Association of the plasma riboflavin levels and riboflavin transporter (C20orf54) gene statuses in Kazak esophageal squamous cell carcinoma patients

To evaluate the association of the plasma riboflavin level in Kazak esophageal cancer patients and their riboflavin transporter (C20orf54) gene statuses. Plasma riboflavin levels were detected by high performance liquid chromatography in Kazak patients with esophageal squamous cell carcinoma (ESCC) and healthy controls. C20orf54 mRNA and protein expression were analyzed by real-time fluorogenic quantitative polymerase chain reaction and immunohistochemistry in samples from 61 ESCC patients consisting of both tumor and normal tissue, respectively. C20orf54 mRNA expression was decreased in ESCC (0.279 ± 0.102) than in normal counterpart tissue (0.479 ± 0.287; P = 0.049) significantly. Tumors exhibited low C20orf54 protein expression (42.6, 26.2, 18.0 and 13.1 % for no C20orf54 staining, weak staining, medium staining and strong staining, respectively), which was significantly lower than that in the normal mucous membrane (13.1, 26.2, 41.0 and 19.7 % for no C20orf54 staining, weak staining, medium staining and strong staining, respectively). Defective expression of C20orf54 in tumor cells was significantly associated with poor differentiation. However, other parameters such as depth of invasion and lymph node metastasis had no significant relationship with C20orf54 expression. The average blood concentration of riboflavin was 2.6468 ± 1.3474 ng/ml in ESCC patients lower than control group (4.2960 ± 3.2293 ng/ml, P = 0.015). A positive correlation of plasma riboflavin levels with defective expression of C20orf54 protein was found in ESCC patients (F = 8.626; P = 0.038). Defective expression of C20orf54 is associated with the development of Kazak esophageal squamous cell carcinoma and this may represent a mechanism underlying the decreased plasma riboflavin levels in ESCC.     

Down-regulation of platelet-derived growth factor-D expression blockades NF-κB pathway to inhibit cell proliferation and invasion as well as induce apoptosis in esophageal squamous cell carcinoma

Substantial evidence has demonstrated that platelet-derived growth factor-D (PDGF-D) is tightly associated with the development and progression of tumors. However, its biological functions in esophageal squamous cell carcinoma (ESCC) remain to be delineated. In this study, we found that expressions of PDGF-D mRNA and protein in ESCC tissues and cells were significantly higher than that in normal esophageal epithelial tissues (P < 0.05), further investigation showed that PDGF-D protein level in EC1 cells was obviously higher than those in EC9706 and Eca109 cells (P < 0.05). Elevated PDGF-D level was closely associated with TNM staging, tumor differentiation and lymph node metastasis (P < 0.05), but not related to the patients’ age and gender (P > 0.05). In addition, down-regulation of PDGF-D expression markedly inhibited proliferation, reduced invasion and induced apoptosis in EC1 cells. More importantly, reduced PDGF-D level evoked the down-regulation of p65 and p-IκBα proteins and elevation of IκBα protein of NF-κB pathway, accompanied with the decreases of bcl-2 and MMP-9 protein expressions and increases of bax protein level and caspase-3 activities. Correctively, our data suggest that PDGF-D plays pivotal roles in the development and progression of ESCC, and combinations with PDGF-D and NF-κB pathway may be effective and feasible molecular targets for therapy of ESCC.     

MicroRNA-143 functions as a tumor suppressor in human esophageal squamous cell carcinoma

Esophageal cancer is one of the most common cancers worldwide with a poor prognosis. MicroRNAs(miRNAs) are a class of naturally occurring small noncoding RNAs and play an important role in cancer initiation and development. In this study, we demonstrate that the expression levels of miR-143 and miR-145 were significantly decreased in ESCC tissues in comparison with adjacent normal esophageal squamous tissues(NESTs). Furthermore, an inverse correlation between miR-143 and tumor invasion depth and lymph node metastasis was observed. The enforced expression of miR-143 induced growth suppression and apoptosis of ESCC cells. Rescue of miR-143 significantly suppressed the ESCC cells migration and invasion capabilities. Moreover, we show that functions of miR-143 in ESCC are mediated at least in part by the inhibition of extracellular signal regulated kinase-5(ERK-5) activity. These results prove that miR-143 may act as a tumor suppressor in ESCC.     

H2-Calponin 在食管鳞癌中的表达及其与食管磷癌预后的关系

目的:探讨H_2-Calponin在食管磷癌组织中的表达及其与食管磷癌患者临床病理特征及预后的相关性。方法:利用免疫组织化学染色技术检测73例食管磷癌及相应癌旁组织中H_2-Calponin的表达情况,并分析其与食管磷癌患者临床病理参数及预后的关系。结果:H_2-calponin在食管磷癌组织中的阳性表达率为95.8%(70/73),显著高于癌旁组织(35.8%,24/67),差异具有统计学意义(p0.0001)。H_2-calponin的表达与食管癌患者的性别、年龄、肿瘤大小、浸润深度及淋巴结转移均无显著相关性,但与AJCC分期显著相关(P0.05)。H_2-calponin的表达水平影响食管癌患者预后及生存期。结论:H_2-Calponin在食管磷癌组织中呈高表达并可预示食管磷癌预后不良。     

食管癌组织中HIF-1α、Survivin、CyclinD 1蛋白的表达及其意义

目的：探讨缺氧诱导因子-1α（HIF-1α）、生存蛋白（survivin）、细胞周期蛋白D1（cyclinD 1）在食管癌组织中的表达及其临床意义。方法：应用免疫组化技术检测50例食管癌组织和10例手术切除的远端正常食管组织中HIF-1α、Survivin、CyclinD 1蛋白的表达。结果：食管癌组织中HIF-1α、Survivin、CyclinD 1蛋白阳性表达率均与肿瘤浸润深度以及淋巴结转移相关（P〈0.05）,Survivin阳性表达率与肿瘤分级相关（P〈0.05）,HIF-1α与CyclinD 1的表达呈显著正相关（P〈0.05）。结论：检测HIF-1α、Survivin、CyclinD 1的蛋白的表达有助于判断食管癌的恶性程度以及推断其临床预后。     

TWIST1, MMP-21, and HLAG-1 co-overexpression is associated with ESCC aggressiveness

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive types of cancer, requiring reliable biomarkers for prognosis and therapeutic responsiveness. TWIST1, as an important factor responsible for metastasis of several cancers, is involved in tumor invasion and metastasis through indirectly regulation of MMP-21 expression. On the other hand, NF-ĸβ which is a regulator of HLAG-1 has direct interaction with TWIST1 protein. In this retrospective study we investigated the clinical significance of TWIST1, MMP-21, and HLAG-1 expression in ESCC, and the possible correlation between these genes and progression of the disease. The gene expression analyses of TWIST1, MMP-21, and HLA-G1 were performed by relative comparative real-time polymerase chain reaction in 58 ESCCs compared with corresponding margin-normal esophageal tissues. Significant overexpression of HLAG-1, TWIST1, and MMP-21 messenger RNA was observed in 22.4%, 41.4%, and 60.3% of tumor samples, respectively. Concomitant overexpression of TWIST1/MMP-21 and TWIST1/HLAG-1 were significantly correlated to each other in various clinicopathological features, including depth of tumor invasion, stage of tumor progression, lymphatic invasion, and grade of tumor cell differentiation ( P < 0.05). The current study is the first report of coexpression of TWIST1, MMP-21, and HLAG-1 in ESCC. Such findings suggest an oncogenic role for concomitant expression of these genes in ESCC invasion and metastasis.     

LMP gene promoter hypermethylation is a mechanism for its down regulation in Kazak’s esophageal squamous cell carcinomas

Abnormal hypermethylation of CpG islands not only associated with tumor suppressor genes can lead to repression of gene expression, but also contribute to escape of the tumor from immune surveillance and contribute significantly to tumorigenesis. In the present study, we studied the hypermethylation of low molecular-weight protein (LMP) gene and its regulation on protein expression in biopsies from resected tissues from Kazak’s esophageal squamous cell carcinoma (ESCC) patients and their neighboring normal tissues. LMP2 and LMP7 genes promoter region methylation sequences were maped in esophageal cancer cell line Eca109 by bisulfite-sequencing PCR and quantitative detection of methylated DNA from 30 pairs of Kazak’s ESCC and adjacent normal tissues by MassARRAY (Sequenom, San Diego, CA, USA) and LMP2 and LMP7 protein expression were analyzed with immunohistochemistry. In Eca109, we identified 6 CG sites methylated from all of 22 CpG sites of LMP7 gene. However, no methylation was found for LMP2. The analysis of the data resulted from the quantitative analysis of single CpG site methylation by Sequenom MassARRAY platform, has shown that the methylation level between two groups CpG sites (CpG_5, CpG_9, CpG_20, CpG_21 and CpG_20) from CpG_1, CpG_2, CpG_3, CpG_4, CpG_5, CpG_6, CpG_7, CpG_8, CpG_9, CpG_10.11, CpG_12.13.14, CpG_15.16.17.18, CpG_19, CpG_20, CpG_21 and CpG_22 significant differences between ESCC and neighboring normal tissues. The analysis of methylation level of whole target CpG fragment indicated that the methylation level of LMP7 was significant higher in ESCC (0.0517 ± 0.0357) than in neighboring normal tissues (0.0380 ± 0.0214, P < 0.05). there was a tendency of decreasing the LMP7 proteins expression as the increasing the methylation level of LMP7 gene promoter regions (F = 7.69, P = 0.041). The LMP7 gene promoter methylation and protein downregulation were correlated at high extent in Kazakh’s ESCC patients, and may explain the epigenetic regulation on gene expression.     

SASH1 regulates proliferation, apoptosis, and invasion of osteosarcoma cell

SASH1, a member of the SLY-family of signal adapter proteins, is a candidate tumor suppressor in breast and colon cancer. The SASH1 protein possesses both the SH3 and SAM domains, indicating that it may play an important role in intracellular signal transduction. Reduced expression of SASH1 is closely related to tumor growth, invasion, metastasis, and poor prognosis. However, the biological role of SASH1 remains unknown in osteosarcoma. To unravel the function of SASH1, we explored the expression of SASH1 in osteosarcoma tissues and its correlation to the clinical pathology of osteosarcoma and analyzed the relationship between SASH1 expression and cell cycle, apoptosis and invasion of osteosarcoma MG-63 cells, using the flow cytometry analysis and transwell invasion chamber experiments. Furthermore, the effect of SASH1 on the expression of cyclin D1, caspase-3, matrix metalloproteinase (MMP)-9 were observed by western blot. Our results showed that the expression rate of SASH1 mRNA in osteosarcoma tissues was significantly lower than that in normal bone tissue (p = 0.000), that the expression rate of SASH1 mRNA in the carcinoma tissues from patients with lung metastasis was significantly lower than that from patients without lung metastasis (p = 0.041), and that the expression rate of SASH1 mRNA also decreased with increasing Enneking stage (p = 0.032). However, the mRNA expression of SASH1 in osteosarcoma was independent of the patient’s gender, age, and tumor size (p = 0.983, 0.343, 0.517, respectively). The SASH1 protein displayed a down-regulation in osteosarcoma tissues compared to normal bone tissue (p = 0.000), displayed a down-regulation in osteosarcoma tissues from patients with lung metastasis compared to from patients without lung metastasis (p = 0.000), and displayed a gradual decrease with increasing Enneking stage (p = 0.000). In addition, the MG-63 cells from pcDNA3.1-SASH1 group exhibited significantly reduced cell viability, proliferation, and invasive ability compared to the empty vector group and blank control group (p = 0.023, 0.001, respectively), and there was no difference between the empty vector group and blank control group. The pcDNA3.1-SASH1 group displayed significantly more apoptotic cells than the empty vector group and blank control group (p = 0.004). The expression of cyclin D1, MMP-9 displayed a down-regulation in MG-63 cells from pcDNA3.1-SASH1 group compared to the empty vector group and blank control group (p = 0.000, 0.001, respectively) and the expression levels of caspase-3 displayed an up-regulation in MG-63 cells from pcDNA3.1-SASH1 group compared to the empty vector group and blank control group (p = 0.000). Taken together, these data indicated that the overexpression of SASH1 might be associated with the inhibition of growth, proliferation, and invasion of MG-63 cells and the promotion of apoptosis of MG-63 cells.     

The oncogenic RNA-binding protein Musashi1 is regulated by HuR via mRNA translation and stability in glioblastoma cells

Musashi1 (Msi1) is an evolutionarily conserved RNA-binding protein (RBP) that has profound implications in cellular processes such as stem cell maintenance, nervous system development, and tumorigenesis. Msi1 is highly expressed in many cancers, including glioblastoma, whereas in normal tissues, its expression is restricted to stem cells. Unfortunately, the factors that modulate Msi1 expression and trigger high levels in tumors are largely unknown. The Msi1 mRNA has a long 3' untranslated region (UTR) containing several AU- and U-rich sequences. This type of sequence motif is often targeted by HuR, another important RBP known to be highly expressed in tumor tissue such as glioblastoma and to regulate a variety of cancer-related genes. In this report, we show an interaction between HuR and the Msi1 3'-UTR, resulting in a positive regulation of Msi1 expression. We show that HuR increased MSI1 mRNA stability and promoted its translation. We also present evidence that expression of HuR and Msi1 correlate positively in clinical glioblastoma samples. Finally, we show that inhibition of cell proliferation, increased apoptosis, and changes in cell-cycle profile as a result of silencing HuR are partially rescued when Msi1 is ectopically expressed. In summary, our results suggest that HuR is an important regulator of Msi1 in glioblastoma and that this regulation has important biological consequences during gliomagenesis.     

WISP1 silencing confers protection against epithelial–mesenchymal transition of renal tubular epithelial cells in rats via inactivation of the wnt/β-catenin signaling pathway in uremia

Uremia can affect hepatic metabolism of drugs by regulating the clearance of drugs, but it has not been clarified whether gene silencing could modulate the epithelial–mesenchymal transition (EMT) process in uremia. Hence, we investigated the effect of WISP1 gene silencing on the renal tubular EMT in uremia through the wnt/β-catenin signaling pathway. Initially, microarray-based gene expression profiling of uremia was used to identify differentially expressed genes. Following the establishment of uremia rat model, serum creatinine, and urea nitrogen of rats were detected. Renal tubular epithelial cells (TECs) were transfected with shRNA-WISP1 lentivirus interference vectors and LiCI (the wnt/β-catenin signaling pathway activator) to explore the regulatory mechanism of WISP1 in uremia in relation to the wnt/β-catenin signaling pathway. Then, expression of WISP1, wnt2b, E-cadherin, α-SMA, c-myc, Cyclin D1, MMP-2, and MMP-9 was determined. Furthermore, TEC migration and invasion were evaluated. Results suggested that WISP1 and the wnt/β-catenin signaling pathway were associated with uremia. Uremic rats exhibited increased serum creatinine and urea nitrogen levels, upregulated WISPl, and activated wnt/β-catenin signaling pathway. Subsequently, WISP1 silencing decreased wnt2b, c-myc, Cyclin D1, α-SMA, MMP-2, and MMP-9 expression but increased E-cadherin expression, whereas LiCI treatment exhibited the opposite trends. In addition, WISP1 silencing suppressed TEC migration and invasion, whereas LiCI treatment promoted TEC migration and invasion. The findings indicate that WISP1 gene silencing suppresses the activation of the wnt/β-catenin signaling pathway, thus reducing EMT of renal TECs in uremic rats.     

Copper transporter Ctr1 contributes to enhancement of the sensitivity of cisplatin in esophageal squamous cell carcinoma

Increasing evidence has demonstrated that Ctr1 plays a crucial role in the regulation of cisplatin uptake in a variety of tumors. The purpose of this study was to investigate its role in mediating cisplatin sensitivity in ESCC cells. Immunohistochemistry (IHC), In situ hybridization (ISH) and semi-quantitative RT-PCR were used to detect Ctr1 expressions in ESCC tissues. qRT-PCR and Western blot was performed to investigate the levels of Ctr1 mRNA and protein in ESCC cells. CCK-8, Flow cytometry and Transwell chamber assay were carried out to examine cell proliferation, apoptosis, migration and invasion abilities in ESCC cells. We found that ESCC tissues and cells had higher Ctr1 level than normal tissues and Het-1A cell. Ctr1 expression was correlated with histological grade, invasion depth, TNM staging and lymph node metastasis in ESCC patients. Ctr1 depletion reduced the suppressive role of proliferation, migration and invasion as well as the inductive role of cell apoptosis and Caspase-3 activity evoked by cisplatin, whereas Ctr1 upregulation combined with cisplatin exerted the synergistic role in regulation of proliferation, apoptosis, Caspase-3 activity, migration and invasion in ESCC. In conclusion, Ctr1 is implicated in ESCC development and progression and its expression may be a novel predictor for assessment of cisplatin sensitivity in ESCC.     

Correlation of STAT1 with Apoptosis and Cell-Cycle Markers in Esophageal Squamous Cell Carcinoma

We recently found evidence that STAT1 in esophageal squamous carcinoma (ESCC) cells exerts tumor suppressor function, and it regulates five key regulators of apoptosis or cell-cycle progression, including Bcl-2, Bcl-xL, survivin, cyclin D1 and p21. In this study, we confirmed these findings in four ESCC cell lines. Using immunohistochemistry, we also assessed the expression of these proteins in 62 primary tumors. The expression of these markers was heterogeneous, ranging 39 to 69% of the cohort. Significant correlation was found between STAT1 and three proteins (p21, Bcl-xL and survivin), whereas only a trend was identified for cyclin D1 and Bcl-2. We then correlated the expression of these proteins with several clinicopathologic parameters including lymph node metastasis, depth of invasion, clinical stage and overall survival. Significant correlations were found between Bcl-2 and deep invasion (p = 0.033), survivin and lymph node metastasis (p = 0.006), as well as cyclin D1 and clinical stage (p = 0.014). Patients with p21-positive tumors had a significantly longer survival compared to those with p21-negative tumors (p = 0.031). To conclude, our findings support the concept that STAT1 exerts its tumor suppressor effects in ESCC via modulating the expression of key regulators of apoptosis and cell-cycle progression.     

Targeted Knockdown of IQGAP1 Inhibits the Progression of Esophageal Squamous Cell Carcinoma In Vitro and In Vivo

IQGAP1 is a scaffolding protein that can regulate several distinct signaling pathways. The accumulating evidence has demonstrated that IQGAP1 plays an important role in tumorigenesis and tumor progression. However, the function of IQGAP1 in esophageal squamous cell carcinoma (ESCC) has not been thoroughly investigated. In the present study, we showed that IQGAP1 was overexpressed in ESCC tumor tissues, and its overexpression was correlated with the invasion depth of ESCC. Importantly, by using RNA interference (RNAi) technology we successfully silenced IQGAP1 gene in two ESCC cell lines, EC9706 and KYSE150, and for the first time found that suppressing IQGAP1 expression not only obviously reduced the tumor cell growth, migration and invasion in vitro but also markedly inhibited the tumor growth, invasion, lymph node and lung metastasis in xenograft mice. Furthermore, Knockdown of IQGAP1 expression in ESCC cell lines led to a reversion of epithelial to mesenchymal transition (EMT) progress. These results suggest that IQGAP1 plays crucial roles in regulating ESCC occurrence and progression. IQGAP1 silencing may therefore develop into a promising novel anticancer therapy.     

食管鳞状细胞癌中DICKKOPF-3基因的甲基化状态及表达

目的检测食管鳞状细胞癌(esophageal squamous cell cancer,ESCC)中Wnt通路拮抗基因DICKKOPF-3(DKK3)的甲基化状态,探讨其与ESCC发生的关系。方法应用甲基化特异性PCR(methylation specificPCR,MSP)及RT-PCR的方法检测78例ESCC及相应癌旁非肿瘤组织中DKK3基因的甲基化状态及mRNA表达情况,应用免疫组化的方法检测通路中心因子-βcatenin蛋白及下游靶基因cyclinD1的表达,并分析其与食管癌发生的关系。结果在ESCC组织中,DKK3基因的甲基化频率为37.2%(29/78),明显高于癌旁非肿瘤组织(χ2=35.622,P=0.000);癌组织中该基因的甲基化率与肿瘤患者临床分期相关(χ2=4.705,P=0.030),而与组织学分级无关;食管癌中DKK3基因mRNA的阳性表达率为65.4%(51/78),明显低于癌旁非肿瘤组织(χ2=13.298,P=0.000)。在发生甲基化的食管癌组织中该基因的mRNA表达缺失及-βcatenin蛋白的异质表达率均明显高于未发生甲基化的癌组织,且差异有统计学意义(χ2mRNA=29.141,P=0.000;χ2-βcatenin=6.245,P=0.012)。食管癌中cyclinD1的表达明显高于癌旁组织,且癌组织中该蛋白的表达与DKK3基因的甲基化状态相关(χ2=4.921,P=0.027)。结论 ESCC组织中DKK3基因高甲基化导致的转录沉默可能与食管癌的发生有关,并可能通过活化Wnt/-βcatenin信号转导通路促进下游靶基因cyclinD1的过表达发挥作用。     

Expression of a putative stem cell marker,Musashi 1, in mammary glands of ewes

Several recent studies demonstrated that development, function and remodelling of mammary glands involved multipotent cells, but no specific molecular markers for mammary epithelial stem cells were revealed. These studies principally concerned human and mouse mammary tissue, but mammary stem cells could be a valuable tool in agricultural production and bioengineering in farm animals. The Musashi-1 (Msi 1) gene encodes an RNA binding protein, which is likely to be associated with self-renewal of neural, intestinal and mammary progenitor cells and is believed to influence the Notch signalling pathway. In this study Musashi-1 expression was detected using immunohistochemistry and in situ hybridisation analysis on mammary glands of ewes at different developmental stages. The protein expression was observed in the epithelial cells at all stages examined. In situ hybridization analysis showed that Msi 1 mRNA has an expression pattern similar to the encoded protein, with positive staining in both nuclei and cytoplasm of ductal, secretory and stromal cells. Ultrastructural in situ analysis confirmed the nuclear and cytoplasmatic expression of Msi. Quantitative analysis of Msi 1 gene expression showed a strong correlation with that of Ki-67, that is a marker of cell proliferation. This is the first report outlining expression of Msi 1 in ovine mammary glands during a complete cycle of lactation.