实时荧光定量PCR方法检测人癌组织中RFP蛋白的表达

目的检测RFP(Ret finger protein)蛋白在人正常组织及人癌组织中的分布。方法采用实时荧光定量PCR方法,以18 S rRNA为内对照,检测RFP在人正常组织及人癌组织中的表达水平。结果RFP表达水平子宫颈鳞状细胞癌组织高于正常子宫颈组织,子宫内膜腺癌组织高于正常子宫内膜组织,胃腺癌组织高于正常胃组织,食管鳞状细胞癌组织高于正常食管组织,而子宫内膜腺癌组织高于子宫颈鳞状细胞癌组织,胃腺癌组织高于食管鳞状细胞癌组织,脑癌组织高于正常脑组织。结论RFP可能成为治疗恶性肿瘤的一个潜在的分子靶点。     

实时荧光定量PCR方法检测人、鼠正常组织及脑癌组织中CD109蛋白的表达

目的分析CD109基因在正常组织及脑癌组织中的分布。方法采用实时荧光定量PCR方法,分析CD109在人、鼠正常组织及脑癌中的表达,以18SrRNA作为内标。结果CD109蛋白的mRNA丰度在睾丸组织中最高,在其它组织中较低,在12份脑癌标本中,检测到9份CD109表达上调。结论CD109基因在多数正常组织中表达很少,有可能是治疗恶性肿瘤的一个潜在的分子靶点。     

子宫颈腺癌中ILK与E-Cadherin蛋白表达的关系

目的探讨子宫颈腺癌组织中整合素连接激酶(ILK)与E-Cadherin的表达关系。方法采用免疫组织化学方法检测106例子宫颈腺癌组织中和22例慢性子宫颈炎组织中ILK与E-Cadherin的表达。结果106例子宫颈腺癌组织ILK的阳性表达率为86.8%,高于慢性子宫颈炎组织(P<0.01);E-Cadherin蛋白表达率为58.5%,低于慢性子宫颈炎组织81.8%(P<0.05)。ILK在子宫颈腺癌病理分级,G3组阳性表达率均明显高于G1组(P<0.05)。E-Cadherin表达与子宫颈腺癌组织类型有关,透明细胞腺癌阳性表达率低于子宫颈内膜腺癌和子宫内膜样腺癌(P<0.05)。ILK阳性表达强度与E-Cadherin的阳性表达强度呈负相关(P<0.05)。结论ILK与E-Cadherin在子宫颈腺癌组织中的表达密切相关,E-Cadherin低表达与ILK增强表达在子宫颈腺癌的发生发展中起重要作用,三者可能作为子宫颈腺癌恶性化的分子标志。     

LHRH-PE40与宫颈癌组织结合特性的研究

目的观察LHRH-PE40对各种病理型宫颈癌组织的结合能力,为其临床应用提供理论依据。方法采用免疫荧光法检测LHRH-PE40与正常宫颈组织及宫颈癌组织的结合能力。结果宫颈结缔组织、鳞状上皮的荧光结合率为0%。宫颈癌组织荧光结合率为74%,弱阳性及阴性表达病例中,高分化鳞状细胞癌占67%,中低分化鳞状细胞癌占15%,两者差异有显著意义(P<0.05)。中强阳性表达病例中,高分化鳞状细胞癌仅占37%,中低分化鳞状细胞癌占86%,两者差异有显著意义(P<0.05)。5例强阳性表达病例均为高分化腺癌。结论高分化鳞状细胞癌与LHRH-PE40结合能力最弱,子宫颈腺癌与LHRH-PE40结合能力最强。     

鱼精蛋白1在结肠腺癌组织中的表达及其临床意义

目的比较鱼精蛋白1(Protamines 1,PRM1)在结肠腺癌组织中的表达及其临床意义。方法通过实时荧光定量PCR法检测53例结肠腺癌及正常结肠组织中PRM1基因mRNA水平,并分析PRM1 mRNA检测对于结肠腺癌诊断的敏感性;SP免疫组化染色法检测组织中PRM1蛋白的表达水平。结果结肠腺癌组织中PRM1基因mRNA水平明显高于正常结肠组织,差异有统计学意义(P<0.05),总体阳性率为96.23%(51/53);结肠腺癌组织高表达PRM1,且能够分泌入腺腔。结论结肠腺癌细胞在基因及蛋白水平均高表达PRM1,PRM1 mRNA检测对结肠腺癌的诊断具有参考价值。     

米非司酮对异位子宫内膜基质金属蛋白酶表达的影响

目的探讨米非司酮对子宫内膜异位症(endometriosis,EMS)患者子宫内膜基质金属蛋白酶(MMP-2,MMP-9mRNA)表达的影响,以探讨米非司酮治疗EMS的可能机制。方法收集子宫内膜异位症患者38例,用宫腔镜分别取患者米非司酮治疗前和治疗后子宫内膜标本38份。采用RT-PCR半定量方法检测内膜标本中MMP-2,MMP-9 mRNA表达率及表达强度。结果所有内膜标本均有MMP-2、MMP-9 mRNA表达,治疗前和治疗后标本中MMP-2、MMP-9mRNA的表达率分别为(97%,68%)和(95%,66%);治疗后子宫内膜中MMP-2、MMP-9mRNA的表达强度低于治疗前(P<0.05)。结论米非司酮可以有效抑制子宫内膜症患者内膜中MMP-2、MMP-9mRNA的表达,降低了内膜的侵袭性,减少了转移到发生,为子宫内膜异位症的治疗提供了新的思路。     

胃黏液腺癌组织中HIF-1α的表达及临床意义

目的探讨胃黏液腺癌组织中缺氧诱导因子-1α(HIF-1α)的表达及意义。方法应用免疫组织化学方法检测和分析63例胃黏液腺癌癌区组织、癌旁区组织、手术切缘区正常组织各区域HIF-1α蛋白的表达。结果 HIF-1α蛋白在胃黏液腺癌组织的阳性率明显高于癌旁区组织、正常组织,差异具有显著性(P<0.05);在胃黏液腺癌组织中,随着组织学病理分级的增加、临床分期的进展和淋巴结的转移,HIF-1α蛋白表达阳性率逐渐上升,差异具有显著性(P<0.05);HIF-1α蛋白表达具有相关性。结论 HIF-1α可能调控胃癌血道转移,可为胃黏液腺癌的早期诊断、治疗、预后判断提供必要的理论依据。     

核输入受体蛋白在子宫内膜异位症和子宫内膜癌中的表达

目的探讨核输入受体蛋白13(Importin 13,IPO13)在子宫内膜异位症和子宫内膜癌中的表达及其临床意义。方法随机选取20份正常增殖期子宫内膜和20份正常分泌期子宫内膜标本为对照组A、B,20例子宫内膜异位症和20例子宫内膜癌患者的子宫内膜标本为实验组C、D。采用免疫组化法检测IPO13在各组中是否表达;免疫荧光法检测IPO13和干/祖细胞标记在各组中的共表达;荧光定量PCR法检测各组中IPO13基因mRNA转录水平;Western blot法检测IPO13蛋白在各组中的表达水平。结果 IPO13在各组子宫内膜腺上皮细胞和间质细胞的胞浆及胞核中均有表达。对照组B的IPO13阳性细胞数明显低于对照组A和实验组(P<0.01)。IPO13与CD34、CD45、端粒酶、OCT4、C-KIT、PDGF-Rβ、CD90和CD146共表达于各组子宫内膜的上皮细胞和间质细胞;实验组及对照组A的IPO13基因mRNA和IPO13蛋白表达量明显高于对照组B(P<0.01)。结论 IPO13在正常子宫内膜中有表达,且增殖期表达高于分泌期;在子宫内膜异位症和子宫内膜癌中的表达明显高于正常分泌期子宫内膜,此结果可能与子宫内膜干/祖细胞的表达异常相关;IPO13可作为子宫内膜干/祖细胞的一个候选的标记。     

CENP-E变异体在不同肿瘤细胞株及癌组织中的表达差异

目的研究CENP-E变异体(CENP-EⅠ)在不同肿瘤细胞株及癌组织和相应的癌旁组织中的表达情况。方法应用巢式RT-PCR检测14个细胞株和17份癌组织及相应癌旁组织标本中CENP-EⅠmRNA(缺失第38个外显子)的表达情况,并比较变异体和野生型CENP-E(CENP-EWT) mRNA的表达差异;应用巢式PCR检测上述标本中CENP-E在DNA水平上的变异;并通过细胞增殖试验、细胞周期检测和染色体核型鉴定,分析CENP-EⅠ与细胞恶性程度的相关性。结果 RT-PCR结果显示,实验样本中CENP-E mRNA均有CENP-EWT和CENP-EⅠ两种形式同时存在于一种组织或细胞中,且在正常细胞株和癌旁组织中CENP-EWT表达量较高,在肿瘤细胞株和癌组织中CENP-EⅠ表达量较高,且差异有统计学意义(P<0.05);样本中CENP-E在DNA水平上不存在第38个外显子缺失,且细胞间CENP-E表达量的差异无统计学意义(P>0.05);CENP-EⅠ与细胞的恶性程度有一定的相关性。结论同一细胞内存在2种或2种以上形式的CENP-E mRNA,CENP-E变异体可能与肿瘤的发生有关。     

黑色素瘤抗原-4基因在结肠腺癌中表达的检测

目的检测黑色素瘤抗原-4(MAGE-4)基因在结肠腺癌中的表达情况。方法采用RT-PCR方法检测44份结肠腺癌组织及15份癌旁正常组织标本中MAGE-4基因的表达。结果44份结肠腺癌标本中,MAGE-4基因的阳性表达率为38.64%,而在15份癌旁正常组织中均不表达,两组比较差异有显著意义。对阳性标本的RT-PCR扩增产物中目的基因片段进行DNA测序,证实为MAGE-4全长编码基因。结论MAGE-4基因在结肠腺癌组织中呈特异性表达,提示该基因可用于结肠癌疫苗的开发。     

胃癌相关miRNAs的筛选及其作用靶点的验证

目的筛选胃癌中相关miRNAs,并验证其作用靶点。方法应用基因芯片技术检测3份正常胃组织标本、24份胃癌组织标本、胃癌细胞SGC7901和正常胃黏膜细胞GES-1中328个miRNAs的表达情况。采用实时荧光定量PCR对结果进行验证,并用基因克隆和Western blot方法分析miR-9的作用靶点。结果共有26个miRNAs在胃癌标本(包括24份胃癌组织和SGC7901细胞)中异常表达。其中19个下调,7个上调。实时荧光定量PCR检测出miR-9在胃癌标本中的表达水平显著下调,该结果与基因芯片检测结果一致。miR-9与RAS癌基因家族成员RAB34的表达呈负相关。结论已初步筛选了胃癌相关miRNAs。miR-9可能是胃癌中的标记性miRNAs之一,RAB34是其作用靶点。     

A Novel Protein Is Lower Expressed in Renal Cell Carcinoma

Engrailed-2 (EN2) has been identified as a candidate oncogene in breast cancer and prostate cancer. It is usually recognized as a mainly nuclear staining in the cells. However, recent studies showed a cytoplasmic staining occurred in prostate cancer, bladder cancer and clear cell renal cell carcinoma. The inconsistency makes us confused. To clarify the localization and expression of EN2 in renal cell carcinoma, anti-EN2 antibody (ab28731) and anti-EN2 antibody (MAB2600) were used for immunohistochemistry (IHC) respectively. Interestingly, we found that EN2 detected by ab28731 was mainly presented in cytoplasm while EN2 detected by MAB2600 was mainly presented in nucleus. To further investigate the different patterns observed above, lysates from full-length EN2 over expression in HEK293T cells were used to identify which antibody the EN2 molecule bound by western blot. Results showed ab28731 did not react with the lysates. For this reason, the novel specific protein detected by ab28731 was not the EN2 molecule and was named nonEN2. Then using the renal carcinoma tissue microarray and renal tissues, we found that the protein expression levels of nonEN2 in kidney tumor tissues was significantly lower than that in kidney normal tissues (p < 0.05), so was in renal cell lines. Taken together, nonEN2 is lower expressed and may play an important role in renal cell carcinoma.     

Anti-Tumor Effects of Mfn2 in Gastric Cancer

Mitofusin-2 (Mfn2) is a mitochondrial outer membrane protein involved in mitochondrial fusion. Its mutation can cause Charcot-Marie-Tooth disease. Recent studies of Mfn2 in cancer research have not included gastric cancer. We confirmed that Mfn2 expression was lower in tumor tissue than in normal gastric mucosal tissue and that it was negatively correlated with tumor size, indicating an anti-tumor role for Mfn2. In vitro experiments showed that Mfn2 overexpression suppressed gastric cancer cell proliferation and colony formation, weakened the invasion and migratory ability of cancer cells by downregulating MMP-2 and MMP-9, halted the cell cycle and induced apoptosis. Western blotting indicated the likely involvement of P21 and PI3K/Akt signaling. Therefore, Mfn2 is a potential anti-tumor gene and a potential therapeutic target for treating gastric cancer. The progress of gastric cancer may be delayed by controlling Mfn2 expression.     

Enhanced Chemosensitivity by Targeting Nanog in Head and Neck Squamous Cell Carcinomas

Chemo-resistance is the major cause of high mortality in head and neck squamous cell carcinomas (HNSCC) in which HNSCC-derived cancer stem cells (CSCs) may be involved. Previously, we enriched a subpopulation of HNSCC-derived spheroid cells (SC) (HNSCC-SC) and identified Nanog as a CSCs marker. The aim of this study was to determine the role of Nanog in the chemosensitivity of HNSCC. The functional and clinicopathological studies of Nanog were investigated in HNSCC cells and specimens. Nanog expression was increased in HNSCC cell lines as compared to a normal oral epithelial cell line. Nanog upregulation in clinical tissues from HNSCC patients with recurrent and metastatic specimens relative to the mRNA levels in the samples from normal or primary tissues were examined. Targeting Nanog in HNSCC-SC significantly inhibited their tumorigenic and CSCs-like abilities and effectively increased the sensitivity of HNSCC-SC to chemotherapeutic drug cisplatin treatment. Targeting Nanog in HNSCC-SC showed a synergistic therapeutic effect with cisplatin. Our results suggest that targeting Nanog may have promising therapeutic potential for HNSCC.     

Tissue Microarray-Based Evaluation of Chromatin Assembly Factor-1 (CAF-1)/p60 as Tumour Prognostic Marker

In this study we aimed to confirm the emerging role of Chromatin Assembly Factor 1 (CAF-1 p60) as a new proliferation and prognostic marker for cancer and to test the usefulness of the tissue microarray technique (TMA) for CAF-1 p60 rapid screening in several human malignancies. CAF-1 is a histone chaperone, regulating chromatin dynamics during DNA replication and repair in eukaryotics. TMA is a powerful high-throughput methodology in the study of cancer, allowing simultaneous assessment of different biomarkers within large numbers of tissue specimens. We generated TMA taking 3 mm diameter-core biopsies from oral squamous cell carcinoma, prostate cancer, salivary gland tumours and skin melanoma specimens, which had been previously tested for CAF-1 p60 on routine tissue sections. We also analysed, for the first time, 30 larynx and 30 skin squamous cell carcinomas. CAF-1 p60 resulted over-expressed in both the tissue sections and the TMA specimens, with the highest levels of expression in tumours which were more aggressive and metastasizing. Notably, a high degree of agreement was found between the CAF-1 p60 assessment on TMAs and on routine tissue sections. Our findings confirm the prognostic role of CAF-1 p60 and indicate TMA as a really advantageous method for CAF-1 p60 immunohistochemical screening, allowing savings on both tissue quantity and operator-time.