miR-93在结肠癌中的表达及其意义

目的研究miR-93在结肠癌组织中的表达与临床病理特征的关系,并探讨其对结肠癌细胞的增殖及细胞周期的影响。方法收集108例结肠癌患者的癌组织及癌旁组织,应用原位杂交和荧光定量PCR检测结肠癌组织及癌旁组织中miR-93的表达水平,分析miR-93表达与结肠癌临床病理特征之间的关系。采用反义miR-93转染降低结肠癌细胞SW480和LoVo中miR-93的表达,采用MTT比色法检测结肠癌细胞增殖的变化情况,利用流式细胞仪检测结肠癌细胞周期的变化情况。结果原位杂交检测显示,结肠癌组织miR-93阳性表达率高于癌旁组织(P<0.05);荧光定量PCR检测显示,61.11%(66/108)的结肠癌组织miR-93表达明显高于癌旁组织(P<0.05);随着结肠癌临床分期的进展,miR-93的表达量逐渐升高,临床Ⅲ/Ⅳ期、Ⅰ/Ⅱ期结肠癌miR-93的表达与癌旁组织相比都显著增高(P<0.05);有淋巴结转移的结肠癌患者的miR-93的表达比无淋巴结转移者显著增加(P<0.05);反义miR-93转染结肠癌细胞SW480和LoVo后,miR-93的表达明显降低,SW480和LoVo结肠癌细胞生长受到明显抑制,其生长主要停滞在G0/G1期,而S期和G2/M期细胞的比例下降。结论 miR-93与结肠癌的发生发展密切相关,其可以通过调节细胞周期中G1/S期的转换而影响结肠癌细胞的生长,为结肠癌治疗提供新的靶点。     

miR-20a-5p在胰腺癌中的表达及作用机制研究

目的检测并分析胰腺癌组织和胰腺癌细胞株中miR-20a-5p的表达情况及对胰腺癌细胞发生、发展的影响。方法收集16对手术切除胰腺癌患者的癌组织及对应癌旁组织、5种胰腺癌细胞株及1株正常胰腺导管上皮细胞,通过实时荧光定量聚合酶链反应(quantitative realtime polymerase chain reaction,qRT-PCR)检测临床标本及胰腺癌细胞株中miR-20a-5p的表达水平;通过转染miR-20a-5p mimics及miR-20a-5p inhibitor分别上调及下调胰腺癌细胞(AsPC-1、BxPC-3)中miR-20a-5p的表达水平,采用CCK-8、EdU及流式凋亡实验检测过表达及敲低miR-20a-5p后对胰腺癌细胞活力、增殖及凋亡能力的影响。结果胰腺癌组织中miR-20a-5p的表达量显著高于癌旁组织(P0.01),5种胰腺癌细胞系中miR-20a-5p的表达量较正常胰腺细胞也明显升高(P0.05)。CCK-8及EdU实验结果显示,过表达miR-20a-5p促进胰腺癌细胞增殖能力,反之,干扰miR-20a-5p抑制胰腺癌细胞增殖。凋亡检测结果显示,过表达miR-20a-5p抑制胰腺癌细胞凋亡能力,反之,干扰miR-20a-5p促进胰腺癌细胞凋亡。结论胰腺癌组织和胰腺癌细胞系均高表达miR-20a-5p,上调miR-20a-5p能促进胰腺癌细胞增殖及抗凋亡能力。     

miR-101在胰腺癌组织中的表达以及对胰腺癌细胞ASPC-1增殖的影响

目的 观察miR-101在胰腺癌组织中的表达,探讨其对胰腺癌细胞增殖的影响.方法 采用实时定量PCR方法 检测miR-101在胰腺癌组织、癌旁组织和胰腺癌细胞株ASPC-1中的表达.通过基因重组技术构建miR-101的表达载体peGFPc1-miR-101,应用脂质体将其转染到ASPC-1细胞,荧光显微镜检测转染效率;实时定量PCR检测转染细胞miR-101的表达水平,以癌旁正常胰腺组织为1,折算成相对倍数;MTT法检测转染细胞的增殖率.利用在线软件targetScan预测miRNA可能的靶基因.结果 miR-101在胰腺癌组织和胰腺癌细胞株ASPC-1中相对表达量分别为55%和39%,较癌旁正常胰腺组织显著降低(P＜0.01).peGFPc1-miR-101转染ASPC-1细胞后,miR-101表达增加,为对照组的19.8倍(P＜0.01),癌细胞增殖率明显降低,最低仅为原代细胞的26%(P＜0.01).EZH2基因是miR-101影响胰腺癌细胞增殖活力的可能靶基因.结论胰腺癌组织miR-101低表达,它可能通过抑制EZH2的表达调控细胞的增殖.     

miR-155在肝细胞癌中的表达及对肝癌细胞增殖的影响

目的:检测微小RNA-155(miR-155)在肝癌组织中的表达并分析其对肝癌细胞增殖和细胞凋亡的影响.方法:采用TagMan MGB探针法荧光定量P C R分析42例原发性肝癌及对应的癌旁组织miR-155的表达;利用miR-155反义寡核苷酸(ASO-miR-155)降低肝癌细胞HepG2和SMMC7721中miR-155的表达;利用MTT比色法检测肝癌细胞增殖的变化,并通过流式细胞技术检测肝癌细胞早期凋亡情况.结果:42例肝癌及癌旁组织标本中,miR-155在52%(22/42)肝癌组织中的表达明显高于癌旁组织(P<0.05);利用脂质体将ASO-miR-155转染肝癌细胞HepG2和SMMC7721后,miR-155的表达明显降低,肝癌细胞HepG2和SMMC7721生长受到明显抑制;并且细胞的早期凋亡明显增加.结论:miR-155在肝癌组织中过表达,降低其表达能明显抑制肝癌细胞的生长并诱导细胞早期凋亡,miR-155有可能成为肝癌治疗的新靶点.     

反义端粒酶寡核苷酸对胰腺癌细胞株生长的体外抑制作用

目的探讨反义端粒酶寡核苷酸片段对胰腺癌细胞株端粒酶活性及细胞株生长的抑制作用.方法人工合成端粒酶反义寡核苷酸片段,电转移法将反义端粒酶序列导入胰腺癌细胞株Aspc-1、Bxpc-3中,RT-PCR-ELISA法检测端粒酶活性,MTT法检测细胞活性.结果 Aspc-1、Bxpc-3细胞株的端粒酶活性分别由0.551±0.013、0.540±0.022降低至0.222±0.021、0.224±0.024,两株细胞株的A580值分别由0.420±0.006、0.322±0.013降低至0.169±0.025、0.129±0.026,P＜0.01;细胞生长显著受抑.结论反义端粒酶寡核苷酸片段具有抑制胰腺癌端粒酶活性的作用,并抑制胰腺癌细胞的生长.     

人胰腺癌组织中微RNA-134-5p的异常表达及其作用研究

目的:研究微RNA-134-5p(microRNA-134-5p,miR-134-5p)在人胰腺癌组织中的异常表达及其对癌细胞生物学行为、RAN基因表达的影响。方法:于我院消化科、普外科收集30例确诊胰腺癌患者,以癌旁正常组织为对照,采用TaqMan实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,q RT-PCR)方法检测胰腺癌组织中miR-134-5p的异常表达情况;体外实验采用蛋白质印迹、双荧光素酶实验证实RAN基因是miR-134-5p的直接靶基因,流式细胞仪检测miR-134-5p对人胰腺癌细胞凋亡的影响,细胞计数试剂盒(cell counting kit-8,CCK8)法和Transwell小室试验检测miR-134-5p对人胰腺癌细胞增殖、迁移能力的影响。结果:与癌旁正常胰腺组织相比,人胰腺癌组织中miR-134-5p的表达水平明显降低; RAN基因为miR-134-5p的直接靶基因;瞬时转染增高miR-134-5p的表达可促进人胰腺癌细胞的凋亡,抑制其增殖和迁移能力;瞬时转染降低miR-134-5p的表达则获得相反的效果。结论:人胰腺癌组织中miR-134-5p的表达水平较正常胰腺组织明显降低,miR-134-5p可促进胰腺癌细胞的凋亡、抑制其增殖和迁移、抑制RAN的表达从而发挥抗肿瘤作用。     

异常表达的miRNA对胰腺癌细胞增殖和凋亡的影响

下调miR-221改变细胞周期,增加G0/G1期细胞,减少S期细胞,但对细胞活性无明显影响;miR-221和miR-21共同下调后,细胞活力则明显下降.其他miR转染对细胞生长和凋亡的影响不明显.结论 部分在胰腺癌中异常表达的miRNA对胰腺癌细胞的生长有一定影响,选用一定组合的联合转染,对细胞生长和凋亡的影响比单独转染时加强.     

miR-346/DKK3信号轴在结肠癌中的细胞增殖的调控

目的研究miR-346/DKK3信号轴在结肠癌中的调控作用和机制.方法 RT-PCR实验检测miR-346在正常结肠上皮细胞和结肠癌细胞中的表达水平,随后检测其在结肠癌组织和对应的癌旁组织中的表达水平. MTT实验检测miR-346对结肠癌细胞增殖能力的影响.流式细胞周期检测miR-346对结肠癌细胞周期的影响.双荧光素酶报告基因实验验证miR-346和DKK3之间的结合关系.利用siR NA和pcD NA-DKK3转染结肠癌细胞研究DKK3对结肠癌细胞功能的影响.结果 miR-346在结肠癌细胞中的表达水平显著上调.过表达miR-346后,结肠癌细胞的增殖能力变强, G1期细胞比例降低, S期和G2/M期细胞比例增加.双荧光素酶报告基因实验显示miR-346能够和DKK3直接结合.转染siRNA抑制DKK3的表达后,结肠癌细胞增殖能力变强, G1期细胞比例降低, S期和G2/M期细胞比例增加.过表达DKK3后,能够部分抵消miR-346对结肠癌细胞的促增殖作用.结论 miR-346通过靶向结合并抑制DKK3进而促进结肠癌细胞的增殖.     

Hsa-miR-93在肝癌中的作用机制研究

目的观察hsa-miR-93对肝癌细胞生长和细胞周期的影响，以分析其在肝癌中的作用机制。方法用real time RT-PCR法分析hsa-miR-93在人肝癌和癌旁肝组织的表达情况。此后构建hsa-miR-93过表达载体，同时合成二甲氧修饰的hsa-miR-93的反义核苷酸序列，二者转染肝癌细胞株观察它们对肝癌细胞生长及细胞周期的影响，分别以空载体和无关寡核苷酸序列为对照。转染后CCK-8法检测肝癌细胞生长情况，通过流式细胞仪分析细胞周期。结果hsa-miR-93在肝癌组织中表达明显高于癌旁肝组织。hsa-miR-93过表达载体可以促进肝癌细胞生长，促进细胞周期的G1／S期转换，而hsa-miR-93反义序列抑制肝癌细胞生长，使肝癌细胞阻滞于G1期（P〈0．05）。结论hsa-miR-93通过促进细胞周期的G1／S期转换而促进肝癌细胞生长，提示hsa-miR-93可能是肝癌的发病机制中一个重要分子。     

TSLC1和MPP3在胰腺癌中的表达及其意义

目的:探讨TSLC1和MPP3两种蛋白在胰腺癌中的表达和临床病理意义.方法:采用免疫组织化学S-P法检测37例胰腺癌组织、12例胰腺炎组织和10例正常胰腺组织中TSLC1和MPP3两种蛋白的表达.结果:TSLC1、MPP3蛋白在胰腺癌组织中的阳性表达率均明显低于在正常胰腺组织和胰腺炎组织中的表达(21.62%vs70.00%,75.00%;27.03%vs80.00%,66.67%,P<0.05或0.01).TSLC1和MPP3蛋白的异常表达均与胰腺癌的分化程度、淋巴结转移和TNM分期相关(均P<0.05),而与患者的性别、年龄、部位和病理分型无关.在37例胰腺癌中TSLC1与MPP3蛋白表达呈显著正相关(rs=0.715,P<0.01).结论:胰腺癌中存在TSLC1和MPP3基因的失活和蛋白表达下调,两者可能通过TSLC1-MPP3级联反应共同参与胰腺癌的发生、发展和转移.     

MicroRNA expression pattern in intraductal papillary mucinous neoplasm

Background/Aims: Intraductal papillary mucinous neoplasms (IPMN) are precursor lesions of fatal pancreatic cancer. Physiological function of microRNA is to regulate the stability and translation of mRNA. The aberrant microRNA expression is commonly observed in many cancers. The aim of this study was to analyze the expression pattern of microRNA in IPMN and evaluate the role of the microRNA.Methods: Using two paraffin-embedded IPMN tissues, microRNA expression of normal tissue, IPMN adenoma and carcinoma were compared by cDNA-mediated annealing, selection, extension and ligation microarray assay. Using real time PCR, expression levels of aberrantly up-regulated microRNAs were assessed in another 20 IPMNs, four pancreatic cancer cell lines (Panc1, MiaPaCa-2, XPA-3, BxPC-3) and immortalized pancreatic ductal cell line (HPNE). Effect of suppressing highly over-expressed two microRNAs in pancreatic cancer cell lines with anti-microRNA inhibitors were evaluated using CCK-8 assay.Results: Among aberrantly expressed 122 microRNAs in IPMN, miR-552, miR-25*, miR-183, miR-1300, miR-196a, miR-182*, and miR-30c-1* were consistently increased more than 3-fold. On average, miR-196a and miR-183 increased 10,824 folds and 26,519 folds in four pancreatic cancer cell lines compared with HPNE. These two microRNAs were also over-expressed in 20 IPMNs compared with HPNE. After applying anti-miRNA inhibitors, cell survival of four pancreatic cancer cell lines decreased by 24.5% with anti-miR-196a and by 14.2% with anti-miR-183 on average.Conclusions: Aberrant expression of 122 microRNAs was observed in IPMN. Two microRNAs, miR-196a and miR-183-increased in IPMN and pancreatic cancer cell lines compared with immortalized dancreatic ductal cell line. The inhibitions of these microRNAs repressed cell proliferation of pancreatic cancer cell lines. (Korean J Gastroenterol 2011;58:190-200).     

miR-224 Regulates the Aggressiveness of Hepatoma Cells Through the IL-6/STAT3/SMAD4 Pathway

Background: Previous studies have shown that miR-224 regulates the progression of liver cancer. The aim of this study was to investigate the underlying mechanisms.Methods: The miR-224, p-STAT3 and SMAD4 expression levels were checked with tissue or/and serum samples of HCC patients by qRT-PCR or IHC methods. The regulatory role of IL-6 in p-STAT3 and SMAD4 was investigated by Western-blot. The targeted gene of miR-224 was verified by both Western-blot and luciferase reporter assay. Furthermore, the carcinogenesis of miR-224 in HCC was investigated by cell experiments in vitro and mouse xenograft model and in vivo imaging in vivo.Results: It was found miR-224 was elevated in both tissue and serum of HCC patients. The p-STAT3 expression was higher but the SMAD4 was lower in the HCC tumor tissues. Moreover, IL-6 can induce the p-STAT3/STAT3 and miR-224 expression in HCC cells and STAT3 played the bridge role between IL-6 and miR-224. Target gene studies found miR-224 targeted the 3ʹUTR of SMAD4. Finally, the promoting roles of miR-224in the growth, proliferation, invasion and migration of HCC were discovered by in vitro and in vivo studies.Conclusion: It implies that miR-224 may potentially represent a new target for developing novel anti-HCC therapeutics.     

胰腺癌组织c-MET表达与循环miR-34a、miR-449水平的相关性

目的 探讨胰腺癌组织织间质表皮转化因子(c-MET)的表达与循环miR-34a、miR-449水平的相关性及其临床意义。方法 收集2015年3月至2017年3月间嘉兴医学院附属第二医院行手术治疗并经病理证实的41例胰腺癌患者的临床资料。通过免疫组织化学染色检测胰腺癌组织及其匹配的癌旁正常胰腺组c-MET表达,根据测定结果将患者分为c-MET阳性组与c-MET阴性组。采集患者手术前和手术后3个月外周血,应用荧光定量PCR法检测循环miR-34a和miR-449水平。分析癌组织c-MET表达与患者临床病理参数、预后及循环miR-34a、miR-449水平的关系。分析循环miR-34a和miR-449水平对胰腺癌TNM分期、淋巴结转移和预后的评估价值。结果 胰腺癌组织c-MET阳性率明显高于癌旁正常组织(63.4%比24.4%),差异有统计学意义(P<0.05)。与c-MET阴性患者比较,c-MET阳性患者TNMⅢ/Ⅳ期者居多(73.1%比33.4%),淋巴结转移率高(76.9%比46.7%),生存时间短(29.5个月比35.0个月),生存率低(38.5%比53.3%),差异均有统计学意义(P值均<0.05)。术前c-MET阳性患者循环miR-34a、miR-449水平明显低于c-MET阴性患者(0.228±0.068比0.524±0.106、0.252±0.063比0.432±0.094,P<0.05),术后c-MET阳性患者循环miR-449水平仍明显低于c-MET阴性患者(0.414±0.088比0.512±0.114,P<0.05),但两组间miR-34a水平的差异无统计学意义。术前循环miR-34a、miR-449水平对胰腺癌TNM分期、淋巴结转移及预后有预测价值(P<0.05)。结论 miR-34a、miR-449靶向结合胰腺癌组织c-MET,有可能成为潜在的胰腺癌标志物。     

肿瘤-睾丸抗原SSX在胰腺癌中的表达及其生物意义

目的探讨肿瘤-睾丸抗原(CTA)SSX及其亚型在胰腺癌的表达情况,为胰腺癌的免疫治疗提供实验依据。方法应用免疫组织化学S-P法对52例胰腺癌组织及癌旁组织中SSX的表达情况进行检测,并应用逆转录聚合酶链反应(RT-PCR)方法检测SSX的亚型SSX-2、SSX-4的表达情况。结果 SSX在胰腺癌的阳性表达率为48.08%;SSX的阳性表达与患者的年龄、性别、病变部位、分化程度均无明显相关关系(P>0.05);SSX的表达与肿瘤的浸润转移呈负相关(r=-0.306,P=0.028)。在SSX的亚型中,SSX-2表达率较高。结论胰腺癌中SSX的高表达与肿瘤的转移呈明显的负相关关系;SSX的阳性表达在胰腺癌中呈现簇生现象,SSX-2的表达较高。     

Initial study of microRNA expression profiles of colonic cancer without lymph node metastasis

OBJECTIVE: To investigate the difference of microRNA expression profiles between colonic cancer without lymph node metastasis and the para-cancerous control, to identify the specific microRNA associated with the cancer and to predict the carcinogenetic mechanism of microRNA on the basis of these results. METHODS: The microRNA (miRNA) were extracted and isolated from six specimens, including colonic cancerous and para-cancerous ones, all of which were confirmed to be without lymph node metastasis. Agilent microRNA microarrays consisting of 723 probes were used for screening the expression differences of microRNA. Data were analyzed using feature extraction software. The expression level of differentially expressed microRNA using quantitative real-time polymerase chain reaction (RT-PCR) was validated. RESULTS: A total of 14 miRNAs were found to be associated with colonic cancer, in which the expression of miR-106b, miR-135b, miR-18a, miR-18b, miR-196b, miR-19a, miR-224, miR-335, miR-424, miR-20a*, miR-301b and miR-374a were up-regulated and the expression of miR-378 and miR-378* were downregulated in colonic cancer tissues, compared with the para-cancerous control. The expression level of miR-18a and miR-135b were validated in accordance with the results of RT-PCR. CONCLUSION: The miRNAs are differentially expressed between colonic tumor tissues and para-cancerous tissues. Many of these miRNAs are expected to participate in the process of multiple tumorigenesis. These miRNAs could play an important role in the carcinogenesis of colon. These results provide new insights in human colorectal cancer genesis.     

血小板衍化内皮细胞生长因子在胰腺癌组织中的表达及其临床意义

目的:观察血小板衍化内皮细胞生长因子(PD-ECGF)在胰腺癌组织中的表达,探讨其临床意义.方法:利用Elivison免疫组织化学法检测36例手术切除的胰腺癌及21例癌旁正常胰腺组织,以及胃癌、食管癌、肝癌、结肠癌、肺癌及乳腺癌组织各10例中PD-ECGF的表达.分析PD-ECGF与胰腺癌大小、分化程度及淋巴结转移的相关性.结果:PD-ECGF在胰腺癌组织中的表达阳性率显著高于在癌旁正常胰腺组织(88.9% vs 28.6%,P<0.01).PD-ECGF在结肠癌、肝癌、乳腺癌、食管癌、胃癌及肺癌组织中的表达阳性率为60%、70%、80%、90%、90%及80%.Ⅱ-Ⅳ期胰腺癌组织中PD-ECGF的表达阳性率显著高于Ⅰ期胰腺癌组织(100% vs 75%,P<0.05).结论:PD-ECGF为一种非特异性的肿瘤相关因子,其可能与胰腺癌的病程进展有关.     

AKT2在胰腺癌组织中的表达及意义

目的 探讨胰腺癌组织中蛋白激酶B（AKT）2的表达及其在胰腺癌发生、发展中的作用。方法 采用免疫组化SABC法检测63例胰腺癌组织和23例胰腺良性病变组织中AKT2的表达,分析其与胰腺癌临床病理因素的关系。结果 AKT2在胰腺癌组织中的阳性表达率为39．7％（25／63）,明显高于胰腺良性病变组织的13％（3／23）,P〈0．05。AKT2表达与胰腺癌组织学分级、淋巴结转移、TNM分期有关（P〈0．05）。结论 AKT2在胰腺癌组织中表达增高,可能在胰腺癌发生、发展、转移中起重要作用。