应用SYBR实时荧光定量Real—TimePCR法检测人宫颈癌MCPH1 mRNA表达

目的 应用SYBR实时荧光定量RT-PCR法检测MCPH1/BRIT1 mRNA的表达.方法 提取人宫颈癌总RNA,经逆转录PCR获得靶基因（MCPH1）及管家基因（GAPDH）的CDNA,采用SYBR Green 荧光实时定量法检测,以GAPDH基因作为内参,计算各组MCPH1 mRNA的相对表达量.结果 在31例宫颈癌标本中,MCPH1基因mRNA的表达,19例癌比正常低,1 例癌比正常高,其余标本无统计学意义;6例癌比癌旁低,1例癌比癌旁高,其余无统计学意义.结论 利用SYBR实时荧光定量RT-PCR检测出人宫颈癌中MCPH1基因mRNA的表达下调,为进一步研究MCPH1在宫颈癌中的作用及功能奠定了基础.     

UCA1及其剪接变异体与膀胱癌

尿路上皮癌胚抗原1 (urothelial carcinoma antigen 1,UCA1) 在人膀胱移行细胞癌细胞系 BLZ-211 中包含3个剪接变异体：UCA1、UCA1a 和 UCA1b. 我们以往的研究表明, UCA1、UCA1a 均属于长非编码 RNA (long non-coding RNA,lncRNA),它们之间具有一段长 1 265 bp 的共同序列. 组织表达谱分析表明,它们具有相似的组织表达模式,提示它们可能与胚胎发育和膀胱癌发生发展密切相关. 异位表达UCA1基因、UCA1a 基因均可以促进人膀胱癌细胞生长,增强细胞的恶性表型,使其体外增殖、迁移、侵袭、抗凋亡能力明显增加,裸鼠致瘤能力明显增强,表明它们在膀胱癌的发生发展中均起到了重要的促进作用. 本文将从基因结构、组织表达谱及机制功能等不同角度系统地阐述UCA1基因及其剪接变异体在膀胱癌中的研究现状与进展.     

新基因BCAPP2Ac在膀胱癌中的表达 及其对膀胱癌细胞增殖的影响

为探讨膀胱癌相关蛋白磷酸酶2A催化亚单位（BCAPP2Ac）新基因在膀胱癌组织中的表达及其对膀胱癌细胞增殖的影响,通过合成抗原多肽免疫家兔获得抗BCAPP2Ac多克隆抗体及通过慢病毒感染方式获得稳定表达BCAPP2Ac的膀胱癌细胞.采用实时PCR及免疫组化染色方法检测BCAPP2Ac mRNA和蛋白在膀胱癌组织、癌旁组织及其它多种肿瘤组织中的表达;用细胞增殖实验检测BCAPP2Ac对膀胱癌细胞增殖的影响.实时PCR及免疫组化结果显示,BCAPP2Ac mRNA及蛋白在膀胱癌组织较癌旁组织表达明显下调;过表达BCAPP2Ac能抑制膀胱癌EJ、T24细胞的增殖, 蛋白磷酸酶2A催化结构域缺失（△PP2Ac）能逆转BCAPP2Ac的增殖抑制作用.研究结果提示,新基因BCAPP2Ac能抑制膀胱癌细胞的增殖,PP2Ac结构域在其抑制细胞增殖作用中发挥重要作用.     

MKi67-siRNA可抑制QGY-7703细胞的生长

为了探讨MKI67在肝癌细胞发生发展中的作用,采用实时定量 PCR 方法检测人肝细胞癌 QGY 7703 细胞中MKI67 基因表达水平, 以及 MKI67在肝细胞癌组织和癌旁正常组织中的表达情况,设计并合成针对MKI67 的siRNA,利用脂质体转染法将其转入QGY-7703 细胞内,通过MTT和细胞集落形成实验观察MKI67-siRNA 对QGY-7703细胞生长活性和增殖能力的影响.实时定量PCR结果表明,MKI67在肝细胞癌组织中的表达水平明显高于癌旁正常组织（P< 0.01）. MTT和细胞集落形成实验结果显示,转染MKI67-siRNA 的QGY-7703细胞生长活性和集落形成率明显低于对照组（P< 0.01）.由此得出结论：MKI67 在肝癌细胞系QGY-7703细胞中的表达水平较高,且它在肝癌组织中的表达水平明显上调. 同时,MKI67-siRNA 可以有效抑制QGY-7703细胞的生长活性和增殖能力,提示MKI67可能与肝细胞癌的发生、发展相关.     

肿瘤微环境中ROS介导IgG表达对膀胱癌细胞增殖和侵袭能力的影响

摘要 目的：探讨肿瘤微环境（TME）中活性氧（ROS）介导免疫球蛋白G（IgG）表达对膀胱癌EJ细胞增殖、迁移和侵袭能力的影响。方法：临床收集的18例膀胱癌患者样本,通过Western blot法检测膀胱癌和癌旁正常组织样本中IgG表达量。利用免疫荧光染色（IF）技术分别对膀胱癌组织和癌旁正常组织中ROS和IgG分子进行共定位和相对定量分析。将活性氧清除剂N-乙酰基-L-半胱氨酸（NAC）加入膀胱癌细胞EJ中,实验分为3组：空白组（EJ细胞）、阴性对照组（EJ+PBS）、实验组（EJ+PBS+NAC）,10 mM NAC药物处理48小时后,运用DHE-ROS荧光探针技术和Western blot实验检测药物NAC对ROS和IgG相对表达水平的影响;通过克隆集落形成实验、划痕实验、Transwell实验检测去除ROS后对膀胱癌细胞增殖、迁移和侵袭的影响。结果：人体膀胱癌组织中ROS和IgG分子表达水平显著高于癌旁正常组织（P<0.001）;荧光显微镜显示膀胱肿瘤组织中正常膀胱尿路上皮细胞组织被肿瘤细胞严重破坏,结构紊乱不规则,IgG和ROS表达水平均升高,而癌旁组织膀胱尿路上皮组织的结构均匀规则;NAC药物处理EJ细胞后,与空白组和阴性对照组相比ROS和IgG表达显著降低,同时实验组细胞的增殖、迁移和侵袭能力明显下降（P<0.01）。结论：ROS和IgG在临床膀胱癌组织和体外膀胱癌细胞株EJ中均显著高表达,在肿瘤微环境中ROS通过调控IgG表达,从而促进膀胱癌细胞的增殖、迁移、侵袭。ROS和IgG可能成为膀胱癌早期诊断和生物治疗的临床新靶点。     

STAT3及下游基因Cyclin D1在膀胱癌中的表达及意义

目的 探讨STAT3及下游基因CyclinD1在膀胱癌中的表达及临床意义.为膀胱癌的基因治疗提供理论依据.方法 收集武汉大学人民医院病理科2004-2006年膀胱移行细胞癌存档蜡块50例,其中男性38例,女性12例,平均年龄50.3岁,另取5例癌旁组织做对照.标本均经HE染色,光镜观察诊断,按WHO病理分级标准,G1:30例,G2:15例,G3:5例;运用免疫组织化学染色方法检测STAT3和CyclinD1在以上各组中的表达.利用HPIAS-2000图像分析系统测定STAT3和CyclinD1在以上各组中表达的平均光密度和平均阳性面积率.结果 1.STAT3的表达:STAT3在膀胱移行细胞癌中呈高表达;STAT3在癌旁组织中呈低表达.膀胱移行细胞癌与癌旁组织比较,STAT3的表达差异有显著性(P＜0.05);2.CyclinD1的表达:CyclinD1在膀胱移行细胞癌中呈高表达;CyclinD1在癌旁组织中呈低表达.膀胱移行细胞癌与癌旁组织比较,CyclinD1的表达差异有显著性(P＜0.05).结论 1.STAT3可能参与了膀胱癌细胞的增殖分化、细胞周期等的调节;2.STAT3的活化促进了抗凋亡基因CyclinD1的表达;3.STAT3及其下游基因CyclinD1在膀胱癌的形成中起了一定作用.     

Cyclin D1在膀胱癌中的表达及意义

目的探讨细胞因子CyclinD1在膀胱癌中的表达及临床意义。方法收集武汉大学人民医院病理科2000-2006年有完整临床和病理资料的膀胱癌存档蜡块50例和5例癌旁组织,采用免疫组织化学S-P法检测50例膀胱癌和5例癌旁组织中CyclinD1的表达水平,分析了CyclinD1与临床分期,病理分级的关系。采用HPIAS-1000高清晰度彩色病理图文报告管理系统,对CyclinD1的表达进行定量分析,并用SPSS13.0软件对各组免疫组织化学反应阳性颗粒的平均光密度、阳性面积率做单因素方差分析和SNK(q)检验。结果 CyclinD1在膀胱癌中呈高表达,癌旁组织中呈低表达,差异有显著性(P<0.05)。图像分析结果显示:膀胱癌与癌旁组织之间,CyclinD1的平均光密度及阳性面积率有显著性差异(P<0.05),经统计学分析,CyclinD1表达与膀胱癌病理分级临床分期无显著性差异(P>0.05)。结论 CyclinD1在膀胱癌中的异常表达可能参与膀胱癌的发生、发展过程。     

miR-448通过抑制上皮间质转化抑制肺癌细胞增殖和运动能力

探讨mi R-448对肺癌细胞增殖和运动的影响及其分子机制。采用实时荧光定量PCR(polymerase chain reaction)检测原发肺癌组织和癌旁正常组织mi R-448表达水平。转染mi R-448 mimic和inhibitor至肺癌A549细胞系,通过MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoliumbromide)、平板克隆形成和Transwell实验观察mi R-448表达对A549增殖和运动能力的影响;利用Western blot检测EMT(epithelial-mesenchymal transition)标志物蛋白表达水平,通过实时荧光定量PCR检测EMT相关转录因子m RNA表达水平。实时荧光定量PCR显示较癌旁正常组织相比,mi R-448在原发肺癌组织中表达降低。MTT和平板克隆形成实验显示,过表达mi R-448抑制A549细胞增殖和运动能力;降表达mi R-448增强A549细胞增殖和运动能力。Western blot显示降表达mi R-448能下调上皮标志物E-cadherin,上调间质标志物Vimentin表达水平。实时荧光定量PCR显示降表达mi R-448能上调EMT相关转录因子Twist1和ZEB1 m RNA表达水平。mi R-448可通过抑制EMT抑制肺癌进展。     

miR-15a-5p和miR-16-5p在骨肉瘤组织中的表达分析

目的:使用microRNAs基因芯片及实时定量PCR法测定骨肉瘤组织中miR-15a-5p和miR-16-5p的相对表达含量,并与瘤旁组织对比,分析骨肉瘤细胞内miR-15a-5p和miR-16-5p的表达变化。方法:选取34例骨肉瘤组织蜡块样本,使用microRNAs基因芯片观察miR-15a-5p和miR-16-5p在骨肉瘤和瘤旁组织内的表达差异;实时定量PCR法测定骨肉瘤组织和瘤旁组织中miR-15a-5p和miR-16-5p的相对表达含量,并将两种结果对比分析。结果:microRNAs基因芯片结果显示,在骨肉瘤组织中,miR-15a-5p在肿瘤中的表达较瘤旁组织低1.79倍,miR-16-5p较瘤旁组织低1.62倍。实时定量PCR实验结果表明,miR-15a-5p和miR-16-5p表达较瘤旁组织降低,差异有统计学意义(P0.05)。经过统计学计算,miR-15a-5p在肿瘤中的表达较瘤旁组织低3.14倍,miR-16-5p较瘤旁组织低5.65倍。结论:在骨肉瘤中,miR-15a-5p和miR-16-5p表达含量降低,提示这两种microRNAs在骨肉瘤中可能做为抑癌因子存在。     

节律基因CRY1在肝癌中的表达及对肝癌细胞生长的作用

目的:探讨节律基因CRY1(cryptochrome circadian clock 1)在肝癌组织中的表达情况及对肝癌细胞生长的作用。方法:利用实时荧光定量PCR、Western blotting及免疫组织化学染色法,检测CRY1在肝癌组织及细胞中的表达;利用肝癌公共数据信息,分析和验证CRY1在肝癌组织中的表达;真核表达质粒将肝癌细胞CRY1过表达后,采用MTS和Annexin V-FITC/PI双染法,分别检测CRY1对细胞增殖和凋亡的影响。结果 CRY1在癌组织中阳性率为57.5%(23/40),癌旁组织中阳性率为87.5%(35/40),癌中CRY1表达低于癌旁的占80%(32/40),与癌旁组织比较,CRY1蛋白在肝癌组织中的表达明显降低,差异有统计学意义(t=7.53,P0.001)。CRY1在3个肝癌公共数据信息显示,与对应癌旁组织相比,其在癌组织中表达水平显著降低,差异均具有统计学意义(TCGA:413.62 vs 335.17,P=0.0155;GSE22058:397.67 vs 360.08,P=0.0207;GSE25097:1.627 vs 1.433,P0.0001)。同时CRY1在被检测的4株肝癌细胞系中表达也显著低于永生化的正常肝细胞。与对照组比,过表达CRY1组细胞增殖能力明显降低,两组间差异有统计学意义(1.100 vs 0.662,P0.0001);与对照组比,过表达CRY1组细胞凋亡显著增加(凋亡细胞百分比:7.9 vs 17.5,P=0.0014)。结论:节律基因CRY1在肝癌中低表达,同时CRY1抑制肝癌细胞增殖和促进细胞凋亡。     

Myocyte enhancer factor-2 and cardiac muscle gene expression during hibernation in thirteen-lined ground squirrels

Long non-coding RNA urothelial carcinoma associated 1 (UCA1) promotes human bladder cancer cell proliferation, but the underlying mechanism remains unknown. After knocking down of UCA1 in BLZ-211 cells, several cell cycle-related genes (CDKN2B, EP300 and TGFβ-2) were screened by microarray assay and validated by real-time PCR. Interestingly, in western blot analysis, p300 (encoded by EP300) and its coactivator cAMP response element-binding protein (CREB) level were significantly down-regulated. Both suppression of UCA1 expression by shRNA in BLZ-211 cells and ectopic expression of UCA1 in UMUC-2 cells showed that UCA1 alteration paralleled to the expression and phosphorylation of CREB, and UCA1 obviously influenced AKT expression and activity. Furthermore, in BLZ-211 cells, cell cycle progression was greatly reduced after PI3-K pathway was blocked by LY294002, indicating that UCA1 affected cell cycle progression through CREB. Taken together, we concluded that UCA1 regulated cell cycle through CREB via PI3K-AKT dependent pathway in bladder cancer.     

Molecular factors regulating E-cadherin expression in urothelial bladder cancer and their correlations with the clinicopathological features

This study aimed to assess the expression of S100A4, Twist and E-cadherin (mRNA and protein) in urothelial bladder cancer, investigate the correlation between them and evaluate their association with the clinicopathological features of the disease. The study included 54 patients diagnosed as urothelial bladder cancer of different stages and grades. The expression levels of S100A4, Twist and E-cadherin (mRNA and protein) in tissue samples were determined by quantitative RT-PCR and immunohistochemistry. The expression of S100A4 and Twist was significantly upregulated while E- cadherin was significantly downregulated in urothelial bladder cancer tissues compared to the adjacent surrounding normal bladder tissues at both mRNA and protein levels (p?<?0.001). Expression levels of S100A4 and Twist were significantly higher in recurrent tumor than in non-recurrent tumors (p?<?0.001) while the expression level of E-cadherin was significantly lower in recurrent tumors than in non-recurrent tumors at both mRNA and protein levels (p?<?0.001). There was a significant positive correlation between S100A4 and Twist expressions (r?=?0.875, p?<?0.001) while significant negative correlations were found between E- cadherin and S100A4 expressions(r=- 0.803, p?<?0.001) and between E-cadherin and Twist (r?=??0.809, p?<?0.001). Up-regulation of S100A4 and Twist and down-regulation of E-cadherin in urothelial bladder cancer tissues compared to adjacent normal tissues were observed. There was a significant negative correlation between S100A4 and E- cadherin and between E- cadherin and Twist expression. However, there was a significant positive correlation between S100A4 and Twist expressions. Furthermore, the alterations in the gene expression were associated with disease stage and grade.     

LncRNA-UCA1 modulates progression of colon cancer through regulating the miR-28-5p/HOXB3 axis

Emerging evidence has shown that the long noncoding RNA urothelial carcinoma–associated 1 (UCA1) plays a tumor-promoting role in colorectal cancer, while miR-28-5p shows tumor-inhibitory activity in several tumor types. However, the mechanisms both of these in colon cancer progression are still unknown. In this work, the detailed roles and mechanisms of UCA1 and its target genes in colon cancer were studied. The results showed that UCA1 was upregulated in colon cancer tissues when compared with the adjacent nonhumorous tissues, as well as in the various colon cancer cell lines, but the expression of miR-28-5p showed an opposite trend. Furthermore, a high UCA1 level in colon cancer tissues is positively associated with the tumor size and advanced tumor stages. Functional assays revealed that both UCA1 knockdown and miR-28-5p overexpression could inhibit colon cancer cell growth and migration. Further mechanistic studies indicated that UCA1 knockdown played tumor suppressive roles in SW480 and HT116 cells through binding with miR-28-5p. We also, for the first time, identified HOXB3 as the target gene of miR-28-5p and that HOXB3 overexpression could mediate the functions of UCA1 in cell proliferation and migration of colon cancer cells. In conclusion, our data provided evidence for the regulatory network of UCA1/miR-28-5p/HOXB3 in colon cancer, suggesting that UCA1, miR-28-5p, and HOXB3 are the potential targets for colon cancer therapy.     

膀胱尿路上皮癌组织CRMP2、NUSAP1、hMSH2表达与临床病理参数和预后的关系研究

目的:探讨膀胱尿路上皮癌组织坍塌反应调节蛋白2(CRMP2)、核仁和纺锤体相关蛋白l(NUSAP1)、人类错配修复基因2(h MSH2)表达与临床病理参数和预后的关系。方法:选取2018年2月至2020年3月我院收治的85例膀胱尿路上皮癌患者,比较手术切除的癌组织和癌旁粘膜组织中CRMP2、NUSAP1、hMSH2表达情况。比较不同病理参数癌组织中CRMP2、NUSAP1、hMSH2阳性表达率差异,分析CRMP2、NUSAP1、hMSH2与膀胱尿路上皮癌患者预后的关系。结果:与癌旁组织比较,膀胱尿路上皮癌患者癌组织中CRMP2、NUSAP1阳性表达率增高(P<0.05),hMSH2降低(P<0.05)。T2-T4、高级别肿瘤患者癌组织中CRMP2、NUSAP1阳性表达率高于Tis-T1、低级别肿瘤患者(P<0.05),多发癌组织中CRMP2阳性表达率高于单发(P<0.05),T2-T4癌组织中hMSH2阳性表达率低于Tis-T1(P<0.05)。总生存率、无复发生存率、无进展生存率比较,CRMP2阳性表达者低于CRMP2阴性表达者(P<0.05),hMSH2阴性表达者低于hMSH2阳性表达者(P<0.05),NUSAP1阳性表达者无复发生存率、无进展生存率低于NUSAP1阴性表达者(P<0.05)。Cox风险比例回归分析结果显示复发、进展、CRMP2阳性表达、NUSAP1阳性表达、hMSH2阴性表达是膀胱尿路上皮癌患者不良预后的危险因素(P<0.05)。结论:CRMP2、NUSAP1阳性表达,hMSH2阴性表达与膀胱尿路上皮癌临床病理参数、复发转移以及不良预后有关。     

SATB1 Overexpression Regulates the Development and Progression in Bladder Cancer through EMT

The global gene regulator Special AT-rich sequence-binding protein-1 (SATB1) has been reported to induce EMT-like changes and be associated with poor clinical outcome in several cancers. This study aims to evaluate whether SATB1 affects the biological behaviors of bladder transitional cell carcinoma (BTCC) and further elucidate if this effect works through an epithelial-mesenchymal transition (EMT) pathway. The expression of SATB1, E-cadherin (epithelial markers), vimentin (mesenchymal markers) in BTCC tissues and adjacent noncancerous tissues, as well as in two cell lines of bladder cancer were investigated. Whether the SATB1 expression is associated with clinicopathological factors or not was statistically analyzed. Cell invasion and migration, cell cycle, cell proliferation and apoptosis were evaluated in SATB1 knockdown and overexpressed cell lines. Our results showed that the expression of SATB1 was remarkably up-regulated both in BTCC tissues and in bladder cancer cell lines with high potential of metastasis. The results were also associated with EMT markers and poor prognosis of BTCC patients. Moreover, SATB1 induced EMT processes through downregulation of E-cadherin, upregulation of E-cadherin repressors (Snail, Slug and vimentin). SATB1 also promoted cell cycle progression, cell proliferation, cell invasion and cell migration, but did not alter cell survival. In conclusion, our results suggest that SATB1 plays a crucial role in the progression of bladder cancer by regulating genes controlling EMT processes. Further, it may be a novel therapeutic target for aggressive bladder cancers.     

细胞信号分子Tiaml和Racl在膀胱尿路上皮癌中的表达及意义

目的 探讨细胞信号分子Tiaml和Racl在膀胱尿路上皮癌中的表达及临床意义.方法 收集武汉大学人民医院病理科2006-2010年有完整临床和病理资料的膀胱尿路上皮癌存档蜡块50例和5例癌旁组织,采用免疫组织化学S-P法检测50例膀胱尿路上皮癌和5例癌旁组织中Tiaml和Racl的表达水平.应用彩色病理图文报告管理系统,对Tiaml和Racl的表达进行定量分析,并用SPSS13.0 软件对各组免疫组织化学反应阳性颗粒的平均光密度、阳性面积率做单因素方差分析和SNK（q）检验.结果 （1）Tiaml的表达 Tiaml在膀胱尿路上皮癌中呈高表达,而在癌旁组织中呈低表达.膀胱尿路上皮癌与癌旁组织相比,差异有显著性 （P〈0.05 ）;（2）Racl的表达 Racl在膀胱尿路上皮癌中呈高表达,而在癌旁组织中呈低表达.膀胱尿路上皮癌与癌旁组织相比,差异有显著性 （P〈0.05 ）;（3）膀胱尿路上皮癌中Tiaml与Racl的表达呈正相关,它们相互作用、相互调控.结论 Tiaml和Racl在膀胱尿路上皮癌中的表达异常可能是膀胱尿路上皮癌的早期分子事件,在膀胱尿路上皮癌的发生和发展中发挥了重要作用,并Tiaml与Racl的表达呈正相关,它们相互作用、相互调控,在膀胱尿路上皮癌的发生、发展过程中,两者起协同作用.