Primary application of a real-time quantitative polymerase chain reaction for the detection of human breast cancer related novel gene-Metadherin expression

Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to explore the relationship between expression of Metadherin gene in the patients peripheral blood and the clinic-pathological features in breast cancer. Methods:Real-time fluorescence quantitative polymerase chain reaction was employed to determine the expression level of Metadherin gene in 80 peripheral blood samples of breast cancer patients and healthy donors. Results:The expression of Metadherin gene in breast cancer patients peripheral blood were positive,in which 34 breast cancer patients were highly expressed,accounting for 55.7%,while the expression of Metadherin gene in normal females peripheral blood were negative,there was statistical significance (Ratio = 2.02±0.81,P < 0.05); Ratio of the Metadherin expression in breast cancer patients peripheral blood and the glyceraldehyde-3-phosphate dehydrogenase expression was 1.15 ± 0.36. REST software analysis showed that the expression of Metadherin gene was significantly up-regulated in breast cancer. Conclusion:The SYBR Green I quantitative real-time polymerase chain reaction method can successfully detect the expression level of Metadherin gene. Expression level of Metadherin gene in breast cancer patients peripheral blood is closely related to survival,and it maybe involved in the development of breast cancer and used as an indicator of prognosis.

The quantitative expressions of lymphangiogenic factors and metastasis in human colorectal cancers

Objective： The purpose of this paper was to study the expression levels of newly described lymphatic endothelial markers – LYVE-1, Prox-1, podoplanin and 5’-nucleotidase, and their correlation with metastasis of human colorectal cancers. Methods： Tumor and corresponding tumor-side normal tissue samples were obtained from resected specimens immediately after operation. Expression level of each factor was determined by quantitative real-time PCR （RT-PCR） and Western blot technique. Results： Expression levels of lymphatic endothelial markers LYVE-1, Prox-1, podoplanin and 5’-nucleotidase were significantly different in tumor and tumor-side normal groups. Expression levels of Prox-1 and podoplanin were higher in patients with positive lymph node metastasis than those without metastasis. LYVE-1, but not 5’-nucleotidase expression level was higher in both cancer and normal groups. Conclusion： These results indicate that combined quantitative analysis of lymphangiogenic markers LYVE-1, Prox-1 and podoplanin in colorectal cancer specimens may be useful in predicting metastasis of colorectal cancer to regional lymph nodes. However, the role of 5’-nucleotidase in predicting metastasis of colorectal cancer still remains to be further analyzed.

Quantitative analysis of a panel of gene expression in prostate cancer——with emphasis on NPY expression analysis

Objective： To investigate molecular alterations associating with prostate carcinoma progression and potentially provide information toward more accurate prognosis/diagnosis. Methods： A set of laser captured microdissected （LCM） specimens from 300 prostate cancer （PCa） patients undergoing radical prostatectomy （RP） were defined. Ten patients representing ＂aggressive＂ PCa, and 10 representing ＂non-aggressive＂ PCa were selected based on prostate-specific antigen （PSA） recurrence, Gleason score, pathological stage and tumor cell differentiation, with matched patient age and race between the two groups. Normal and neoplastic prostate epithelial cells were collected with LCM from frozen tissue slides obtained from the RP specimens. The expressions of a panel of genes, including NPY, PTEN, AR, AMACR, DD3, and GSTP1, were measured by quantitative real-time RT-PCR （TaqMan）, and correlation was analyzed with clinicopathological features. Results： The expressions of AMACR and DD3 were consistently up-regulated in cancer cells compared to benign prostate epithelial cells in all PCa patients, whereas GSTP1 expression was down regulated in each patient. NPY, PTEN and AR exhibited a striking difference in their expression patterns between aggressive and non-aggressive PCas （P=0.0203, 0.0284, and 0.0378, respectively, Wilcoxon rank sum test）. The lower expression of NPY showed association with ＂aggressive＂ PCas based on a larger PCa patient cohort analysis （P=0.0037, univariate generalized linear model （GLM） analysis）. Conclusion： Despite widely noted heterogeneous nature of PCa, gene expression alterations ofAM,4CR, DD3, and GSTP1 in LCM-derived PCa epithelial cells suggest for common underlying mechanisms in the initiation of PCa. Lower NPY expression level is significantly associated with more aggressive clinical behavior of PCa; PTEN and AR may have potential in defining PCa with aggressive clinical behavior. Studies along these lines have potential to define PCa-associated gene expression alterations and likely co-regulation of genes/pathways critical in the biology of PCa onset/progression.

Expression of Stanniocalcin 2 in Breast Cancer and Its Clinical Sigmiicance

This study aims to explore the expression of stanniocalcin 2(STC2)gene in breast cancer and its clinical significance.Female patients with breast cancer from Zhongnan Hospital of Wuhan University admitted during March 2014 to October 2014 were enrolled in this study.All the tissues used in this experiment included 50 cases of breast cancer tissues and corresponding 50 cases of paracancer normal breast tissues with complete patients'information.The real-time quantitative polymerase chain reaction(qPCR)was applied to detect the expression of STC2 gene in 50 cases of breast cancer and paracancer normal breast tissues.The results showed that the expression level of STC2 gene in 50 cases of breast cancer tissues was significantly higher than that in paracancer normal breast tissues(P<0.001).The expression of STC2 gene was correlated with lymph node metastasis,distant metastasis,TNM stage and histological grade(P<0.001).The expression level of STC2 gene was significantly higher in breast cancer tissues with higher expression of Ki-67(P<0.001).The expression level of STC2 gene was significantly higher in estrogen receptor(ER)positive breast cancer tissues than in ER negative ones(P<0.001).However,different groups of age,pathological type,tumor size,PR expression and human epidermal growth factor receptor-2(HER2)expression did not show significant differences in STC2 expression(P>0.05).In conclusion,the abnormal overexpression of STC2 gene may play a role in the development and progression of breast cancer,and it can be used as an independent metastasis and prognostic factor of breast cancer.In addition,STC2 gene probably promotes the development and metastasis of breast cancer by interacting with estrogen and ER,and it may become a new direction for breast cancer endocrine therapy.

BCRP基因在乳腺癌组织中的表达及意义

目的 研究乳腺癌耐药蛋白 (BCRP)基因在乳腺癌组织中的表达水平 ,探讨其与肿瘤病理分期、淋巴结转移的关系。方法 采用实时荧光定量 RT- PCR检测 5 1例乳腺癌患者癌组织和癌旁组织中 BCRP基因表达。结果 BCRP基因在乳腺癌组织中的阳性表达率、表达水平均显著高于癌旁组织 (P<0 .0 1、<0 .0 5 ) ;BCRP基因在 、 、 期乳腺癌组织中的表达水平无显著性差异 (P>0 .0 5 ) ;有淋巴结转移者的 BCRP基因表达水平显著高于无淋巴结转移者 (P<0 .0 5 )。结论 BCRP基因表达水平与肿瘤病理分期无关 ,与淋巴结转移有关 ,BCRP基因过表达可能在乳腺癌的多药耐药中起较重要的作用。

STAT3基因在乳腺癌组织中的表达及意义

背景与目的:STAT3是近期研究较多的一类癌基因,在多种实体瘤如乳腺癌、头颈部癌、胃癌等中均有异常表达和活性增强。本实验研究乳腺肿瘤组织中癌基因STAT3mRNA丰度及其蛋白的表达阳性率,探讨其与组织类型、淋巴结转移、雌激素受体(ER)、孕激素受体(PR)及癌胚表达抗原(CerbB-2)表达的关系。方法:采用实时荧光定量RT-PCR技术检测32例乳腺癌和10例乳腺良性病变中STAT3 mRNA的丰度表达,EnVision免疫组化法检测45例乳腺癌和12例癌旁乳腺组织标本中p-STAT3蛋白的表达阳性率,并进行相关统计分析。结果:EnVi-sion免疫组化法检测45例乳腺癌和12例癌旁乳腺组织标本中,pSTAT3在癌旁组织和癌组织中的阳性表达率分别为16.7%(2/12)和60%(27/45),两者相比统计学差异有显著性(P<0.05);在无淋巴结转移和有淋巴结转移乳腺癌组织中分别为48.1%(13/27)和77.8%(14/18),两者相比统计学差异有显著性(P<0.05);在孕激素阴性和孕激素阳性乳腺癌组织中分别为37.5%(6/16)和72.3%(21/29),两者相比统计学差异有显著性(P<0.05)。实时荧光定量RT-PCR技术检测32例乳腺癌和10例乳腺良性病变中,STAT3 mRNA的相对丰度在乳腺良性病变和乳腺癌中的分别为:6×10-3±3×10-3和34×10-3±17×10-3,两者相比统计学差异有显著性(P<0.05);在无淋巴结转移和有淋巴结转移乳腺癌组织中分别为26×10-3±13×10-3和39×10-3±18×10-3,两者相比统计学差异有显著性(P<0.05);在孕激素阴性和孕激素阳性乳腺癌组织中分别为28×10-3±10×10-3和39×10-3±19×10-3,两者相比统计学差异有显著性(P<0.05)。pSTAT3蛋白阳性率和mRNA的丰度均与乳腺癌患者年龄、肿瘤大小及ER、CerbB-2阳性与阴性组未见相关性(P>0.05)。结论:STAT3基因表达增强可能与乳腺癌淋巴结转移以及孕激素有关。

Dissemination profile of perioperative tumor cells in peripheral blood of colorectal cancer patients detected by multiple marker genes

This work proposes a method to assess the molecular profile of perioperative circulating tumor cells in peripheral blood (PB) of colorectal cancer patients for differentiating the dissemination process of tumor cells. Two-point quantification of multiple marker genes was designed for describing the profile. The expression levels of cytokeratin 20 (CK20),carcino-embryonic antigen (CEA) and survivin mRNA in PB and tumor tissue samples in 37 colorectal cancer patients from 1 d pre-operation to 2 h postoperation were detected with real-time quantitative reverse transcription-polymerase chain reaction. β-Actin mRNA was used as internal control to standardize the results of different mRNA expression levels. The data analysis using Stata statistical packages,Chi-Square test and Mann-Whitney test indicated the expression level of CEA mRNA in PB increased significantly,while those of CK20 and survivin mRNA decreased significantly. Quantitative comparison with tumor tissues indicated that the increase of CEA mRNA level in PB coincided with the decrease of CK20 and survivin mRNA levels in different tumor cells. These results showed surgical manipulation caused tumor cells shedding into blood from primary tumor tissue and significant increase of CEA mRNA level,while occult tumor cells with high expression levels of CK20 and survivin mRNA before surgery decreased after surgery.