hsa_circ_0006950 调控miR-124-3p/EZH2 轴促进胰腺癌细胞增殖与迁移的机制研究

目的　检测环状核糖核酸（circular RNA ,cicrRNA）hsa_circ_0006950 在胰腺癌（pancreatic cancer,PC）中的表达,探讨其对胰腺癌细胞增殖、迁移的影响及其潜在分子作用机制。方法　利用实时荧光定量PCR(qRT-PCR) 检测hsa_circ_0006950 在胰腺癌组织及细胞中的相对表达水平;转染hsa_circ_0006950 干扰载体构建低表达细胞系,通过CCK-8实验、克隆斑点形成实验和Transwell 实验检测hsa_circ_0006950 对胰腺癌细胞增殖、克隆形成及迁移的影响;利用生物信息学网站预测hsa_circ_0006950 的miRNA 分子靶标及其调控网络（hsa_circ_0006950-miR-124-3p-EZH2）,双荧光素酶基因报告实验验证hsa_circ_0006950 和miR-124-3p,miR-124-3p 和EZH2 的靶向结合关系;转染过表达/ 干扰miR-124-3p 和过表达EZH2 细胞系,探究miR-124-3p 和EZH2 及hsa_circ_0006950 调控miR-124-3p/EZH2 轴对胰腺癌细胞克隆形成及迁移的影响。结果　胰腺癌组织中hsa_circ_0006950 表达明显高于癌旁正常组织（3.57±0.52 vs 1.01±0.03）,差异有统计学意义（t=21.980,P ＜ 0.001）。胰腺癌细胞PANC-1（7.51±0.62）,AsPC-1（5.26±0.45）,Capan-2（3.69±0.38）,SW1990（3.25±0.32）and BXPC-3（3.86±0.35）中hsa_circ_0006950 相对表达明显高于正常胰腺导管上皮细胞HPDE6-C7（1.00±0.01）中的表达,差异有统计学意义（F=88.585,P ＜ 0.001）。与对照组相比,干扰hsa_circ_0006950 表达组细胞增殖能力（0.79±0.17 vs 1.83±0.42）,克隆形成率（51.42%±5.84% vs 78.76%±13.65%）和迁移数目（104.64±24.73 vs 218.21±31.57）明显降低,差异均有统计学意义（t=3.976,3.190,4.905,P=0.016,0.033,0.008）。miR-124-3p 是hsa_circ_0006950 的下游靶基因,EZH2 是miR-124-3p 的直接靶标。hsa_circ_0006950 靶向负调控miR-124-3p,miR-124-3p 靶向负调控EZH2。与对照组相比,过表达miR-124-3p 组PC 细胞增殖（0.21±0.16 vs1.75±0.47）,克隆形成率（47.85%±4.13% vs 81.54%±2.33%）和细胞迁移数目（118.74±24.65 vs 202.36±31.45）明显降低,差异均有统计学意义（t=5.378, 14.317, 3.390, 均P ＜ 0.001）;共转染过表达EZH2 后,miR-124-3p 对细胞增殖、克隆形成率及迁移能力的抑制作用被逆转。在干扰hsa_circ_0006950 组细胞中同时下调miR-124-3p 或过表达EZH2 后,hsa_circ_0006950 对细胞克隆形成及迁移的抑制作用被逆转恢复。结论　胰腺癌中hsa_circ_0006950 显著高表达,抑制其表达可以抑制胰腺癌细胞的增殖及迁移;hsa_circ_0006950 可能通过靶向下调miR-124-3p 表达,进而上调EZH2 表达来发挥作用,参与胰腺癌的发生发展。     

吉西他滨联合奥沙利铂治疗进展期胰腺癌

目的:本研究观察了吉西他滨(健择)联合奥沙利铂(艾恒)组成GO方案治疗进展期胰腺癌的有效性及安全性。方法:自2004年1月~2006年1月,30例进展期胰腺癌患者接受GO方案治疗。GO方案:健择800mg/m2,静脉滴入第1,第8天,艾恒60 mg/m2第2第9天,28天为1个周期,共进行6周期,所有患者均接受了至少2个周期的治疗。结果:30例患者平均年龄62岁,所有患者共进行了99周期治疗,平均每个患者接受3.3周期治疗。30例患者中PR 6例,SD11例,PD13例,总有效率为20%,TCR为56.7%。中位进展时间4.37个月,中位生存时间7.14个月,一年生存率为20%。临床受益率显著,CBR达73.3%。具有较好的耐受性,主要毒副反应是血液学毒性。结论:健择联合艾恒治疗进展期胰腺癌疗效较好,毒副反应可以耐受。     

Pulsed High-Intensity Focused Ultrasound Enhances Apoptosis of Pancreatic Cancer Xenograft with Gemcitabine

We sought to investigate whether concurrent exposure to pulsed high-intensity focused ultrasound (HIFU) and the chemotherapeutic drug gemcitabine would enhance apoptosis in pancreatic cancer. A pancreatic cancer xenograft model was established using BALB/c nude mice and human pancreatic cancer cells (PANC-1). In the first study, mice were randomly allocated into one of four groups: control (n = 4), HIFU alone (n = 4), gemcitabine (GEM) alone (n = 28) and concurrent treatment with HIFU and gemcitabine (HIGEM) (n = 28). The GEM and HIGEM groups were subdivided into four subgroups (16 mice) according to the drug dose injected (50–200 mg/kg) and another four subgroups (16 mice) according to the time interval between drug injection and HIFU treatment (each subgroup, n = 4). Apoptosis rates were evaluated using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay and percentage of necrosis, as evaluated with Harris' hematoxylin solution and eosin Y stain, 3 d after treatment. The second study was performed to evaluate tumor growth rates of the four groups. Each group was treated weekly for 3 wk, and tumor size was periodically measured for up to 4 wk from the beginning of treatment. In the first study, overall rates of apoptosis were significantly higher in the HIGEM group than in the GEM group (p = 0.02). In a subgroup analysis, HIGEM was superior to GEM in enhancing apoptosis at gemcitabine dosages of 150–200 mg/kg gemcitabine and intervals between gemcitabine and HIFU less than 2 h (p = 0.01). In the second study, HIGEM treatment resulted in the slowest tumor growth. However, despite a visible distinction, none of the differences found between the HIGEM and GEM groups were statistically significant (p > 0.05). Treatment with both HIFU and gemcitabine might enhance cell apoptosis and reduce tumor growth in pancreatic carcinoma. For this concurrent treatment, a high dosage of gemcitabine and a short-term delay before HIFU are recommended to maximize the therapeutic effect.     

吉西他滨联合奥沙利铂对中晚期胰腺癌患者生存期的影响

目的研究吉西他滨联合奥沙利铂对中晚期胰腺癌患者生存期的影响。方法采用Log-rank单因素分析方法研究影响中晚期胰腺癌生存期的独立性相关因素,并对两组生存期进行分析。结果年龄、KPS、手术方式、是否联合用药为影响中晚期胰腺癌生存期的显著性相关因素(P=0.000),治疗组OS较对照组延长(P=0.000),且治疗组l,2,3年生存率优于对照组。结论吉西他滨联合奥沙利铂是影响中晚期胰腺癌的相关因素,可显著改善患者的生存率。     

Effect of Endostar Combined with Gemcitabine on the Mouse Model of Human Pancreatic Cancer

Objective To investigate the effects of recombinant human endostatin, that is, endostar combined with gemcitabine on the mouse model of human pancreatic cancer. Methods We use the cell line PANC-1 and the severe combined immune deficient mice to set up the mouse model of human pancreatic cancer, then devide them into three groups, treat them with gemcitabine, gemcitabine combined with endostar, and 0.9 % saline water respectively. We observe the change of the tumor volumn, use ELISA method to detect the serum VEGF level, stain the micro vessel in the tumor tissue with immunohistochemistry method, and compare the data among the different groups respectively. Results On the twenty-eighth day, the tumor volume of the control group, the monotherapy group and the combination group, averaged 1 700 mm^3, 19. 2 mm^3, 10.4 mm^3, serum VEGF level 88.6 L, 35.5, 26.3 pg/mL and MVD 43.9, 30.3, 19. 2 respectively, which had significant difference. Conclusion Endostar can strengthen the lethal effect of gemcitabine on the mouse model of human pancreatic cancer.     

Effect of Endostar Combined with Gemcitabine on the Mouse Model of Human Pancreatic Cancer

IntroductionPancreatic canceris an unsolved health problem.The 5-year survival rateis only 3%and median over-all survival is<6 months,a situationthat has remained unchangedfor the past three decades.Surgical re-sectionis the only potentially curative ther…     

Comparison of Gemcitabine Combined With Targeted Agent Therapy Versus Gemcitabine Monotherapy in the Management of Advanced Pancreatic Cancer

Purpose

Targeted therapy has brought great clinical benefits for patients with multiple solid tumors, but its effects in patients with locally advanced/metastatic pancreatic cancer (LA/MPC) are disputed. This systematic evaluation compared the efficacy and safety profiles of gemcitabine combined with targeted agents (GEM + TA) versus gemcitabine administered as monotherapy or combined with placebo (GEM ± PLC) in LA/MPC patients.

Methods

PubMed and EMBASE were searched for relevant randomized controlled trials published on or before April 30, 2013. The primary end points were overall survival (OS) and progression-free survival (PFS); the secondary end points were 1-year survival rate, objective response rate (ORR), and toxicity rates (TRs), defined as the prevalence of grade 3/4 adverse events. The systematic evaluation was performed by using Review Manager version 5.1.7.

Findings

A total of 10 randomized controlled trials involving 3899 patients (2195 males; mean age, 63.6 years) were included in the systematic evaluation. The results reported that there was no significant difference in OS (hazard ratio [HR] = 0.97 [P = 0.85]), PFS (HR = 0.95 [P = 0.14]), or ORR (odds ratio [OR] = 0.95 [P = 0.69]) between GEM + TA and GEM ± PLC. However, a marginal difference in 1-year survival rate (OR = 0.80 [P = 0.05]) between the 2 groups was observed. The grade 3/4 TRs of anemia, diarrhea, nausea, neutropenia, thrombocytopenia, and vomiting were not significantly different between the 2 groups. However, the prevalence of grade 3/4 rash was significantly greater in the GEM + TA group (OR = 8.31 [P < 0.01]).

Implications

Based on the results from this analysis, the addition of targeted agents to a regimen of gemcitabine treatment does not bring survival benefits except 1-year survival rate to patients with LA/MPC.     

吉西他滨联合奥沙利铂双周方案治疗中晚期胰腺癌临床观察

目的观察吉西他滨联合奥沙利铂双周方案对中晚期胰腺癌病人的近期疗效及毒副作用。方法吉西他滨1000mg／m^2于第1天，第8天各静脉滴注1次：奥沙利铂100mg／m^2于第2天，第9天各静脉滴注1次，每21天重复，观察该方案的毒副作用及近期疗效。结果23例病人接受治疗，完全缓解（CR）1例（4．3％），部分缓解（PR）6俩1（26．1％），稳定（SD）10例（43.5％），疾病进展（PD）6例（26.1％）。近期疗效显著，疾病相关症状有所改善，该联合方案毒副作用较小。结论吉西他滨联合奥沙利铂双周方案对中晚期胰腺癌病人的近期疗效较好，毒副作用可以耐受。     

食管癌Survivin mRNA、Caspase-3的表达及其分子机制

【目的】探讨survivin mRNA、caspase-3蛋白在食管鳞癌中的表达及其分子机制。【方法】采用RT—PCR和免疫组化技术检测38例食管鳞癌及其对应的正常组织中survivin mRNA和caspase-3蛋白的表达。【结果】73．68％食管鳞癌组织呈survivin mRNA阳性表达,显著高于正常组织,并与食管鳞癌的分化程度、临床分期有关,与年龄、性别、淋巴结转移无关。caspase-3蛋白在食管鳞癌和正常组织中的阳性表达率分别为26．32％和89．47％,差异有显著性意义,并与食管鳞癌的分化程度有关,与年龄、性别、淋巴结转移和临床分期无关。在食管鳞癌组织中,survivin mRNA和Caspase-3蛋白表达呈负相关。【结论】survivin、caspase-3参与了食管鳞癌的发生、发展,可以作为食管鳞癌预后的指标,survivin通过抑制caspase-3的活性而发挥其抑制细胞凋亡的作用可能是其主要的分子机制。     

VPA和As2O3对Molt-4细胞的协同作用及可能机制

本研究探讨丙戊酸钠（sodium valproate，VPA）单药或与亚砷酸（arsenie trioxide，As2O，3）联用对人急性T细胞白血病细胞株Molt-4增殖活力的抑制、去甲基化作用及其可能机制。用MTT法检测药物对细胞增殖的抑制作用，利用金氏公式观察两药联用体外抗癌的协同作用。采用半巢式甲基化特异PCR（hnMS—PCR）检测p15INK4B基因甲基化，RT—PCR方法检测p15基因、DNA甲基转移酶DNMT-1、DNMT3A、DNMT3B的表达。结果表明：VPA和As2O3均可明显抑制细胞增殖，二者联用在体外有相加作用（Q值均大于0．85），在5mmol／LVPA与10μmol／LAs2O3联用时抑制率达（70．31±2．54）％。Molt-4细胞p15基因因高甲基化而失表达，经VPA和As2O3联合作用后p15基因甲基化程度明显下降，p15基因表达增强，两药联用时DNMT-1、DNMT-3A、DNMT3B mRNA的表达都有下降。结论：VPA可能通过抑制DNA甲基转移酶DNMT-1和（或）DNMT3B使p15INK4B基因去甲基化，使p15基因表达上调，恢复其活性。VPA和As2O3均可明显抑制Mol-4细胞增殖，两药合用有协同相加作用，可减少砷剂的副作用。     

吉西他滨联合腹腔热灌注化疗治疗晚期胰腺癌疗效观察

目的观察吉西他滨联合腹腔热灌注化疗治疗晚期胰腺癌的临床疗效及安全性。方法选择晚期胰腺癌患者44例,随机化分为两组,其中试验组22例,接受吉西他滨1000 mg/m2,d1、d8,腹腔热灌注生理盐水500 mL＋顺铂40 mg/m2,d2、d5、d9;对照组22例,接受吉西他滨1000 mg/m2,静脉滴注,d1、d8＋顺铂40 mg/m2,静脉滴注,d1~3,两组均21 d为1周期,接受治疗2个周期后评价疗效。结果41例患者可进行疗效评估,两组临床疗效比较,试验组稳定率61.90%显著高于对照组稳定率45.00%,差异有统计学意义（P＜0.05）;临床收益反应比较,实验组有效率85.7%也明显高于对照组为50.0%,差异有统计学意义（P ＜0.05）。结论吉西他滨联合腹腔热灌注化疗治疗晚期胰腺癌疗效较好,毒副反应小,止痛效果明显。     

三维适形放射治疗联合吉西他滨与替加氟化学治疗对局部晚期胰腺癌的疗效

目的：评价三维适形放射治疗（three dimensional conformal radiation therapy,3DCRT）联合吉西他滨与替加氟治疗局部晚期胰腺癌的疗效及安全性.方法：对35例经病理检查证实或符合临床诊断标准的局部晚期胰腺癌患者,行3DCRT,180cGy/次,共28次（5.5周内）,总剂量为5040 cGy;同时第1天及第8天静脉滴注吉西他滨（1000 mg/m2）,第1～5天静脉滴注替加氟（1000 mg/d）;每4周为1个周期,至少2个周期,治疗结束4周后评价疗效及安全性.结果：35例患者均完成联合治疗.治疗有效率为37.1％（13/35）,肿瘤控制率为71.4％（25/35）.最常见的不良反应为恶心（26/35,74.3％）,无3度及以上骨髓抑制,患者可耐受所有不良反应.结论：3DCRT同步吉西他滨联合替加氟治疗局部晚期胰腺癌疗效较好,患者能够耐受其不良反应.     

5,7-二甲氧基黄酮抑制人胰腺癌PANC-1细胞系胰腺癌干细胞特性的体外研究

[目的]探讨5,7-二甲氧基黄酮(DMF)对人胰腺癌干细胞特性的影响.[方法]体外培养人胰腺癌PANC-1细胞系得到微球细胞,流式细胞仪检测;划痕法分析不同浓度DMF对胰腺癌干细胞自我更新、体外细胞迁移和运动能力影响;Western Blot分析不同浓度DMF干预后PANC-1细胞系胰腺癌干细胞表面标志物CD133、CD44、ALDH1、Bmil、E-cad、N-cad蛋白表达变化.[结果]DMF以浓度依赖方式抑制人胰腺癌PANC-1细胞系胰腺癌干细胞肿瘤球形成能力及抑制细胞体外迁移能力;DMF以浓度依赖方式使胰腺癌干细胞标志物CD133、CD44、ALDH1、Bmi1、N-cad蛋白表达水平降低,而E-cad蛋白水平升高.[结论]DMF以浓度依赖方式显著抑制人胰腺癌PANC-1细胞系胰腺癌样干细胞自我更新、分化后细胞迁移、细胞标志物CD133、CD44、ALDH1、Bmi1、N-cadherin蛋白表达,而增强E-cadherin蛋白表达.     

The Long-Term Fate of the Sonoporated Pancreatic Cancer Cells is Uncorrelated With the Degree of Model Molecular Loading

Studies have determined that ultrasound-activated microbubbles can increase the membrane permeability of tumor cells by triggering membrane perforation (sonoporation) to improve drug loading. However, because of the distinct cavitation events adjacent to each cell, the degree of drug loading appeared to be heterogeneous. The relationship between the long-term fate trend and the degree of drug loading remains unclear. To investigate the time-lapse viability of diversity loading cells, fluorescein isothiocyanate–dextran (FITC-dextrans) was used as a molecular model mixed with 2% v/v SonoVue microbubbles (Bracco, Milan, Italy) and exposed to various peak negative pressures (0.25 MPa, 0.6 MPa, 1.2 MPa), 1 MHz frequency and 300 μs pulse duration. To select a suitable parameter, the cavitation activity was measured, and the cell analysis was performed by flow cytometry under these acoustic pressures. The sonoporated cells were then categorized into 3 sub-groups by flow cytometry according to the various fluorescence intensity distributions to analyze their long-term fate. We observed that the stable cavitation occurred at 0.25 MPa and microbubbles underwent ultra-harmonic emission, and obvious broadband signals were observed at 0.6 MPa and 1.2 MPa, suggesting the occurs of inertial cavitation. The cell analysis further showed the maximum delivery efficiency and cell viability at 0.6 MPa, and it was selected for the following experiment. The categorization displayed that the fluorescence intensity of FITC-dextrans in sub-groups 2 and 3 were approximate 5.62-fold and 19.53-fold higher than that in sub-group 1, respectively. After separation of these sub-groups, the apoptosis and necrosis ratios in all 3 sub-groups of sonoporated cells gradually increased with increasing culture time and displayed no significant difference in either the apoptosis (p > 0.05) or necrosis (p > 0.05) ratio after 6 h and 24 h of culture, respectively. Further analysis using Western blot verified that the long-term fate of sonoporated cells involves the mitochondrial signaling proteins. These results provide better insight into the role of cavitation-enhanced permeability and a critical guide for acoustic cavitation designs.     

反应停对胆囊癌细胞的作用机制研究

目的初步探讨反应停对胆囊癌细胞的作用机制。方法应用RT-PCR技术检测经反应停处理后胆囊癌细胞株GBC-SD中survivin、VEGF、MMP-9的表达。结果 survivin、VEGF、MMP-9在经反应停处理的人胆囊癌细胞株GBC-SD中表达下降,且与反应停的浓度有关。结论反应停可能通过促进细胞凋亡和     

蛋白酶体抑制剂协同组蛋白去乙酰化酶抑制剂诱导T细胞淋巴瘤凋亡及其与蛋白聚集体关系的研究

本研究探讨蛋白酶体抑制剂硼替唑米（bortezomib）和组蛋白去乙酰化酶抑制剂辛二酰苯胺异羟肟酸（suberoylanilide hydroxamic acid,SAHA）对T淋巴瘤细胞株Jurkat和Hut78的协同诱导凋亡作用,以及两药联合对蛋白聚集体形成的影响。硼替唑米（10nmol／L）单药或硼替唑米（10nmol／L）联合SAHA（2μmol／L）处理Jurkat和Hut78细胞,应用台盼蓝拒染法计数细胞生长抑制率;细胞涂片观察细胞形态学改变;流式细胞术检测细胞凋亡变化;透射电子显微镜观察细胞凋亡和聚集体形成的超微结构特征。研究结果表明,与对照组和单用硼替唑米组比较,两药合用能够显著抑制Jurkat和Hut78细胞生长,促进细胞凋亡。流式结果显示,两药舍用组的Jurkat和Hub78细胞凋亡细胞比例分别为（41．8±4．7）％和（72．7±11．7）％,显著高于对照组[（3．6±1．3）％和（7．0±1．9）％]和单用硼替唑米组[（6．3±2．3）％和（18．7±9．2）％]（P均〈0．01）。超微结构显示,单用硼替唑米组细胞内多为典型的聚集体结构,两药合用组细胞内聚集体密度降低,数量减少甚至消失,且凋亡细胞明显增多。结论：蛋白酶体抑制剂联合组蛋白去乙酰化酶抑制剂能有效诱导T淋巴瘤细胞凋亡,两药具有协同作用。硼替唑米促进聚集体形成,SAHA能破坏聚集体结构,是增强硼替唑米促凋亡效应的可能机制之一。     

α-辅肌动蛋白4对卵巢癌细胞增殖、凋亡及化疗敏感性的影响及其机制

目的:探讨体外沉默α-辅肌动蛋白4(alpha-actinin-4,ACTN4)基因的表达对卵巢癌细胞增殖、凋亡及化疗敏感性的影响及其可能的机制。方法:化学合成靶向ACTN4基因的ACTN4-siRNA,体外转染至人卵巢癌细胞株SKOV3和OVCA429中,采用real-time PCR检测ACTN4 mRNA的表达,采用MTT法检测卵巢癌细胞的增殖能力及存活率,采用流式细胞术检测卵巢癌细胞的凋亡情况,采用Western blotting检测凋亡相关蛋白的表达。结果:ACTN4-siRNA转染SKOV3/OVCA429细胞后,ACTN4的mRNA及蛋白的表达均明显下降;细胞增殖能力明显受到抑制。在不同浓度紫杉醇作用下,与转染CtrlsiRNA的对照组相比,ACTN4-siRNA转染组SKOV3/OVCA429的细胞存活率均显著降低,即转染ACTN4-siRNA后的SKOV3/OVCA429细胞对紫杉醇的敏感性明显增加(P0.01)。SKOV3细胞瞬时转染ACTN4-siRNA48 h后,细胞凋亡率[(7.22±0.31)%]明显高于转染Ctrl-siRNA的对照组[(2.07±0.08)%];OVCA429细胞的情况与之一致,转染ACTN4-siRNA后细胞凋亡率为[(23.37±0.18)%],对照组为[(19.49±0.19)%],差异均有统计学意义(P0.05)。转染ACTN4-siRNA后,SKOV3/OVCA429中p-Akt和p-FAK蛋白表达明显减少,抗凋亡蛋白Bcl-2表达明显减少,而促凋亡蛋白Bid和Bim表达则明显增加。结论:ACTN4可以降低卵巢癌细胞的化疗敏感性,这种作用可能通过抑制细胞凋亡实现。