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[en] To address whether hepcidin functions in bone metabolism. This study was carried out in the Laboratory of Radiation Medicine and Public Health of Soochow University, and the Laboratory of the Second Affiliated Hospital of Soochow University, Suzhou, China, from September 2009 to July 2010. The positive expression of ferroportin-1 (Fpn-1) was detected by reverse transcriptase-polymerase chain reaction. After the treatment with distilled water (control group) and hepcidin (25noml/L, 50noml/L, 100noml/L), the fluorescence intensity related to intracellular iron concentration of a human fetal osteoblast cell line (hFOB 1.19) was measured by a confocal laser scanning microscope. A 3-(4,5- dimethylthiazol-2-yl) -2-5-diphenyltetrazolium bromide assay, and Von Kossa staining was performed to evaluate cell proliferation and mineralization in cultured hFOB 1.19 cells. This study revealed a high level expression of Fpn-1 in hFOB 1.19. On the basis of which, it was found that 25noml/L, 50noml/L, 100noml/L hepcidin could promote the fluorescence intensity related to intracellular iron concentration and mineralization in hFOB 1.19 in a dose-dependent manner (p<0.05), but hepcidin had no effect on FOB 1.19 proliferation (p>0.05). The hepcidin-ferroportin signal pathway may function in the osteoblast cell line of hFOB 1.19 cells. It is also suggested that cross-talk between iron and calcium homeostasis may play a role in bone metabolism in responding to hepcidin activation (Author).
[en] Full text: The achievement of whole genome sequencing in various species has accelerated the development of genome-wide gene expression profiling techniques. Micro-array procedures based on hybridization technology have been used exclusively for this purpose. However, some problems have been raised in actual execution. Here, we report a new expression-profiling method based on PCR technology. This procedure allowed us to analyze more than 80% of expressed genes in certain cells and to investigate species in which genome studies have not yet been carried out, because the procedure does not require any genome information. Furthermore, the method can discern even 1.5-fold gene expression differences, facilitating the narrowing down of candidates isolated by genome-wide approaches to a few strong candidates
[en] The cytogenetic hallmark of myxoid type and round cell type liposarcoma consists of reciprocal translocation of t(12;16)(q13;p11) and t(12;22)(q13;q12), which results in fusion of TLS/FUS and CHOP, and EWS and CHOP, respectively. Nine structural variations of the TLS/FUS-CHOP chimeric transcript have been reported, however, only two types of EWS-CHOP have been described. We describe here a case of myxoid liposarcoma containing a novel EWS-CHOP chimeric transcript and identified the breakpoint occurring in intron 13 of EWS. Reverse transcription-polymerase chain reaction and direct sequence showed that exon 13 of EWS was in-frame fused to exon 2 of CHOP. Genomic analysis revealed that the breaks were located in intron 13 of EWS and intron 1 of CHOP
[en] Cholangiocarcinoma is characterized by late diagnosis and a poor survival rate. MicroRNAs have been involved in the pathogenesis of different cancer types, including cholangiocarcinoma. Our aim was to identify novel microRNAs regulating cholangiocarcinoma cell growth in vitro and in vivo. A functional microRNA library screen was performed in human cholangiocarcinoma cells to identify microRNAs that regulate cholangiocarcinoma cell growth. Real-time PCR analysis evaluated miR-9 and XIAP mRNA levels in cholangiocarcinoma cells and tumors. The screen identified 21 microRNAs that regulated >50 % cholangiocarcinoma cell growth. MiR-410 was identified as the top suppressor of growth, while its overexpression significantly inhibited the invasion and colony formation ability of cholangiocarcinoma cells. Bioinformatics analysis revealed that microRNA-410 exerts its effects through the direct regulation of the X-linked inhibitor of apoptosis protein (XIAP). Furthermore, overexpression of miR-410 significantly reduced cholangiocarcinoma tumor growth in a xenograft mouse model through induction of apoptosis. In addition, we identified an inverse relationship between miR-410 and XIAP mRNA levels in human cholangiocarcinomas. Taken together, our study revealed a novel microRNA signaling pathway involved in cholangiocarcinoma and suggests that manipulation of the miR-410/XIAP pathway could have a therapeutic potential for cholangiocarcinoma. The online version of this article (doi:10.1186/s12885-016-2384-0) contains supplementary material, which is available to authorized users
[en] The incidence of and mortality from colorectal cancers (CRC) can be reduced by early detection. Currently there is a lack of established markers to detect early neoplastic changes. We aimed to identify the copy number variations (CNVs) and the associated genes which could be potential markers for the detection of neoplasia in both ulcerative colitis-associated neoplasia (UC-CRN) and sporadic colorectal neoplasia (S-CRN). We employed array comparative genome hybridization (aCGH) to identify CNVs in tissue samples of UC nonprogressor, progressor and sporadic CRC. Select genes within these CNV regions as a panel of markers were validated using quantitative real time PCR (qRT-PCR) method along with the microsatellite instability (MSI) in an independent cohort of samples. Immunohistochemistry (IHC) analysis was also performed. Integrated analysis showed 10 overlapping CNV regions between UC-Progressor and S-CRN, with the 8q and 12p regions showing greater overlap. The qRT-PCR based panel of MYC, MYCN, CCND1, CCND2, EGFR and FNDC3A was successful in detecting neoplasia with an overall accuracy of 54 % in S-CRN compared to that of 29 % in UC neoplastic samples. IHC study showed that p53 and CCND1 were significantly overexpressed with an increasing frequency from pre-neoplastic to neoplastic stages. EGFR and AMACR were expressed only in the neoplastic conditions. CNVs that are common and unique to both UC-associated and sporadic colorectal neoplasm could be the key players driving carcinogenesis. Comparative analysis of CNVs provides testable driver aberrations but needs further evaluation in larger cohorts of samples. These markers may help in developing more effective neoplasia-detection strategies during screening and surveillance programs. The online version of this article (doi:10.1186/s12885-016-2303-4) contains supplementary material, which is available to authorized users
[en] MicroRNAs (miRNAs) regulate gene expression by binding to mRNA, and can function as oncogenes or tumor suppressors depending on the target. In this study, using qRT-PCR, we examined the expression of six miRNAs (miR-21, miR-31, miR-92a, miR-101, miR-106a and miR-145) in tumors from 193 prospectively recruited patients with colorectal cancer, and associations with clinicopathological parameters and patient outcome were analyzed. The miRNAs were chosen based on previous studies for their biomarker potential and suggested biological relevance in colorectal cancer. The miRNA expression was examined by qRT-PCR. Associations between miRNA expression and clinicopathological variables were explored using Mann–Whitney U and Kruskal-Wallis test while survival was estimated using the Kaplan-Meier method and compared using the log-rank test. MiR-101 was hardly expressed in the tumor samples, while for the other miRNAs, variable expression levels and expression ranges were observed, with miR-21 being most abundantly expressed relative to the reference (RNU44). In our study cohort, major clinical significance was demonstrated only for miR-31, as high expression was associated with advanced tumor stage and poor differentiation. No significant associations were found between expression of the investigated miRNAs and metastasis-free or overall survival. Investigating the expression of six miRNAs previously identified as candidate biomarkers in colorectal cancer, few clinically relevant associations were detected in our patient cohort. Our results emphasize the importance of validating potential tumor markers in independent patient cohorts, and indicate that the role of miRNAs as colorectal cancer biomarkers is still undetermined
[en] In a model of human neuroblastoma (NB) cell lines persistently infected with human cytomegalovirus (HCMV) we previously showed that persistent HCMV infection is associated with an increased malignant phenotype, enhanced drug resistance, and invasive properties. To gain insights into the mechanisms of increased malignancy we analyzed the global changes in cellular gene expression induced by persistent HCMV infection of human neuroblastoma cells by use of high-density oligonucleotide microarrays (HG-U133A, Affymetrix) and RT-PCR. Comparing the gene expression of different NB cell lines with persistently infected cell sub-lines revealed 11 host cell genes regulated in a similar manner throughout all infected samples. Nine of these 11 genes may contribute to the previously observed changes in malignant phenotype of persistently HCMV infected NB cells by influencing invasive growth, apoptosis, angiogenesis, and proliferation. Thus, this work provides the basis for further functional studies
[en] MicroRNAs (miRNAs) are small noncoding RNAs that potentially play a critical role in tumorigenesis. Mounting evidence indicates that one specific miRNA: miR-320b is down regulated in numerous human cancers, including colorectal cancer (CRC); making the hypothesis that miR-320b may play a key role in tumorigenesis plausible. However, its role in carcinogenesis remains poorly defined. The goal of this study is to better clarify the role of miR-320b in tumor growth of CRC. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was conducted to detect the expression of miR-320b in CRC tissues and 5 CRC cell lines. The effect of miR-320b on cell proliferation was analyzed in vitro and in vivo. Furthermore, a luciferase reporter assay was performed to measure the target effects of miR-320b. Lastly, the messenger RNA (mRNA) and protein levels of the gene c-MYC were measured in CRC cell lines and tissues by qRT-PCR, and confirmed via Western blot and Immunohistochemical (IHC) staining. The results presented here showed that miR-320b expression was down regulated in both CRC tissues and cells. Overexpression of miR-320b in CRC cells was statistically correlated with a decrease of cell growth in vitro and in vivo, while c-MYC was identified as a target gene of miR-320b in CRC. Furthermore, it was found that up-regulation of c-Myc can attenuate the effects induced by miR-320b. Our identification of c-MYC as a target gene of miR-320b provides new insights into the pathophysiology of CRC proliferation, and identifies miR-320b as a novel therapeutic target for the treatment of CRC
[en] Highlights: • Voltammetric behaviour of 5-(4-azidophenyl)-2′-deoxycytidine (dCAZP) at HMDE in aqueous media. • Nanomolar concentrations of dCAZP and ss and dsDNA labeled by azidophenyl detectable at HMDE. • Precise sequence-specific incorporation of dCAZP into DNA by primer extension. • Polymerase chain reaction produces an about 350-bp double-stranded DNA fragment globally modified with dCAZP. • Terminal deoxynucleotidyl transferase tailing reaction generates dCAZP end-labeled single stranded oligonucleotides - Abstract: Voltammetric determination of a redox labeled nucleoside 5-(4-azidophenyl)-2′-deoxycytidine (dCAZP) and various polymerase-synthesized dCAZP-labeled DNAs in aqueous buffers is presented. Influence of: i) pH (2–12), ii) scan rates (0.02–10 V s−1), and iii) dCAZP concentration (0.02–10 μmol l−1), on voltammograms of dCAZP were systematically studied for the first time using CV at a hanging mercury drop electrode. Electrode potential-controlled adsorption driven process allowed sensitive determination of dCAZP at nanomolar concentrations using adsorptive stripping voltammetry. Transfer stripping voltammetry (TSV) was used for the detection of dCAZP-labeled DNA in femtomole quantities. Precise sequence-specific incorporation of dCAZP into DNA by primer extension was used to demonstrate a perfect correlation between the number of incorporated AZP moieties and TSV responses. In addition, for the first time we used polymerase chain reaction to prepare an about 350-bp double-stranded DNA fragment globally modified with dCAZP, and of terminal deoxynucleotidyl transferase tailing reaction to generate end-labeled single stranded oligonucleotides. Effects of DNA structure on the AZP-modified DNA TSV responses are discussed.
[en] The prognosis of patients with Ewing sarcoma (ES) has improved over the course of the last decades. However, those patients suffering from metastatic and recurrent ES still have only poor chances of survival and require new therapeutic approaches. Interleukin-6 (IL6) is a pleiotropic cytokine expressed by immune cells and a great variety of cancer cells. It induces inflammatory responses, enhances proliferation and inhibits apoptosis in cancer cells, thereby promoting chemoresistance. We investigated expression of IL6, its receptors and the IL6 signal transduction pathway in ES tumor samples and cell lines applying reverse transcriptase PCR, immunoblot and immunohistochemistry. The impact of IL6 on cell viability and apoptosis in ES cell lines was analyzed by MTT and propidium iodide staining, migration assessed by chorioallantoic membrane (CAM) assay. Immunohistochemistry proved IL6 expression in the stroma of ES tumor samples. IL6 receptor subunits IL6R and IL6ST were expressed on the surface of ES cells. Treatment of ES cells with rhIL6 resulted in phosphorylation of STAT3. rhIL6 protected ES cells from serum starvation-induced apoptosis and promoted migration. IL6 blood serum levels were elevated in a subgroup of ES patients with poor prognosis. These data suggest that IL6 contributes to ES tumor progression by increasing resistance to apoptosis in conditions of cellular stress, such as serum starvation, and by promotion of metastasis. The online version of this article (doi:10.1186/s12885-015-1564-7) contains supplementary material, which is available to authorized users