Angiostatin gene therapy inhibits the growth of murine squamous cell carcinoma in vivo

Tumor growth is an angiogenesis-dependent process and therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive. Angiostatin has been shown to potently inhibit endothelial proliferation in vitro and tumor growth in vivo. We now show that a shift in the balance of tumor angiogenesis by gene transfer of a cDNA coding for mouse angiostatin into mouse squamous cell carcinoma NRS-1 and SCC-VII cells suppresses tumor growth in vivo. The inhibition of an angiostatin-transfected tumor was accompanied by a marked reduction in vascularity and the presence of many apoptotic tumor cells. However, transfected-angiostatin cDNA does not affect the expression of the vascular endothelial growth factor (VEGF) and VEGF-R2 in the vascular endothelium. The inhibition mechanisms of neovascularization may be mediated independent of VEGF:VEGF-R2 complex. Our data may provide a useful approach for human oral cancer therapy by gene therapy with angiostatin.     

肝癌细胞系BEL-7402中siRNA表达载体介导靶向c-myc基因的RNA干涉实验研究

目的:探索经载体介导的RNA干涉对肝癌细胞cmyc基因表达抑制的可行性。方法:设计并合成2条靶向癌基因cmyc的RNA干涉模板片段,分别将其连接至pSilencer1.0U6载体上构建siRNA真核表达载体;脂质体转染法将上述重组质粒转入BEL7402肝癌细胞株。半定量RTPCR分析cmyc基因的表达抑制效率,以及cmyc基因表达下调细胞中CDK4、hTERT和Gadd45β基因表达的改变。结果:获得2个能在细胞内有效转录生成靶向cmyc基因siRNA的重组载体pSicmyc1和pSicmyc2;其中pSicmyc2可有效介导肝癌细胞BEL7402cmyc基因的表达抑制,抑制率高达90%以上;在cmyc基因经RNAi抑制的细胞中,CDK4和hTERT基因的表达分别下调至85%和57%;而Gadd45β的表达上调约110%。结论:肝癌细胞BEL7402中cmyc基因的表达可被载体介导诱发的RNA干涉有效抑制,进而导致细胞中与增殖和凋亡相关基因的表达改变。     

siRNA对卵巢上皮癌细胞HMGA1基因表达抑制的实验研究

目的:探讨小分子干扰RNA对卵巢上皮癌细胞中高迁移率蛋白家族A1 (Hish mobility group A1, HMGA1)基因表达的影响,了解HMGA1基因在卵巢上皮癌发生、发展中的作用.方法:设计并合成HMGA1 siRNA,分为两组,实验组以不同浓度[(1.0,2.5,5.0)μg/L,下同]的HMGA1 siRNA转染HMGA1基因高表达的卵巢上皮癌细胞系OVCAR细胞,对照组以等量阴性对照siRNA转染OVCAR细胞,实时荧光定量RTPCR技术检测转染后OVCAR细胞中HMGA1 mRNA的表达, HMGA1 mRNA的表达以PCR循环数阈值(Ct值)表示;蛋白印迹法测定OVCAR细胞中HMGA1蛋白的表达;显微镜观察并计数转染后存活的OVCAR细胞数.结果: HMGA1 siRNA转染OVCAR细胞后,明显下调OVCAR细胞中HMGA1 mRNA及蛋白的表达水平,实验组不同浓度HMGA1 siRNA转染后, OVCAR细胞的Ct值分别为(19.62±0.02),(20.46±0.02),(20.57±0.03)个循环数,与对照组[分别为(19.30±0.02),(19.45±0.01),(19.58±0.03)个循环数]比较,差异有统计学意义(P<0.05).上述浓度转染后,实验组OVCAR细胞的HMGA1蛋白表达水平(分别为0.75±0.02,0.56±0.02,0.19±0.03)分别与对照组(分别为1.92±0.25,1.80±0.29,1.15±0.18)比较,差异有统计学意义(P<0.01).实验组OCAR细胞生长较对照组明显受到抑制(P<0.05).结论: HMGA1 siRNA可以下调HMGA1 mRNA及其蛋白的表达水平,抑制细胞生长,提示HMGA1基因在卵巢上皮癌的发生、发展中具有重要作用.     

Nuclear DNA content in squamous cell carcinoma, small cell carcinoma, and bronchiolo-alveolar carcinoma of the lung

Nuclear DNA content in individual, morphologically identified tumor cells from 33 squamous lung carcinomas, 20 small cell lung carcinomas, and 10 bronchiolo-alveolar carcinomas were analyzed by means of cytophotometry on Feulgen-stained histologic and cytologic specimens. Twenty-eight of the squamous cell carcinomas and 17 of the small cell carcinomas had high and scattered DNA values, indicative of high malignancy potentials. None of the bronchiolo-alveolar carcinomas showed such high DNA values. These results are in line with clinical experience that squamous cell and small cell carcinoma are associated with rapid progression and death in patients, whereas bronchiolo-alveolar carcinomas have a more indolent course.     

Circulating small non coding RNA signature in head and neck squamous cell carcinoma

The Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common human cancer, causing 350,000 individuals die worldwide each year. The overall prognosis in HNSCC patients has not significantly changed for the last decade. Complete understanding of the molecular mechanisms in HNSCC carcinogenesis could allow an earlier diagnosis and the use of more specific and effective therapies. In the present study we used deep sequencing to characterize small non-coding RNAs (sncRNAs) in serum from HNSCC patients and healthy donors. We identified, for the first time, a multi-marker signature of 3 major classes of circulating sncRNAs in HNSCC, revealing the presence of circulating novel and known miRNAs, and tRNA- and YRNA-derived small RNAs that were significantly deregulated in the sera of HNSCC patients compared to healthy controls. By implementing a triple-filtering approach we identified a subset of highly biologically relevant miRNA-mRNA interactions and we demonstrated that the same genes/pathways affected by somatic mutations in cancer are affected by changes in the abundance of miRNAs. Therefore, one important conclusion from our work is that during cancer development, there seems to be a convergence of oncogenic processes driven by somatic mutations and/or miRNA regulation affecting key cellular pathways.     

Oxic and hypoxic cells in a murine squamous cell carcinoma

[125I]Iododeoxyuridine labeling of a squamous cell carcinoma and follow-up of 125I activity at the tumor in situ revealed that the 125I activity remained at a constant level from the 24th to the 100th hour post-labeling and then decreased with a half time of about 200 hr. Autoradiographic studies with [3H]thymidine showed that the tumor cells were labeled around capillaries, spread through the corded structure (the cord) and finally reached the necrotic regions. One could speculate that the constant 125I period represents the transit time of the labeled cells through the cord and that the decline occurs mostly in the necrotic regions. X-Irradiation shortened the constant period of 125I activity by about 24 hr and accelerated the declining rate in a dose-dependent manner. When tumors were made hypoxic by clamping the legs, the declining rate decreased significantly. When misonidazole (a hypoxic radiosensitizer) was administered before X-rays, the declining rate increased to a level higher than that of the oxic tumors. From the time course studies, it was suggested that the tumor cells immediately after 125I-labeling were oxic, that they became gradually hypoxic during their transit through the cord and that they became anoxic when they reached the necrotic regions.     

靶向VEGF发夹状RNA对宫颈癌HeLa细胞抑制作用的研究

目的:运用RNAi技术下调血管内皮生长因子(VEGF)在HeLa细胞中的表达,观察其对肿瘤细胞凋亡的影响,为人宫颈癌治疗提供理论依据。方法:设计并构建针对VEGF的携带绿色荧光蛋白(GFP)发夹状RNA(shRNA)质粒表达载体(PGPU6/GFP/Neo-shRNA),脂质体法转染HeLa细胞;荧光显微镜观察GFP的表达,并计算转染效率;RT-PCR检测HeLa细胞VEGF的表达,筛选出靶序列;再用流式细胞仪法检测细胞凋亡。结果:构建的PGPU6/GFP/Neo载体成功转入HeLa细胞;转染48h后,HeLa-shVEGF1组HeLa细胞VEGFmRNA表达的抑制率为75.0%;与HeLa组和HeLa-shNC组相比,HeLa-shVEGF1组HeLa细胞凋亡率明显增加,P<0.01。结论:本研究构建的PGPU6-shRNA表达载体携带GFP便于观察细胞的转染情况,且不影响U6启动子的转录,同时有效沉默了VEGF基因,明显增加HeLa细胞的凋亡,为未来肿瘤的治疗提供新途径。     

食管鳞癌DNA和RNA测定及其预后关系的研究

目的　探讨食管鳞癌ＤＮＡ异倍体和ＲＮＡ含量变化与预后的关系。方法　应用流式细胞技术对６２例随访患者的石蜡标本进行研究。结果　术后生存５年以上患者的ＤＮＡ异倍体７８．１％（２５／３２）,ＲＮＡ含量为２０．５３±４．２１。术后半年内死亡者ＤＮＡ为９３．３％ （２８／３０）,ＲＮＡ含量为２４．８４±６．３５,两者均有显著性差异。ＤＮＡ异倍体和ＲＮＡ含量与病期、恶性程度和淋巴结转移有关。结论　ＤＮＡ和ＲＮＡ测定是判断食管鳞癌病期、恶性程度、有无淋巴结转移和预后的重要指标。表２　食管癌不同病期与ＤＮＡ和ＲＮＡ含量的关系生存时间（年） 病期 例数 ＤＮＡ２Ｃ Ａｎ ％ＲＮＡ含量（ｘ±ｓ）＞ ５ Ⅰ ３ ２ １ ５０ １０．６１±０．２４Ⅱ １９ ４ １５ ７８．９ ２１．５６±３．４１Ⅲ ８ １ ７ ８７．５ ２３．９８±２．１１Ⅳ ２ ０ ２ １００ ２６．１２±１．５６＜ ０．５ Ⅰ ０ ０ ０Ⅱ １１ １ １０ ９０．９ ２３．４５±４．２７Ⅲ １３ １ １２ ９２．３ ２４．７６±２．８７Ⅳ ６ ０ ６ １００ ２７．３１±２．５６表３　食管鳞病分化程度与ＤＮＡ和ＲＮＡ含量的关系生存时间（年） 分化 例数 ＤＮＡ２Ｃ Ａｎ ％ＲＮＡ含量（ｘ±ｓ）＞ ５ Ⅰ ５ ３     

质粒表达的短发夹状RNA特异性抑制鼻咽癌细胞的增殖

目的观察pmU6-sibclD重组体在细胞内表达的bcl-xL短发夹状RNA(short hairpin RNA,shRNA)能否特异地抑制人鼻咽癌CNE-2Z细胞的增殖.方法将自行构建的表达短发夹状RNA的重组质粒转染到人鼻咽癌CNE-2Z细胞株、卵巢癌HO-8910细胞株和正常人类肝脏L-O2细胞株中,流式细胞仪检测转染率,MTT比色法检测细胞的生长抑制率.结果bcl-xLshRNA能特异地抑制CNE-2Z细胞株的生长增殖,而对正常人类肝脏细胞株的生长增殖和HO-8910细胞中的绿色荧光蛋白(green fluorescence protein,GFP)的表达无抑制作用.结论pmU6-sibclD重组体在细胞内表达的短发夹状RNA能特异性抑制鼻咽癌细胞的生长增殖,为质粒介导的RNAi技术运用于肿瘤的基因治疗提供一定的理论依据.     

HER2基因沉默抑制人胃癌细胞SGC-7901的生长和侵袭

目的：探讨针对HER2基因的RNA干涉对人胃癌细胞生长和侵袭的影响。方法：构建了针对癌基因HER2的siRNA表达载体,分别命名为pcDNA3-sihe1和pcDNA3-sihe2,将构建的干涉载体与对照载体转染胃癌SGC-7901细胞,通过间接免疫荧光实验确定干涉效果;以软琼脂集落形成实验检测干涉后细胞的集落形成能力,以流式细胞术检测细胞在悬浮培养条件下的凋亡情况;并以Boyden chamber法检测转染后细胞的侵袭能力。结果：成功构建针对癌基因HER2的siRNA表达载体,siRNA表达载体转染SGC-7901细胞后HER2表达水平明显降低,证实siRNA成功抑制HER2蛋白的表达。HER2低表达的细胞株集落形成能力降低,流式细胞术检测发现HER2下调可引起胃癌细胞的凋亡,Boydenchamber实验表明针对HER2的RNA干涉可抑制胃癌细胞SGC-7901的侵袭能力。结论：RNA干涉有效地抑制了HER2分子的表达,进而影响SGC-7901细胞的生长和侵袭,本实验为进一步研究HER2分子与肿瘤细胞生长和转移的关系奠定了基础。     

siRNA抑制VEGF基因表达对鼻咽癌细胞生物学行为的影响

目的:应用RNA干扰(RNAi)技术抑制人鼻咽低分化鳞状上皮细胞癌细胞株CNE-2中血管内皮生长因子(VEGF)的表达,研究VEGF对鼻咽癌细胞生物学行为的影响.方法:构建针对VEGF的siRNA真核表达载体pU-VEGF-siRNA,将其表达载体经脂质体LipofectamineTM2000转染至CNE-2细胞,荧光显微镜下观察转染效率,采用RT-PCR、Western blot方法检测转染后CNE-2细胞中VEGF mRNA和蛋白的表达.通过流式细胞仪分析细胞周期的分布,并应用平板克隆形成实验、Transwell侵袭小室模型观察转染后CNE-2细胞恶性生物学行为的变化.结果:pU-VEGF-siRNA转染后CNE-2细胞株中VEGF mRNA和蛋白表达较pU-siCONT组、空白对照组显著下降(P＜0.05),细胞周期被阻滞在G1期,细胞生长缓慢,体外侵袭能力下降.结论:通过RNAi技术阻断VEGF的表达,可抑制CNE-2细胞的生长、增殖、迁徙,提示VEGF在鼻咽癌的发生、发展过程中起重要作用.     

食管鳞癌DNA和RNA含量测定的临床意义

目的:为探讨食管鳞癌DNA异倍体和RNA含量的变化及临床意义。方法:应用流式细胞仪对140例食管鳞癌石蜡标本进行研究。结果:食管鳞癌DNA异倍体检出率为90％(126/140),RNA含量为21.85±6.90,DNA异倍体和RNA含量与病期、恶性程度和淋巴结转移有关。结论:提示DNA和RNA含量测定有助于判断食管鳞癌患者的病期、恶性程度和淋巴结转移情况,为术后有针对性的制定治疗方案提供依据。     

肝癌细胞系BEL-7402中siRNA表达载体介导靶向c-myc基因的RNA干涉实验研究

Objective： To explore the inhibition of the expression of c-Myc in human hepatocellular carcinoma via vector-based RNA interference （RNAi）. Methods： Two RNA interference DNA templates targeting c-Myc oncogene were designed via online software and synthesized. By ligation, the fragments were inserted into pSilencer 1.0-U6 to construct the recombinant siRNA expressing plasmids. The identified recombinants were introduced into BEL-7402 cells with Lipofactamine. The inhibition of c-Myc expression, together with the expression of CDK4, hTERT and Gadd45β in c-Myc down-regulated BEL-7402 cells, were analyzed by semi-quantitative RT-PCR. Results： Two recombinant plasmids pSic-myc-1 and pSic-myc-2, which direct the yields of siRNAs targeting c-Myc in cells, were constructed. Among which, pSic-myc-2 was shown to trigger a RNAi-mediated inhibition of expression of c-Myc in BEL-7402 by up to 90%. In c-Myc knockdown BEL-7402 cells, the expression of CDK4 and hTERT were down-regulated with a ratio of 85% and 57%, respectively, while the expression of Gadd45β was up-regulated by up to 110%. Conclusion： The expression of c-Myc in BEL-7402 could be suppressed by vector-based RNA interference successfully. The knockdown of c-Myc in turn resulted in the changes of expression of genes related to cell proliferation and apoptosis. Thus, our study provided a preliminary data in searching of a c-Myc-targeted RNAi therapy of human hepatocellular carcinoma.     

Prognostic value of VEGF expression in primary esophageal squamous cell carcinoma

Both the morbidity and mortality of esophagus cancer are very high in China. The modern treatment for this disease is surgery plus adjuvant multi-therapies, mainly including chemotherapy and radiotherapy. The postoperative 5-year survival rate, unfortunately, is still low. It is around 30% in China and 10% in the west. More and more evidences have demonstrated that there is a close relationship between treatment effect and bio-behavior of esophagus cancer. To search for better prognostic ind…     

Expression of thymidine phosphorylase and VEGF in esophageal squamous cell carcinoma

The purpose of this study was to determine the role of TP and VEGF in angiogenesis and its clinical significance in prognosis of patients with esophageal carcinoma. Expressions of TP and VEGF, microvascular density and cell proliferation activity were evaluated by using 40 immunohistochemically stained resected esophageal carcinoma tissues, and the survival rate of the patients was analyzed. Significant positive correlation and regression were found between the VEGF expression level of tumor and microvascular density (r=0.73, p<0.0001). Not statistically strong but significant positive correlation and regression were found between the TP expression level of tumor and microvascular density (r=0.32, p=0.046). No significant relationships were found between TP and VEGF expressions. Pathological T-factor and pathological N-factor were significant prognostic factors. Tumor length, site of lesion, gender, age, and Ki67 labeling index were not significant prognostic factors. The VEGF expression level was one of the unfavorable prognostic factors (risk ratio =1.035, 95% CI=1.007-1.065, p=0.01). The patients with high TP expression showed a tendency for unfavorable prognosis, but it was not statistically significant (RR=1.017, 95% CI=0.996-1.042, p=0.1). The prognosis of patients in the TP/VEGF[+/+] group was significantly poorer than that of the patients in the TP/VEGF[-/-] group and TP/VEGF[+/- or -/+] group (RR=0.488 for TP/VEGF[-/-] group, =0.717 for TP/VEGF[+/- or -/+] group, p=0.005). In conclusion, VEGF and TP expression seems to have a relationship with tumor angiogenesis, and co-expression of TP and VEGF seemed to be one of the unfavorable prognostic factors.     

Establishment of a murine model of bone invasion by oral squamous cell carcinoma

This study examines the establishment an animal model of bone invasion by oral squamous cell carcinoma to clarify the mechanisms of osteoclast-mediated bone invasion. C(3)H/HeN mice were inoculated with SCCVII cells into the masseter region. At the end of week 3, all surviving mice were sacrificed and analyzed by three-dimensional imaging using micro-computed tomography, histopathological observation using Hematoxylin-Eosin staining and Tartrate-Resistant Acid Phosphatase staining, and confirmation of mRNA expression of the osteoclast-related cytokines IL-6, TNF-alpha, and PTHrP. SCCVII cells rapidly multiplied in the masseter muscle of the mice. Bone invasion was evident only in the SCCVII transplanted group on micro-computed tomography. The histopathologic findings obtained with H-E and TRAP staining indicated that the tumor cells in the mandible of all animals of the SCCVII transplanted group exhibited funicular invasion and presented a serrated pattern of bone resorption. The mRNA expression of IL-6, PTHrP, and TNF-alpha increased as the control decreased. SCCVII cells were highly invasive into mandibular bone in C(3)H/HeN mice. This model was similar to the invasion of human oral cancer into maxillary and mandibular bone. Our mandibular invasion model may provide a powerful new modality for the diagnosis and treatment of oral cancer with bone invasion.     

APE1shRNA重组质粒的构建及对人乳腺癌细胞生长的影响

目的设计构建靶向脱嘌呤脱嘧啶核酸内切酶1（APE1/ref-1）特异性短发卡RNA的真核表达载体,并转染人乳腺癌T47D细胞。方法设计、合成针对APE1的特异性的短链寡核苷酸,构建APE1特异性shRNA的重组质粒,稳定转染人乳腺癌T47D细胞;采用聚合酶链反应和免疫印迹法检测转染前后T47D细胞中APE1的表达。采用四甲基偶氮唑盐法观察T47D细胞的生长情况。结果经酶切和测序鉴定,成功构建APE1特异性shRNA的重组质粒,并能够转染T47D细胞。转染后,APE1在mRNA和蛋白水平表达分别下降约53.1%和60.5%,T47D细胞增殖活性受到抑制。结论运用pGenesil-1.1质粒载体构建的靶向APE1特异性shRNA的重组质粒可以成功转染T47D细胞,有效抑制APE1的表达,并抑制肿瘤细胞的生长。     

Circular RNA ARHGAP5 inhibits cisplatin resistance in cervical squamous cell carcinoma by interacting with AUF1

Cervical squamous cell carcinoma (CSCC) is one of the leading causes of cancer death in women worldwide. Patients with advanced cervical carcinoma always have a poor prognosis once resistant to cisplatin due to the lack of effective treatment. It is urgent to investigate the molecular mechanisms of cisplatin resistance. Circular RNAs (circRNAs) are known to exert their regulatory functions in a series of malignancies. However, their effects on CSCC remain to be elucidated. Here, we found that cytoplasmic circARHGAP5, derived from second and third exons of the ARHGAP5 gene, was downregulated in cisplatin-resistant tissues compared with normal cervix tissues and untreated cervical cancer tissues. In addition, experiments from overexpression/knockdown cell lines revealed that circARHGAP5 could inhibit cisplatin-mediated cell apoptosis in CSCC cells both in vitro and in vivo. Mechanistically, circARHGAP5 interacted with AU-rich element RNA-binding protein (AUF1) directly. Overexpression of AUF1 could also inhibit cell apoptosis mediated by cisplatin. Furthermore, we detected the potential targets of AUF1 related to the apoptotic pathway and found that bcl-2-like protein 11 (BIM) was not only negatively regulated by AUF1 but positively regulated by circARHGAP5, which indicated that BIM mRNA might be degraded by AUF1 and thereby inhibited tumor cell apoptosis. Collectively, our data indicated that circARHGAP5 directly bound to AUF1 and prevented AUF1 from interacting with BIM mRNA, thereby playing a pivotal role in cisplatin resistance in CSCC. Our study provides insights into overcoming cancer resistance to cisplatin treatment.     

Transition of oxic cells to hypoxic cells in a murine squamous cell carcinoma

(125I) Iododeoxyuridine labeling of a squamous cell carcinoma and follow-up of 125I activity at the tumor in situ revealed that the 125I activity remained at a constant level from the 24th to the 100th hour post-labeling and then decreased with a half time of about 200 hr. Autoradiographic studies with (3H) thymidine showed that the tumor cells were labeled around capillaries, spread through the corded structure (the cord) and finally reached the necrotic regions. One could speculate that the constant 125I period represents the transit time of the labeled cells through the cord and that the decline occurs mostly in the necrotic regions.     

RNA干扰HMGB1基因对食管鳞癌细胞X线照射后增殖与迁移能力影响

目的 RNAi技术干扰人食管鳞癌细胞中HMGB1基因表达,观察X线照射后细胞增殖活性、迁移和存活能力及细胞周期分布。方法 RT-PCR和Western blot法检测HMGB1 mRNA和蛋白表达;分别采用MTS和Transwell方法检测食管鳞癌细胞ECA109、KYSE30细胞增殖活性和迁移能力;克隆形成实验检测各组细胞的存活能力;流式细胞术检测照射后各组的细胞周期分布。结果 照射组ECA109、KYSE30细胞中HMGB1 mRNA和蛋白表达水平较未照射组明显增加(P<0.05),且存在剂量-效应和时间-效应关系;MTS结果显示食管鳞癌细胞的增殖水平在照射后不同时间点均明显降低(P<0.01);HMGB1沉默组细胞中HMGB1 mRNA和蛋白的表达均明显低于对照组和NC组(P<0.01);克隆形成实验结果显示HMGB1表达降低后,肿瘤细胞的放射敏感性明显增加(P<0.01);Tanswell侵袭小室模型检测显示照射后4 h沉默组穿膜细胞数明显降低(P<0.01),且沉默组G0/G1期比例明显高于对照组和NC组,S期比例显著低于其他组,照射后这种趋势更明显(P<0.01)。结论 RNAi干扰技术降低食管鳞癌细胞中HMGB1的表达,降低了细胞增殖和迁移能力,通过诱导照射后G0/G1期阻滞增加了放射敏感性。