miR-181b在结肠炎相关结肠癌发生过程中的表达

背景:miR-181b与多种肿瘤的形成相关,但其在结肠炎相关结肠癌中的表达情况尚不明确。目的:探索miR-181b在结肠炎相关结肠癌发生过程中的变化,并初步筛选其下游可能的靶基因。方法:联合使用氧化偶氮甲烷(AOM)和葡聚糖硫酸钠(DSS)制备结肠炎相关结肠癌小鼠模型。以实时荧光定量PCR(qP CR)检测不同时期小鼠结肠组织miR-181b表达。利用全基因组表达谱芯片结合miRNA靶基因预测软件TargetS can、PicTar和miRDB初步预测miR-181b的下游靶基因,并以qPCR法检测结肠炎相关结肠癌小鼠中靶基因的表达。结果:miR-181b表达在结肠炎相关结肠癌形成过程中逐渐升高,但单独使用AOM或DSS均不会改变miR-181b水平。结合全基因组表达谱芯片和miRNA靶基因预测软件初步筛选出miR-181b可能的下游靶基因为Ipmk、E2f5、Klf6、Prkcd、Bai3和Hic2,qPCR法显示Ipmk、Prkcd、Bai3、Hic2表达明显低于对照组。结论:miR-181b表达随结肠炎相关结肠癌的发展而升高,该变化与单纯慢性炎症无关。     

转移性结肠癌癌旁组织miRNA谱研究

近年来,对miRNA研究不断深入,miRNA表达谱芯片应运而生,其凭借高通量、快速检测miRNA表达水平的优点,迅速成为了研究肿瘤发病机制、早期诊断、病理特征、预测转移及评估预后的全新方法.本研究利用miRNA表达谱芯片技术对比分析结肠癌伴淋巴结转移与无转移患者癌旁组织miRNA表达的差异,并对差异表达的miRNA行实时荧光定量PCR验证,以期在结肠癌的癌旁组织中发现与淋巴结转移相关的miRNA,从中找到特征性的miRNA标记及治疗靶分子.     

miR-106a在大肠癌中的表达及其与肠癌细胞侵袭的关系

目的:分析miR-106a在肠癌中的表达并研究其在肠癌细胞侵袭中的作用.方法:提取52例肠癌手术标本及其癌旁组织中总RNA,PCR法检测miR-106a在肠癌及其癌旁正常组织中的表达量,分析其与临床病理特征关系,并进一步采用Transwell侵袭小室检测其在大肠癌细胞侵袭中的作用.结果:经2-△△Ct计算,肠癌组织miR-106a表达量为2.50(0.017-14.269),其中73%(38/52)大肠癌组织miR-106a高表达(Z=-3.748,P=0.000),miR-106a的高表达与TNM分期(t=2.813,P=0.003)和淋巴结转移(t=-2.635,P=0.008)有关,TNM分期较高和有淋巴结转移者,miR-106a表达较高.Transwell侵袭小室检测表明,过表达miR-106a可促进肠癌细胞侵袭作用(均P=0.000).结论:miR-106a表达上调可能与大肠癌的发生及其转移相关.     

MicroRNA在直肠癌中的表达谱

目的:研究直肠癌microRNA(miRNA)表达谱,以期发现直肠癌发生过程和正常直肠黏膜组织的miRNA差异表达谱,并理论上预测可能受其调控的靶基因.方法:选择2011-01/03于中国人民解放军第306医院肠镜检查的进展期直肠癌患者,对直肠癌及正常组织进行miRNA芯片检测,筛选直肠癌、正常直肠组织差异表达的miRNA,并运用Gomir分析软件筛选TargetScan、PicTar、miRanda3个预测软件同时预测出的靶基因.结果:选择直肠癌组织、正常直肠黏膜组织标本各2例,通过Affymetrix miRNAs芯片检测,发现直肠腺癌与正常直肠黏膜相比,上调的miRNA为miR-15a、miR-18a、miR-18a*、miR-21、miR-24-2*、miR-27a等37种,下调的为miR-139-5p、miR-139-3p、miR-215、miR-200b、miR-133a、miR-150等18种.依据Ratio值,选择其中最高者miR-31和最低者miR-375进行预测其靶基因.预测ZFHX4可能为miR-375的靶基因,预测BACH2、FZD3等可能为miR-31的靶基因.结论:直肠癌和正常直肠黏膜相比有其特异的miRNA表达谱,miRNA在直肠癌的发生中可能起到重要作用,受其调控的靶基因及作用方式值得进一步研究.     

miR-135b、LZTS1、β-catenin在胰腺癌中的表达及临床意义

目的:探讨miR-135b、LZTS1及β-catenin在胰腺癌中的表达意义及三者之间的相关性.方法:分别采用锁定核酸原位杂交技术(LNA-ISH)和免疫组织化学法检测miR-135b、LZTS1和β-catenin蛋白在70例胰腺癌及相应癌旁组织中的表达情况.结果:miR-135b在胰腺癌中的表达率高于癌旁正常组织(71.4%vs 42.9%,P=0.001),LZTS1、β-catenin蛋白在胰腺癌中的表达率均低于癌旁正常组织(34.3%vs 68.6%、34.3%vs 74.3%,P0.05).在胰腺癌组织中miR-135b与LZTS1的表达水平呈负相关(r=-0.61,P0.05),与β-catenin无相关性(r=0.06,P0.05);LZTS1与β-catenin呈正相关(r=0.37,P0.05),且miR-135b、LZTS1和β-catenin的阳性表达水平与胰腺癌的临床分期及淋巴结转移密切相关(P0.05),与胰腺癌患者年龄、性别、肿瘤部位及组织学分级无关(P0.05).结论:miR-135b在胰腺癌中表达升高,LZTS1与β-catenin在胰腺癌中表达减少,并且表达升高的miR135b可能通过下调LZTS1基因表达而参与了胰腺癌的发生发展.     

miR-133b、Bcl-w在老年食管鳞癌中表达及相关机制

目的 探讨microRNA(miR)-133b、B细胞淋巴瘤(Bcl)-w在老年食管鳞状细胞癌中的表达及对食管鳞状细胞癌细胞的影响。方法 使用实时荧光定量聚合酶链反应(qRT-PCR)检测30例老年患者食管鳞状细胞癌组织及其对应癌旁组织中miR-133b及Bcl-w mRNA表达。将miR-133b mimics及阴性对照转染至食管鳞状细胞癌ECA109细胞株，检测转染效率。检测转染后ECA109细胞的增殖和凋亡情况，使用TargetScan和miRDB软件预测miR-133b的靶基因，并通过双荧光素酶报告实验方法验证，Western印迹检测靶基因Bcl-w蛋白表达。结果 食管鳞状细胞癌中miR-133b mRNA表达量明显低于癌旁组织(P<0.001),而食管鳞状细胞癌中Bcl-w蛋白表达量明显高于癌旁组织(P<0.001),miR-133b与Bcl-w表达呈负相关(r=-0.792,P<0.01)。miR-133b mimics转染能明显上调ECA109细胞中的miR-133b水平，并明显降低其增殖能力，明显促进其凋亡(P<0.001)。miR-133b与B...     

胰腺导管腺癌组织miR-196b、TGFβR1表达变化及其意义

目的 观察胰腺导管腺癌组织微小RNA-196b(miR-196b)、转化生长因子β受体1(TGFβR1)表达变化,并探讨其临床意义.方法 选择胰腺导管腺癌患者88例,取手术切除的胰腺癌组织及其配对的癌旁正常组织,采用RT-qPCR法检测miR-196b、TGFβR1表达.比较胰腺癌组织与癌旁正常组织miR-196b、T...     

胰腺导管腺癌microRNA表达谱的研究

目的 应用microRNA( miRNA)高通量生物芯片筛选胰腺导管腺癌及癌旁组织差异表达的miRNA,分析其相关的靶基因.方法 收集9例新鲜的胰腺导管腺癌和3例癌旁组织,运用标记713个miRNA的Agilent miRNA生物芯片筛选胰腺导管腺癌差异表达的miRNA,应用荧光实时定量PCR方法验证表达上调的miRNA.采用TargetScan 5.1和miRandaV5分析软件分析差异表达miRNAs的靶基因.结果 miRNA芯片筛选出11个胰腺导管腺癌相关的差异表达的miRNA,其中miR-194*、miR-192*、miR-602、miR-194表达上调,miR-139-3p、miR-513a-5p、miR-630、miR-30c-1*、miR-887、miR-508-5p、miR-516a-5p表达下调.miR-192、miR-194及其同源体的表达在31例胰腺癌组织中得到验证.经软件分析,miR.192靶基因有ZEB2、CXCL-2、EEF1A1、ERCC3,miR-192*靶基因有DCC、SMAD4、FAS,miR-194靶基因有DACH1、IGSF11、PTPN2、RBBP4,miR-194*靶基因有CD40LG、CIDEB、FHL1.结论胰腺导管腺癌存在11个表达差异的miRNA,这些miRNA可能与胰腺导管腺癌的发生、发展有关.     

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。     

肝癌患者中microRNA-378与FOXO1基因表达的相关关系研究

目的研究microRNA-378a-3p(miRNA-378)与FOXO1基因在肝癌中的表达情况及两者表达的相关性。方法提取8例肝癌患者癌组织及其相对应癌旁组织标本的总RNA,采用实时荧光定量PCR分别检测miR-378和FOXO1基因的相对表达量,通过独立样本t检验比较癌及癌旁组织中miR-378、FOXO1基因表达的差异,并通过双变量相关分析探索癌组织中两者之间的相关关系。结果肝癌组织中miR-378和FOXO1基因的表达量均明显低于癌旁组织中的表达(P=0.0106和P=0.000623),两者相对表达量在肝癌组织中呈正相关关系(r=0.541,P=0.031)。结论肝癌组织中miR-378与FOXO1基因的表达较癌旁组织明显降低,miR-378与FOXO1基因的异常表达及其相互作用网络在肝癌发生发展中可能具有重要的意义。     

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.     

MicroRNA expression profile in non‐cancerous colonic tissue associated with lymph node metastasis of colon cancer

OBJECTIVE: To identify microRNA expression patterns associated with the lymph node metastasis of colon cancer. METHODS: MicroRNA were isolated from six frozen non‐cancerous surrounding colonic tissues derived from stage II–III colon cancer patients with (n = 3) and without (n = 3) lymph node metastasis. We compared the microRNA expression profiles of the six non‐cancerous colonic tissues from two colon cancer patient groups; those with confirmed lymph node metastasis, termed the lymph node positive group, and those without detectable lymph node metastasis, termed the lymph node negative group. MicroRNA expression was analyzed with Agilent microarrays containing 723 human microRNA probes. We validated the expression level of differentially expressed microRNA using quantitative real‐time PCR analysis. RESULTS: Two microRNA (hsa‐miR‐129*, hsa‐miR‐137) were differentially expressed in the lymph node positive group compared with the lymph node negative group. The expression level of hsa‐miR‐137 was quantified via quantitative real‐time PCR analysis for validation. Hsa‐miR‐137 expression was significantly upregulated nearly 6.6‐fold in lymph node positive specimens (P = 0.036). The quantitative real‐time PCR result correlates with the microarray finding. CONCLUSION: The non‐cancerous colonic tissues from colon cancer patients with lymph node metastasis have a significantly different microRNA expression profile compared to that from colon cancer patients without lymph node metastasis. The differentially expressed microRNA could have relevance to the lymph node metastasis of colon cancer and may provide a simple profiling method to assist in identifying patients with lymph node metastasis. Besides, these data might offer new ideas for preventing and controlling lymphatic metastasis in colon cancer.     

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).     

Expression of a novel apoptosis inhibitor-survivin in colorectal carcinoma

AIM: To investigate the role of survivin expression in the pathogenesis of colorectal carcinoma.METHODS: Immunohistochemistry S-P method and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) were used to detect the expression of survivin and apoptotic cell in situ in colorectal cancerous tissues, para-cancerous tissues and normal tissues of 48 cases of colorectal carcinoma.RESULTS: The survivin positive unit (PU) was higher in cancerous tissues (38.76±5.14)than in para-cancerous (25.17±7.26) or normal tissues (0.57±0.03) (P<0.05).The apoptosis index (AI) of para-cancerous tissues was(7.51±2.63%) higher than cancerous tissues (4.65±1.76%).The expression of survivin was associated with pathological grade, lymph node metastasis and Dukes stage of colorectal carcinoma.CONCLUSION: Survivin expression may play an important role in carcinogenesis of colorectal carcinoma and may be associated with malignant biological behaviors of colorectal carcinoma.     

结肠癌相关miRNA的转化机制

结肠癌的发生是多阶段、多步骤的病变过程,是从良性息肉-浸润性腺癌-癌远处转移的演变过程.这些病理变化与编码蛋白质的原癌基因和肿瘤抑制基因的异常激活或失活有关.但最近关于微小RNA(microRNAs,miRNAs)的研究改变了我们对非编码蛋白质基因在肿瘤发生中的作用的理解.在结肠癌组织中多种miRNAs的表达水平不同,...     

粪便microRNAs检测用于胰腺癌筛查诊断的价值评价

目的 检测胰腺癌患者粪便microRNAs,评价其诊断价值.方法 收集29例胰腺癌患者、22例慢性胰腺炎(CP)患者以及13例健康志愿者的粪便标本,抽提粪便总RNA,应用实时定量PCR法检测各组样本miR-21、miR-155、miR-181a、miR-181b、miR-196a、miR-210的表达量,以miR-16作为内参基因.应用接受者操作特征(ROC)曲线(AUC)评估microRNAs对胰腺癌的诊断价值.结果 粪便总RNA抽提及microRNAs检测方法具有稳定及可重复性.胰腺癌组miR-181b、miR-196a、miR-210的表达量分别为2.22±0.64、2.78±0.14、5.55±0.38;CP组为1.42±0.39、3.88±0.85、5.39±0.69;对照组为0.32±0.40、1.14±0.98、4.23±0.99.胰腺癌组和CP组均较对照组显著增加(P值均＜0.05);而胰腺癌组与CP组间无显著差异.胰腺癌组对对照组的miR-181b AUC为0.745(95%CI 0.597～0.894),诊断胰腺癌的敏感性和特异性分别为84.6%和51.7%;miR-210的AUC为0.772(95%CI 0.629～0.914),对胰腺癌的诊断敏感性和特异性分别为84.6%和65.5%.两组比较差异均有统计学意义(P值均＜0.05).miR-196a对胰腺癌无诊断意义,但胰腺癌患者粪便miR-196a的表达与肿瘤直径相关(r=0.516,P=0.041).结论 粪便RNA的抽提和microRNAs检测为无创性,且具有可重复性.miR-181b和miR-210在胰腺癌患者粪便中的表达增高,有可能是胰腺癌潜在的分子标志物.     

Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells

Accumulating evidence suggests a role for microRNAs in human carcinogenesis as novel types of tumor suppressors or oncogenes. However, their precise biological role remains largely elusive. In the present study, we aimed to identify microRNA species involved in the regulation of cell proliferation. Using quantitative RT-PCR analysis, we demonstrated that miR-34a was highly up-regulated in a human colon cancer cell line, HCT 116, treated with a DNA-damaging agent, adriamycin. Transient introduction of miR-34a into two human colon cancer cell lines, HCT 116 and RKO, caused complete suppression of cell proliferation and induced senescence-like phenotypes. Moreover, miR-34a also suppressed in vivo growth of HCT 116 and RKO cells in tumors in mice when complexed and administered with atelocollagen for drug delivery. Gene-expression microarray and immunoblot analyses revealed down-regulation of the E2F pathway by miR-34a introduction. Up-regulation of the p53 pathway was also observed. Furthermore, 9 of 25 human colon cancers (36%) showed decreased expression of miR-34a compared with counterpart normal tissues. Our results provide evidence that miR-34a functions as a potent suppressor of cell proliferation through modulation of the E2F signaling pathway. Abrogation of miR-34a function could contribute to aberrant cell proliferation, leading to colon cancer development.     

Differential expression of microRNA species in human gastric cancer versus non-tumorous tissues

Background and Aim:  MicroRNAs (miRNAs) play important roles in carcinogenesis. The global miRNA expression profile of gastric cancer has not been reported. The purpose of the present study was to determine the miRNA expression profile of gastric cancer.
Methods:  Total RNA were first extracted from primary gastric cancer tissues and adjacent non-tumorous tissues and then small isolated RNAs (< 300 nt) were 3'-extended with a poly(A) tail. Hybridization was carried out on a μParaflo™ microfluidic chip (LC Sciences, Houston, TX, USA). After hybridization detection by fluorescence labeling using tag-specific Cy3 and Cy5 dyes, hybridization images were collected using a laser scanner and digitized using Array-Pro image analysis software (Media Cybernetics, Silver Spring, MD, USA). To validate the results and investigate the biological meaning of differential expressed miRNAs, immunohistochemistry was used to detect the differential expression of target genes.
Results:  The most highly expressed miRNAs in non-tumorous tissues were miR-768-3p, miR-139-5p, miR-378, miR-31, miR-195, miR-497 and miR-133b. Three of them, miR-139-5p, miR-497 and miR-768-3p, were first found in non-tumorous tissues. The most highly expressed miRNAs in gastric cancer tissues were miR-20b, miR-20a, miR-17, miR-106a, miR-18a, miR-21, miR-106b, miR-18b, miR-421, miR-340*, miR-19a and miR-658. Among them, miR-340*, miR-421 and miR-658 were first found highly expressed in cancer cells. The expression of some target genes (such as Rb and PTEN ) in cancer tissues was found to be decreased.
Conclusion:  To our knowledge, this is the first report about these miRNAs associated with gastric cancer. This new information may suggest the potential roles of these miRNAs in the diagnosis of gastric cancer.     

Expression of a novel apoptosis inhibitor-survivin in colorectal carcinoma

AIM: To investigate the role of survivin expression in the pathogenesis of colorectal carcinoma. METHODS: Immunohistochemistry S-P method and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) were used to detect the expression of survivin and apoptotic cell in situ in colorectal cancerous tissues, para-cancerous tissues and normal tissues of 48 cases of colorectal carcinoma. RESULTS: The survivin positive unit (PU) was higher in cancerous tissues (38.76±5.14) than in para-cancerous (25.17±7.26) or normal tissues (0.57±0.03) (P<0.05). The apoptosis index (AI) of para-cancerous tissues was (7.51±2.63%) higher than cancerous tissues (4.65±1.76%). The expression of survivin was associated with pathological grade, lymph node metastasis and Dukes stage of colorectal carcinoma. CONCLUSION: Survivin expression may play an important role in carcinogenesis of colorectal carcinoma and may be associated with malignant biological behaviors of colorectal carcinoma.