Effects of miR-362 in Regulating the Proliferation, Invasion and Apoptosis of Gastric Cancer by Inhibiting the Expression of Tumor-Promoting Factor PRDM2.

Purpose - The high expression of positive regulatory domain zinc finger protein2 (PRDM2) is an important factor in inducing the formation and progression of gastric cancer. The current study was performed to explore the effect of micro-RNA-362 (miR-362) targeting PRDM2 on the proliferation and apoptosis of gastric cancer cells. Methods - The expression of miR-362 in gastric adenocarcinoma and normal gastric mucosa was detected by real-time fluorescence quantitative PCR (qPCR), and the expression of PRDM2 in gastric adenocarcinoma was detected by im-munohistochemical method. Gastric cancer cell line MGC-803 and human normal gastric mucosal epithelial cell line (GES-1) were selected for study. Blank control group, empty vector transfection group, miR-362 transfection group, and miR-362 and PRDM2 co-transfection group were established. CCK-8 assay was utilized to detect cell activity, flow cytometry was used to detect cell cycle and apoptosis, and invasion capability of cells was observed through transwell experiment. The expression of Cyclin D1 and Caspases-3 was detected by western blot method. Results - The expression of miR-362 in gastric adenocarcinoma was significantly lower than that in normal gastric mucosa. The expression of miR-362 in gastric adenocarcinoma was significantly different in the maximum diameter, depth of invasion, and different TNM stages. The expression of miR-362 was linked to prognosis. In addition, there was a negative correlation between miR-362 and PRDM2. The expression of miR-362 in gastric cancer cell line MGC-803 was significantly lower than that in GES-1. Compared with the blank control group and empty vector transfection group, the proliferation level of gastric cancer cells in miR-362 transfection group was remarkably decreased, the cells in S phase and G2/M phase were significantly decreased, the capability for invasion was evidently decreased, while the apoptosis was significantly increased. The expression of Caspases-3 increased significantly and the expression of Cyclin D1 decreased significantly. MiR-362 and PRDM2 co-transfection groups could reverse the abovementioned expression levels. Conclusion - MiR-362 can regulate the proliferation, invasion and apoptosis of gastric cancer by inhibiting the expression of tumor-promoting factor PRDM2. The expression of miR-362 in gastric adenocarcinoma is significantly decreased, which can regulate the formation and development of gastric adenocarcinoma by promoting the expression of PRDM2. Moreover, low expression of miR-362 in gastric adenocarcinoma is an important risk factor for tumor progression and poor prognosis.

Progress in Pretreatment and Analysis of Cephalosporins: An Update Since 2005.

Cephalosporins, one of the classic ß-lactam antibiotics, has been widely concerned by global population as the most commonly used broad-spectrum antibiotic. Serious health hazards are possible to be posed by quality control problems caused by its unstable structure, as well as food and environmental pollution introduced by improper use. Given the reasons above, the sensitive and valid methods for monitor and determination of cephalosporins in different matrices are urgently required. This review aims to provide a comprehensive overview on the current status of the pretreatment and analysis methods of cephalosporins in bulk drug, pharmaceutics, animal-derived foodstuffs including eggs, milk, meat, environment samples, and biological samples. Pretreatment methods including simple steps (protein precipitation, centrifugation, filtration), liquid-liquid extraction, solid phase extraction, QuECHERS, and detection methods covering LC, LC-MS/MS, voltammetric sensor, capillary electrophoresis spectroscopy, biological methods from January 2005 to October 2018 are updated, elaborated and compared here. Moreover, advanced materials and prospects for development are discussed. HighlightsDetermination of cephalosporins in different newly found matrix are represented.Comparisons between different mass analyzers and progress in HRMS methods are in detailed.Optimization of experimental conditions are discussed.Newly emerged eco-friendly methods are introduced.

Pharmacokinetics and Tissue Distribution of Anwuligan in Rats after Intravenous and Intragastric Administration by Liquid Chromatography-Mass Spectrometry.

Anwuligan, a natural 2,3-dibenzylbutane lignan from the nutmeg mace of Myristica fragans, has been proved to possess a broad range of pharmacological effects. A rapid, simple, and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been established and successfully applied to the study of pharmacokinetics and tissue distribution of anwuligan after intravenous or intragastric administration. Sample preparation was carried out through a liquid-liquid extraction method with ethyl acetate as the extraction reagent. Arctigenin was used as the internal standard (IS). A gradient program was employed with a mobile phase consisting of 0.1% formic acid aqueous solution and acetonitrile. The mass spectrometer was operated in a positive ionization mode with multiple reaction monitoring. The transitions for quantification were m/z 329.0→205.0 for anwuligan and m/z 373.0→137.0 for IS, respectively. Calibration curves were linear over the ranges of 0.5-2000 ng/mL for both plasma samples and tissue samples (r > 0.996). The absolute bioavailability is 16.2%, which represented the existing of the obvious first-pass effect. An enterohepatic circulation was found after the intragastric administration. Anwuligan could be distributed rapidly and widely in different tissues and maintained a high concentration in the liver. The developed and validated LC-MS/MS method and the pharmacokinetic study of anwuligan would provide reference for the future investigation of the preclinical safety of anwuligan as a candidate drug.

The utility of measuring the apparent diffusion coefficient for peritumoral zone in assessing infiltration depth of endometrial cancer.

BACKGROUND: The invasion depth of endometrial cancer is one of the most important prognosis factors. The aim of the current study was to investigate the diagnostic value of the apparent diffusion coefficient (ADC) of the peritumoral zone for assessing the infiltration depth of endometrial cancer. METHODS: An institutional review board approved this prospective study, and all study participants provided informed consent. A total of 58 patients (mean age 54 ± 8.3 years, range 34-69 years) with endometrial cancer were prospectively enrolled. Two radiologists assessed all preoperative magnetic resonance images with T1, T2, and diffusion-weighted imaging, and determined the location of the deepest invasion of the tumor. The peritumoral zone was defined as a 5-mm-thick zone surrounding and adjacent to the cancerous endometrium. The mean ADC (ADCm) values of the tumor and the peritumoral zone were measured. Sensitivity, specificity, positive and negative predictive values, and the area under the receiver operating characteristic curve (Az) were calculated for visual inspection, and an ADC cutoff value for the peri-endometrial zone was determined for predicting the myometrial invasion depth. RESULTS: The ADCm values of tumors and peritumoral zones were 0.83 × 10- 3 mm2/sec and 1.06 × 10- 3 mm2/sec, respectively. There was no significant difference between the ADCm values of the tumors in the superficial and deep myometrial invasion groups (P > 0.05). However, the ADCm value at the peritumoral zone in the deep myometrial invasion group (1.23 × 10- 3 mm2/sec) significantly differed from that in the superficial myometrial invasion group (0.99 × 10- 3 mm2/sec) (p = 0.005). In assessments of deep myometrial invasion, the sensitivity, specificity, negative predictive value, and positive predictive value were 0.58, 0.93, 0.84, and 0.77, respectively, for the ADCm cutoff value of the peritumoral zone, and 0.71, 0.80, 0.87, and 0.60. respectively, for visual inspection. The accuracy of myometrial invasion depth assessment using the ADCm cutoff value and visual inspection were 83 and 78%, respectively. The Az for both was 0.76. CONCLUSION: ADCm at the peritumoral zone can predict deep myometrial invasion of endometrial cancer. This value can therefore enhance confidence in preoperative endometrial cancer evaluation, and when tailoring surgical approaches.

Prognostic role of pretreatment neutrophil to lymphocyte ratio in breast cancer patients: A meta-analysis.

BACKGROUND: Inflammation and cancer are closely related to each other. As a parameter that can reflect inflammation and host immune reaction, elevated blood neutrophil to lymphocyte ratio (NLR) has been confirmed to be correlated with poor prognosis in a variety of cancers. However, this remains controversial in breast cancer. Thus, we performed this updated meta-analysis to further clarify whether high NLR could be a predictor of survival in breast cancer patients. METHODS: We searched on PubMed Database and Cochrane Library. Overall survival (OS), disease-free survival (DFS), and cancer-specific survival were used as outcome events, and hazard ratio (HR) was chosen as the parameter to evaluate the correlation. RESULT: Eighteen eligible studies were involved in this meta-analysis. The synthesized analysis demonstrated that elevated NLR was associated with poor DFS [HR = 1.72, 95% confidence interval (95% CI) = 1.30-2.27], OS (HR = 1.87, 95% CI = 1.41-2.48), and cancer-specific survival (HR = 2.09, 95% CI = 1.04-4.21). The correlation was stronger in triple-negative breast cancer (TNBC) (OS: HR = 2.58, 95% CI = 1.63-4.06; DFS: HR = 3.51, 95% CI = 1.97-6.24). CONCLUSION: Higher NLR was correlated to poor prognosis of breast cancer patients. As a clinical parameter that we can easily obtain, NLR might be a potential predictor in patients' survival to assist with physicians' treatment decisions.

SUVmax of 18F-FDG PET/CT correlates to expression of major chemotherapy-related tumor markers and serum tumor markers in gastric adenocarcinoma patients.

The expression of P53 was previously found by us significantly correlated with maximal standardized uptake value (SUVmax) in non-small cell lung cancer (NSCLC) patients. Hence, the aim of this study was to clarify the relationship between SUVmax and the status of the chemotherapy-related tumor marker expression or serum tumor markers in gastric adenocarcinoma patients. Sixty-four gastric adenocarcinoma patients who underwent 18F-FDG PET/CT prior to treatment were enrolled in this study. Immunohistochemistry was performed to detect changes of Her-2, P53 and Survivin in lesions, and electrochemiluminescence (ECL) method was used to quantify expression of serum CA72-4, CA19-9 and CEA of these patients. Then, the relationships between these parameters above were assessed by Spearman correlation analysis. Also, receiver-operating characteristic (ROC) curve was performed to determine the best cut-off value of SUVmax for suggesting chemotherapy resistant tumor markers. Besides, we identified a linear correlation to estimate the equations between SUVmax and the serum tumor markers. Our results showed that higher SUVmax was detected in patients with positive expression of Her-2 and P53, compared with negative groups. The Spearman correlation analysis showed that SUVmax was associated with Her-2 or P53 with the moderate relevant Pearson correlation coefficient. ROC curve analysis showed that the sensitivity and specificity of SUVmax for suggesting Her-2 or P53-positive, when the cut-off value of SUVmax was set at 3.25 or 5.45, respectively. Moreover, the relationship between SUVmax and serum tumor markers were analyzed by linear correlation analysis, and serum CA72-4 and CA19-9 could be used as independent parameters to establish an equation for SUVmax by the linear regression models. These results suggested that SUVmax of 18F-FDG PET/CT could be used to predict and evaluate Her-2 or P53 related chemotherapy resistance of gastric adenocarcinoma patients. However, before PET/CT scanning, serum tumor markers could be used to calculate the SUVmax approximately.

Combined subjective and quantitative analysis of magnetic resonance images could improve the diagnostic performance of deep myometrial invasion in endometrial cancer.

PURPOSE: To evaluate whether the combination of subjective magnetic resonance imaging (MRI) and quantitative analysis by using the exponential ADC (EADC) value of the peri-endometrial zone can improve the diagnostic performance of deep myometrial invasion in endometrial cancer patients. MATERIALS AND METHODS: We prospectively evaluated 111 patients with either cervical cancer (normal endometria group) or endometrial cancer (endometrial cancer group). Two radiologists assessed all preoperative MR images with T1, T2, and diffusion-weighted imaging. The EADC value of the peri-endometrial zone was measured. Sensitivity, specificity, positive and negative predictive values, and the area under the receiver operating characteristic curve (Az) were calculated for Subjective MRI, an EADC cutoff value of the peri-endometrial zone and the combination of the two methods in assessing the prediction of deep myometrial invasion. RESULTS: Specificity for EADC cutoff of the peri-endometrial zone was higher (0.93) than for Subjective MRI (0.80), as were the positive predictive values (EADC, 0.79; visual, 0.60). Sensitivity for the combined test was higher (0.88) than for Subjective MRI (0.71) and the EADC cutoff value (0.65), as were the negative predictive values (the combined test, 0.94; vs. EADC, 0.79; vs. Subjective MRI, 0.60). There were no differences in Az between the three methods (P>0.05), but the combined test had the highest Az. CONCLUSIONS: Combined with conventional Subjective MRI, calculating EADC value of the peri-endometrial zone could improve the accuracy of preoperative assessment of deep myometrial invasion in endometrial cancer patients, and maybe helpful in tailoring a surgical approach for intervention.

High tumor-associated macrophages infiltration is associated with poor prognosis and may contribute to the phenomenon of epithelial-mesenchymal transition in gastric cancer.

BACKGROUND: Recent studies show that epithelial-mesenchymal transition (EMT) and tumor-associated macrophages (TAMs) contribute to the progression and poor prognosis of carcinoma through multiple mechanisms. Both inflammation and changing of epithelium have a close relationship with tumorigenesis of gastric cancer. However, the relevance between EMT and TAMs is still unclear in gastric cancer and needs more scientific research. This study is designed to explore the relationship between EMT and TAMs in gastric cancer. MATERIALS AND METHODS: Immunohistochemistry was used to detect the expression of EMT-related proteins and TAM markers in cancer tissues and normal gastric tissues. RESULTS: High levels of EMT and TAMs infiltration are related to aggressive features and independent prognostic factors in gastric cancer, respectively. In addition, expression of the two indicators is associated with expression of transforming growth factor-ß1 (TGF-ß1). Infiltration of TAMs is also associated with EMT-related marker in gastric cancer. CONCLUSION: Our results suggest that high levels of EMT and TAMs infiltration are related to aggressive features and independent prognostic factors in gastric cancer, respectively. A correlation was found between EMT- and TAM-related indicators, which may be associated with TGF-ß signaling pathway. The level of TAMs infiltration plays an important role in gastric cancer, the markers of which can be used as prognostic indicators.

Expression of RAP1B is associated with poor prognosis and promotes an aggressive phenotype in gastric cancer.

Metastasis is the major cause of death among gastric cancer (GC) patients, and altered expression of Ras-related protein RAP1B is associated with cancer development. The present study assessed RAP1B expression ex vivo and the effect of hypoxia­induced RAP1B overexpression on the promotion of the metastatic potential of GC cells in vitro. Immunohistochemistry was used to detect the expression of RAP1B and hypoxia­inducible factor-1α (HIF-1α) in 178 GC tissue specimens. GC cell lines were used to assess the effects of hypoxia and RAP1B knockdown with RAP1B small interfering RNA (siRNA). Tumor cell viability was detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, invasion capacity was evaluated by a Transwell assay, and gene expression was determined by qRT-PCR and western blotting. The data showed that expression levels of RAP1B and HIF-1α proteins were high in the GC tissue specimens, and RAP1B expression was significantly associated with tumor-node-metastasis (TNM) stage and tumor size, while HIF-1α expression was significantly associated with TNM stage, Borrmann type and tumor differentiation. Moreover, RAP1B expression was associated with HIF-1α expression (r=0.547, P<0.001). The expression of RAP1B and HIF-1α proteins was associated with a shorter overall survival of patients according to the univariate analysis (log-rank test, P<0.01), and RAP1B expression and TNM stage were independent prognostic predictors in patients using a multivariate analysis (P<0.001). In vitro, hypoxia induced the invasion of GC cells and the expression of RAP1B and HIF-1α (P<0.05); whereas knockdown of RAP1B expression using siRNA inhibited the tumor cell invasion capacity even under hypoxic culture conditions (P<0.05). In conclusion, the protein expression of RAP1B and HIF-1α contributed to GC malignant behavior and poor prognosis. Future studies will evaluate whether targeting RAP1B expression can be used as a novel strategy to control GC or as a biomarker for prognosis prediction.

The Combination of Diffusion- and T2-Weighted Imaging in Predicting Deep Myometrial Invasion of Endometrial Cancer: A Systematic Review and Meta-Analysis.

OBJECTIVE: The aim of this study was to explore the diagnostic difference among the combination of diffusion-weighted imaging (DWI) and T2-weighted imaging (T2WI), diffusion-weighted imaging (DWI), and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) in predicting deep myometrial invasion of endometrial cancer. METHODS: A structured search was conducted to identify published studies between January 2005 and April 2014, which assessed depth of myometrial invasion in endometrial cancer by using DCE-MRI or DWI or DWI-T2WI. RESULTS: A total of 15 studies were included. Significant difference was found between DWI-T2WI and DWI in pooled specificity, and also in comparison between DCE-MRI and DWI-T2WI (P < 0.05). In summary, receiver operating characteristic analysis, area under the curve for DWI-T2WI, DWI, and DCE-MRI were 0.94, 0.90, and 0.93, respectively. CONCLUSIONS: Diffusion-weighted imaging-T2WI can improve diagnostic performance in comparison with DWI alone. Meanwhile, DWI-T2WI performs better than DCE-MRI in predicting myometrial invasion of endometrial cancer. It may be an alternative for DCE-MRI in presurgical staging of endometrial cancer.

Fluorodeoxyglucose positron emission tomography and chemotherapy-related tumor marker expression in non-small cell lung cancer.

BACKGROUND: The chemotherapy resistance of non-small cell lung cancer (NSCLC) remains a clinic challenge and is closely associated with several biomarkers including epidermal growth factor receptor (EGFR) ( Drugs 72(Suppl 1):28-36, 012.), p53 ( Med Sci Monit 11(6):HY11-HY20, 2005.) and excision repair cross complementing gene 1 (ERCC1) ( J Thorac Oncol 8(5):582-586, 2013.). Fluorodeoxyglucose positron emission tomography (FDG-PET) is the best non-invasive surrogate for tumor biology with the maximal standardized uptake values (SUVmax) being the most important paradigm. However, there are limited data correlating FDG-PET with the chemotherapy resistant tumor markers. The purpose of this study was to determine the correlation of chemotherapy related tumor marker expression with FDG-PET SUVmax in NSCLC. METHODS: FDG-PET SUVmax was calculated in chemotherapy naïve patients with NSCLC (n=62) and immunohistochemical analysis was performed for EGFR, p53 or ERCC1 on the intraoperative NSCLC tissues. Each tumor marker was assessed independently by two pathologists using common grading criteria. The SUVmax difference based on the histologic characteristics, gender, differentiation, grading and age as well as correlation analysis among these parameters were performed. Multiple stepwise regression analysis was further performed to determine the primary predictor for SUVmax and the receiver operating characteristics (ROC) curve analysis was performed to detect the optimized sensitivity and specificity for SUVmax in suggesting chemotherapy resistant tumor markers. RESULTS: The significant tumor type (P=0.045), differentiation (P=0.021), p53 (P=0.000) or ERCC1 (P=0.033) positivity dependent differences of SUVmax values were observed. The tumor differentiation is significantly correlated with SUVmax (R=-0.327), tumor size (R=-0.286), grading (R=-0.499), gender (R=0.286) as well as the expression levels for p53 (R= -0.605) and ERCC1 (R=-0.644). The expression level of p53 is significantly correlated with SUVmax (R=0.508) and grading (R=0.321). Furthermore, multiple stepwise regression analysis revealed that p53 expression was the primary predictor for SUVmax. When the cut-off value of SUVmax was set at 5.15 in the ROC curve analysis, the sensitivity and specificity of SUVmax in suggesting p53 positive NSCLC were 79.5% and 47.8%, respectively. CONCLUSION: The current study suggests that SUVmax of primary tumor on FDG-PET might be a simple and good non-invasive method for predicting p53-related chemotherapy resistance in NSCLC when we set the cu-off value of SUVmax at 5.15.

[Expression and distribution of Nrf2 in several hepatocellular carcinoma cell lines].

AIM: The aim of this study was to investigate the expression and the distribution of Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) in several human hepatocellular cancer cell lines (HepG2, Hep3B, SMMC-7721). METHODS: Flow cytometry was used to figure out the percentages of Nrf2-positive cells in these three cell lines. The localization of Nrf2 was estimated by the laser confocal microsopy and the expression levels of Nrf2 in hepatocellular cancer cell lines were detected by Western blot analysis. RESULTS: The percentages of Nrf2 positive cells in HepG2, Hep3B, and SMMC-7721 were 99.39%, 99.94%, and 99.98% through the flow cytometry. The laser confocal microsopy showed that Nrf2 mainly localized in the cytoplasm of HepG2 cells, distributed evenly in the cytoplasm and nucleus of Hep3B cells, and mainly localized in the nucleus of SMMC-7721 cells. Western blot analysis confirmed the result by the laser confocal microsopy. CONCLUSION: The data on the expression and localization of Nrf2 will be helpful for the following research on the role of Nrf2 in the drug resistance of hepatocellular carcinoma to chemotherapy.

[Effect of low dose propofol on biological behavior of esophageal squamous cell carcinoma cell line Eca109].

AIM: To detect the effect of low dose propofol on the proliferation, apoptosis, migration, and invasion of esophageal squamous cell carcinoma cell line Eca109. METHODS: The proliferation, apoptosis, migration, and invasion of esophageal squamous cell carcinoma cell line Eca109 were detected by MTT assay, flow cytometry, transwell assay respectively. The effect of low dose propofol on expression of heme oxygenase-1 (HO-1) was confirmed by Real-time quantitative PCR. RESULTS: Low dose propofol could inhibit the proliferation, migration, invasion and promate the apoptosis of esophageal squamous cell carcinoma cell line Eca109. And low dose propofol increased the expression of HO-1 mRNA in a dose-dependment manner. CONCLUSION: Low dose propofol affects the biological behavior of esophageal squamous cell carcinoma cell line Eca109, which has a relationship with increasing the expression of HO-1.

[Effects of antidiabetic drug metformin on human breast carcinoma cells with different estrogen receptor expressing in vitro].

AIM: To research different effects of human breast carcinoma cells with different estrogen receptor expressing by antidiabetic drug metformin, and preliminary explore the possible underlying molecular mechanisms. METHODS: Cells were treated with metformin. Growth inhibition rates of the cells were measured by MTT assay. Cell cycle and apoptosis were detected by flow cytometery (FCM). Expressions of HIF-1α mRNA in the cells were measured by RT-PCR. RESULTS: After drug intervention, the cell proliferation were inhibited by metformin, and reinforced with the concentration and reaction time increase (P<0.05). The growth inhibition rates of MCF-7(ER(+);) breast carcinoma cell were higher than MDA-MB-231(ER(-);) breast carcinoma cell at each concentration group (P<0.05). The results of FCM prompted: MCF-7(ER(+);) breast carcinoma cell was arrested in G1 phase by metformin significantly in a dose-dependent increase(P<0.05). While for MDA-MB-231(ER(-);) breast carcinoma cell, only the 20 and 40 mmol/L groups had significant difference compared with the control group(P<0.05); and the percentage of G1 phase arresting were lower than MCF-7(ER(+);) breast carcinoma cell at the same concentration group(P<0.05). The effect of apoptosis for these two kinds of cells via metformin were not obvious(P<0.05). The expressions of HIF-1α mRNA detected by RT-PCR prompted: the expressions of HIF-1α mRNA for these two breast carcinoma cells were in a dose-dependent decrease(P<0.05). CONCLUSION: The effect of metformin for human breast carcinoma cell with estrogen receptor was better than the one without estrogen receptor. Maybe the molecular mechanism had a relationship with HIF-1α up-regulating.

[Differential expression of hsa-miR-126 and hsa-miR-518b in esophageal squamous carcinoma].

OBJECTIVE: To identify the differentially expressed microRNAs (miRNAs) between esophageal squamous carcinoma (ESC) and adjacent non-tumorous tissue (NT). METHODS: The expression levels of the miRNAs were detected in 3 fresh ESC and NT samples by hybridization with miRNAs microarray chip. Real-time quantitative RT-PCR was performed to confirm the results of the microarray analysis. The expressions of hsa-miR-126 and hsa-miR-518b in ESC were validated by real-time quantitative RT-PCR in another independent 15 matched samples. RESULTS: A total of 11 miRNAs exhibited differential expressions in ESC samples as compared to their expressions in the NT samples, including a 1 up-regulated miRNA and 10 down-regulated miRNAs. Compared with normal esophageal samples, the ESC tissues showed up-regulated hsa-miR-126 and down-regulated hsa-miR-518b expression. CONCLUSION: hsa-miR-126 and hsa-miR-518b are differentially expressed in ESC, and they might play important roles in the carcinogenesis and progression of ESC.

[Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody].

OBJECTIVE: To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system. METHODS: The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody. RESULTS: The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII. CONCLUSION: An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.

Generation of fusion protein EGFRvIII-HBcAg and its anti-tumor effect in vivo.

The epidermal growth factor receptor variant III (EGFRvIII) is the most common variation of EGFR. Because it shows a high frequency in several different types of tumor and has not been detected in normal tissues, it is an ideal target for tumor specific therapy. In this study, we prepared EGFRvIII-HBcAg fusion protein. After immunization with fusion protein, HBcAg or PBS, the titers of antibody in BALB/c mice immunized with fusion protein reached 2.75 x 10(5). Western blot analysis demonstrated that the fusion protein had specific antigenicity against anti-EGFRvIII antibody. Further observation showed fusion protein induced a high frequency of IFN-gamma-secreting lymphocytes. CD4+T cells rather than CD8+T cells were associated with the production of IFN-gamma. Using Renca-vIII(+) cell as specific stimulator, we observed remarkable cytotoxic activity in splenocytes from mice immunized with fusion protein. Mice were challenged with Renca-vIII(+) cells after five times immunization. In fusion protein group, three of ten mice failed to develop tumor and all survived at the end of the research. The weight of tumors in fusion protein were obviously lighter than that in other two groups (t = 4.73, P = 0.044; t = 6.89, P = 0.040). These findings demonstrated that EGFRvIII-HBcAg fusion protein triggered protective responses against tumor expressing EGFRvIII.

[Construction of a prokaryotic expression vector for apoptin and preparation of polyclonal antibody of apoptin].

OBJECTIVE: To construct a prokaryotic expression vector for apoptin and prepare polyclonal antibody of apoptin. METHODS: Apoptin gene amplified from pGEM-T/Apoptin plasmid by PCR was cloned into pET-28a (+). E.coli BL21 (DE3) was transformed by the recombinant plasmid, and apoptin protein expression induced by IPTG was analyzed by SDS-PAGE. BALB/c mice were immunized with the protein and the titer of the antibody was determined using indirect enzyme-linked immunosorbent assay (ELISA). RESULTS: Apoptin gene was successfully cloned into pET-28a (+), and the expression of a protein with relative molecular mass of about 17 000 was identified by SDA-PAGE. After 5 immunizations of the mice with the protein, the blood antibody titer reached 1:5x10(5). CONCLUSION: The prokaryotic expression vector for apoptin is successfully constructed and the polyclonal antibody of apoptin is obtained, which allows further functional study of apoptin.

A meta-analysis of the accuracy of prostate cancer studies which use magnetic resonance spectroscopy as a diagnostic tool.

OBJECTIVE: We aimed to do a meta-analysis of the existing literature to assess the accuracy of prostate cancer studies which use magnetic resonance spectroscopy (MRS) as a diagnostic tool. MATERIALS AND METHODS: Prospectively, independent, blind studies were selected from the Cochrane library, Pubmed, and other network databases. The criteria for inclusion and exclusion in this study referenced the criteria of diagnostic research published by the Cochrane center. The statistical analysis was adopted by using Meta-Test version 6.0. Using the homogeneity test, a statistical effect model was chosen to calculate different pooled weighted values of sensitivity, specificity, and the corresponding 95% confidence intervals (95% CI). The summary receiver operating characteristic (SROC) curves method was used to assess the results. RESULTS: We chose two cut-off values (0.75 and 0.86) as the diagnostic criteria for discriminating between benign and malignant. In the first diagnostic criterion, the pooled weighted sensitivity, specificity, and corresponding 95% CI (expressed as area under curve [AUC]) were 0.82 (0.73, 0.89), 0.68 (0.58, 0.76), and 83.4% (74.97, 91.83). In the second criterion, the pooled weighted sensitivity, specificity, and corresponding 95% CI were 0.64 (0.55, 0.72), 0.86 (0.79, 0.91) and 82.7% (68.73, 96.68). CONCLUSION: As a new method in the diagnostic of prostate cancer, MRS has a better applied value compared to other common modalities. Ultimately, large scale RCT (randomized controlled trial) randomized controlled trial studies are necessary to assess its clinical value.

[Construction and identification of recombinant adeno-associated virus vector harboring fusion gene NT4-Apoptin-HA2-TAT].

AIM: To construct a recombinant adeno-associated virus vector harboring fusion gene NT4-Apoptin-HA2-TAT, laying a foundation for gene therapy research of malignant tumors. METHODS: The Apoptin and HA2-TAT gene were inserted in pUC19/NT4 vector after digested with restriction enzyme. The fusion gene of NT4-Apoptin-HA2-TAT was sub-cloned into the shuttle plasmid of adeno-associated virus; the products were co-transferred into HEK-293 cell line with helper plasmid pAAV/Ad and adeno-plasmid pFG140.The recombinant adeno-associated virus was produced by homologous recombination of above 3 plasmids in HEK-293 cells and its titer was measured by quantitative dot blot hybridization. The effect of AAV-NT4-Apoptin -HA2-TAT on HepG2 cell line was measured by a colorimetric 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. RESULTS: The NT4-Apoptin-HA2-TAT was confirmed by restriction enzyme digestion and DNA sequencing. High titer of recombinant adeno-associated virus was obtained by homologous recombination in HEK-293 cells (3.14 x 10(15) pfu/L). AAV-NT4-Apoptin-HA2-TAT had strong deduce apoptosis effect on HepG2 cells. Compared with Adeno-associated mock virus and in normal cell line NIH3T3, Aden-associated virus NT4-Apoptin-HA2-TAT significantly decreased the survival rate of HepG2 cells. CONCLUSION: The recombinant adeno-associated virus vector encoding gene NT4-Apoptin-HA2-TAT has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques, laying a foundation for further research of gene therapy of cancer.