bub3 mRNA在人口腔鳞癌组织中的表达水平显著高于癌旁组织（P<0.05）;Spindly和Bub3 mRNA表达量与T分期、临床分期、淋巴结转移有关（P<0.05）;Spindly、Bub3、Spindly/Bub3高表达患者的5年生存率高于低表达患者,且Spindly/Bub3高表达患者的5年生存率低于Spindly、Bub3高表达患者,差异有统计学意义（P<0.05）;siRNA-Spindly组的Spindly表达水平低于Spindly阴性对照组、空白对照组,siRNA-Bub3组的Bub3表达水平也低于Bub3阴性对照组、空白对照组,差异具有统计学意义（P<0.05）;siRNA-Spindly组细胞在24、48、72、96 h的增殖能力低于Spindly阴性对照组、空白对照组,siRNA-Bub3组细胞在24、48、72、96 h的增殖能力低于Bub3阴性对照组、空白对照组,差异有统计学意义（P<0.05）;siRNA-Spindly组细胞在24、48 h的迁移能力低于Spindly阴性对照组、空白对照组,siRNA-Bub3组细胞在24、48 h的迁移能力低于Bub3阴性对照组、空白对照组,差异有统计学意义（P<0.05）。结论：Spindly和Bub3在口腔鳞癌中呈高表达,且与临床、病理分期相关;特异性抑制Spindly和Bub3基因表达,可降低癌细胞的增殖和迁移能力,有望作为治疗口腔鳞癌的靶点之一。