MicroRNA target prediction: theory and practice.

The present study is one of the few that includes tissue samples in the evaluation of target prediction algorithms designed to detect microRNA (miRNA) sequences that might interact with particular messenger RNA (mRNA) sequences. Twelve different target prediction tools were used to find miRNA sequences that might interact with CCL20 gene expression. Different algorithms predicted controversial miRNA sequences for CCL20 regulation due to a different weighting of parameters. Hsa-miR-21 and hsa-miR-145 suggested by four or more programs were chosen for further investigation. Possible real interaction of these miRNA sequences with CCL20 gene expression was monitored using luciferase assays and expression analyses of tissue samples of colorectal adenocarcinoma by either qRT-PCR or ELISA. Folding status of seed-binding sites in complete mRNA and 3'UTR of CCL20 was predicted. Prediction of miRNA expression was attempted based on CCL20 expression data. Eight of the target prediction tools forecasted a role for hsa-miR-21 and four mentioned hsa-miR-145 in CCL20 gene regulation. Laboratory experimentation showed that CCL20 may serve as a target of hsa-miR-21 but not hsa-miR-145. Expression of the molecules resulted in no clear assertion. Folding of seed-binding sites was predicted to be relatively constant for the complete mRNA and 3'UTR. Predicting miRNA expression based on target gene expression was impossible. This might be attributable to the fact that effects of miRNA activity may oscillate between gene product repression and activation. Additional systematic studies are needed to address this issue.

Chemokine receptor CCR6 expression is regulated by miR-518a-5p in colorectal cancer cells.

BACKGROUND: Recently, involvement of the chemokine/receptor system CCL20/CCR6 in colorectal cancer (CRC) progression was shown. Here, we analyzed the functional interaction of miRNA-518-5p (miR-518a-5p) with CCR6 and its impact on CCR6 expression in CRC cells. METHODS: MiR-518a-5p was identified by computer software to potentially interact with CCR6. Hence, functional implications of miR-518a-5p with the 3'UTR of CCR6 were analyzed using the Dual Luciferase Reporter assay system. Confirmation of the predicted target site for miR-518a-5p was achieved by site-directed mutagenesis of the seed sequence in the 3'UTR of CCR6 and subsequent application of the mutated seed sequence in a luciferase assay with miR-518a-5p mimics. Accordingly, two CRC cell lines (Caco-2 and HT-29) were transfected with miR-518a-5p miRNA mimics and gene and protein expression of CCR6 was monitored using qRT PCR and immunocytochemistry, respectively. RESULTS: Addition of miR-518a-5p led to significant down-regulation of luciferase activity (P < 0.05), which was significantly reversed in a reporter test system containing the mutated seed sequences in the 3'UTR of CCR6. Following transfection of CRC cell lines with miR-518a-5p mimics and subsequent monitoring of CCR6 expression showed significant down-regulation of CCR6 mRNA and CCR6 protein expression in both CRC cell lines under investigation (P < 0.05). CONCLUSIONS: We have shown that miR-518a-5p functionally interacts with CCR6 and that transfection of CRC cells with miR-518a-5p leads to significant CCR6 down-regulation. Consequently, CCR6 expression is regulated by miR-518a-5p in CRC cells indicating that regulation of CCR6 expression by miR-518a-5p might be a regulatory mechanism involved in CRC pathogenesis.

CCL20/CCR6 expression profile in pancreatic cancer.

BACKGROUND: CCL20 and its receptor CCR6 have been shown to play a role in the onset, development and metastatic spread of various gastrointestinal malignancies. In this study, the expression profile and clinical significance of the CCL20/CCR6 system in distinct benign, pre-malignant and malignant pancreatic tissues was investigated. METHODS: Using RealTime-PCR, enzyme-linked immunosorbent assay (ELISA), Western Blot and immunohistochemistry, we have analyzed the expression profile of CCL20/CCR6 in resection specimens from patients with chronic pancreatitis (CP) (n = 22), pancreatic cystadenoma (PA) (n = 11) and pancreatic carcinoma (PCA) (n = 25) as well as in the respective matched normal pancreatic tissues. RESULTS: CCL20 mRNA and protein was weakly expressed in normal pancreatic tissues and CP and PA specimens but significantly up-regulated in PCA (8-fold) as compared to the matched normal tissue (P < 0.05). Moreover, CCL20 mRNA and protein expression was significantly associated with advanced T-category in patients with PCA (P < 0.05). CCR6 mRNA showed a significant up-regulation in all three disease entities as compared to normal tissues (P < 0.05, respectively). CONCLUSION: CCL20 and CCR6 were significantly up-regulated in PCA as compared to the normal pancreatic tissue and CCL20 was significantly associated with advanced T-category in PCA patients. This suggests that CCL20 and CCR6 play a role in the development and progression of PCA and may constitute potential targets for novel treatment strategies.

ELR+ CXC chemokine expression in benign and malignant colorectal conditions.

BACKGROUND: CXCR2 chemokine ligands CXCL1, CXCL5 and CXCL6 were shown to be involved in chemoattraction, inflammatory responses, tumor growth and angiogenesis. Here, we comparatively analyzed their expression profile in resection specimens from patients with colorectal adenoma (CRA) (n = 30) as well as colorectal carcinoma (CRC) (n = 48) and corresponding colorectal liver metastases (CRLM) (n = 16). METHODS: Chemokine expression was assessed by microdissection, quantitative real-time PCR (Q-RT-PCR), the enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC). RESULTS: In contrast to CXCL6, we demonstrated CXCL1 and CXCL5 mRNA and protein expression to be significantly up-regulated in CRC and CRLM tissue specimens in relation to their matched tumor neighbor tissues. Moreover, both chemokine ligands were demonstrated to be significantly higher expressed in CRC tissues than in CRA tissues thus indicating a progressive increase in the transition from the premalignant condition to the development of the malignant status. Although a comparative analysis of the CXCL1/CXCL5 protein expression profiles in CRC patients revealed that the absolute expression level of CXCL1 was significantly higher in comparison to CXCL5, mRNA- and protein overexpression of CXCL5 in CRC and CRLM tissues was much more pronounced (80- and 60- fold in CRC tissues, respectively) in comparison to CXCL1 (5- and 3.5- fold in CRC tissues, respectively). CONCLUSION: Our results demonstrate a significant association between CXCL1 and CXCL5 expression with CRC and CRLM suggesting for both chemokine ligands a potential role in the progression from CRA to CRC and thus, in the initiation of CRC.

Enhanced ENA-78 and IL-8 expression in patients with malignant pancreatic diseases.

BACKGROUND/AIM: Pancreatic cancer is characterized by perineural invasion, early lymph node and liver metastases, and an extremely dismal prognosis. In the present study we aimed at investigating the expression profile of pro-inflammatory and angiogenic CXC chemokines as potential factors contributing to the aggressive biology of this gastrointestinal malignancy. METHODS: Protein expression profiles of the CXC chemokines growth-related oncogene alpha (GRO-alpha/CXCL1), epithelial cell-derived neutrophil-activating peptide-78 (ENA-78/CXCL5), granulocyte chemoattractant protein-2 (GCP-2/CXCL6), neutrophil-activating protein-2 (NAP-2/CXCL7), and interleukin-8 (IL-8/CXCL8) were assessed by enzyme-linked immunosorbent assay in pancreatic carcinoma, cancer of the papilla of Vater, pancreatic cystadenoma, and chronic pancreatitis specimens. RESULTS: IL-8 and ENA-78 protein expression was most pronounced in pancreatic carcinoma specimens, showing an 11-fold and 17-fold overexpression in comparison with non-affected neighbouring tissues, a 66-fold and 24-fold upregulation compared to pancreatic cystadenoma, and a 6-fold and 9-fold overexpression with respect to chronic pancreatitis, respectively (p < 0.05 between all groups). In addition, a close correlation between IL-8 and ENA-78 protein expression and advanced pancreatic carcinomas in relation to the T category was evident (p < 0.05). CONCLUSION: Our results demonstrate that ELR+ CXC chemokines are differentially expressed in malignant and non-malignant human pancreatic specimens, suggesting a potential contribution of these chemokines to the pathogenesis of pancreatic carcinoma.

Correlation of IL-8 with induction, progression and metastatic potential of colorectal cancer.

AIM: To investigate the expression profile of IL-8 in inflammatory and malignant colorectal diseases to evaluate its potential role in the regulation of colorectal cancer (CRC) and the development of colorectal liver metastases (CRLM). METHODS: IL-8 expression was assessed by quantitative real-time PCR (Q-RT-PCR) and the enzyme-linked immunosorbent assay (ELISA) in resected specimens from patients with ulcerative colitis (UC, n = 6) colorectal adenomas (CRA, n = 8), different stages of colorectal cancer (n = 48) as well as synchronous and metachronous CRLM along with their corresponding primary colorectal tumors (n = 16). RESULTS: IL-8 mRNA and protein expression was significantly up-regulated in all pathological colorectal entities investigated compared with the corresponding neighboring tissues. However, in the CRC specimens IL-8 revealed a significantly more pronounced overexpression in relation to the CRA and UC tissues with an average 30-fold IL-8 protein up-regulation in the CRC specimens in comparison to the CRA tissues. Moreover, IL-8 expression revealed a close correlation with tumor grading. Most interestingly, IL-8 up-regulation was most enhanced in synchronous and metachronous CRLM, if compared with the corresponding primary CRC tissues. Herein, an up to 80-fold IL-8 overexpression in individual metachronous metastases compared to normal tumor neighbor tissues was found. CONCLUSION: Our results strongly suggest an association between IL-8 expression, induction and progression of colorectal carcinoma and the development of colorectal liver metastases.

Chemokine expression in hepatocellular carcinoma versus colorectal liver metastases.

AIM: To evaluate and compare the expression profiles of CXCL12 (SDF-1), CCL19 (MIP-3beta), CCL20 (MIP-3alpha) and CCL21 (6Ckine, Exodus2) and their receptors on RNA and protein levels in hepatocellular carcinoma (HCC) versus colorectal liver metastases (CRLM) and to elucidate their impact on the carcinogenesis and progression of malignant liver diseases. METHODS: Chemokine expression was analyzed by RT-PCR and ELISA in 11 cases of HCC specimens and in 23 cases of CRLM and corresponding adjacent non-tumorous liver tissues, respectively. Expressions of their receptors CXCR4, CCR6 and CCR7 were analyzed by RT-PCR and Western blot analysis in the same cases of HCC and CRLM. RESULTS: Significant up-regulation for CCL20/CCR6 was detected in both cancer types. Moreover, CCL20 demonstrated significant overexpression in CRLM in relation to the HCC tissues. Being significantly up-regulated only in CRLM, CXCR4 displayed an aberrant expression pattern with respect to the HCC tissues. CONCLUSION: Correlation of CXCR4 expression with CRLM suggests CXCR4 as a potential predictive factor for CRLM. High level expression of CCL20 and its receptor CCR6 in HCC and CRLM with marked up-regulation of CCL20 in CRLM in relation to HCC tissues indicates involvement of the CCL20/CCR6 ligand-receptor pair in the carcinogenesis and progression of hepatic malignancies.

Chemokine/chemokine receptor pair CCL20/CCR6 in human colorectal malignancy: An overview.

Chemokines belong to a superfamily of small, cytokine-like proteins, which induce multiple physiological functions, particularly cytoskeletal rearrangement and compartment-specific migration through their interaction with G-protein-coupled receptors. Chemokines and their receptors have been widely acknowledged as essential and selective mediators in leukocyte migration in inflammatory response. It is now established that the chemokine/chemokine receptor system is also used by cancer cells to direct lymphatic and haematogenous spreading and additionally has an impact on the site of metastatic growth of different tumours. In recent years an increasing number of studies have drawn attention to CC-chemokine cysteine motif chemokine ligand 20 (CCL20) and its physiological sole receptor CCR6 to play a role in the onset, development and metastatic spread of various gastrointestinal cancer entities. Among various cancer types CCR6 was also demonstrated to be significantly overexpressed in colorectal cancer (CRC) and stimulation by its physiological ligand CCL20 has been reported to promote CRC cell proliferation and migration in vitro. Further, the CCL20/CCR6 system apparently plays a role in the organ-selective liver metastasis of CRC. Here we review the literature on expression patterns of CCL20 and CCR6 and their physiological interactions as well as the currently presumed role of CCL20 and CCR6 in the formation of CRC and the development of liver metastasis, providing a potential basis for novel treatment strategies.

Text mining, a race against time? An attempt to quantify possible variations in text corpora of medical publications throughout the years.

BACKGROUND: The continuous growth of medical sciences literature indicates the need for automated text analysis. Scientific writing which is neither unitary, transcending social situation nor defined by a timeless idea is subject to constant change as it develops in response to evolving knowledge, aims at different goals, and embodies different assumptions about nature and communication. The objective of this study was to evaluate whether publication dates should be considered when performing text mining. METHODS: A search of PUBMED for combined references to chemokine identifiers and particular cancer related terms was conducted to detect changes over the past 36 years. Text analyses were performed using freeware available from the World Wide Web. TOEFL Scores of territories hosting institutional affiliations as well as various readability indices were investigated. Further assessment was conducted using Principal Component Analysis. Laboratory examination was performed to evaluate the quality of attempts to extract content from the examined linguistic features. RESULTS: The PUBMED search yielded a total of 14,420 abstracts (3,190,219 words). The range of findings in laboratory experimentation were coherent with the variability of the results described in the analyzed body of literature. Increased concurrence of chemokine identifiers together with cancer related terms was found at the abstract and sentence level, whereas complexity of sentences remained fairly stable. CONCLUSIONS: The findings of the present study indicate that concurrent references to chemokines and cancer increased over time whereas text complexity remained stable.

miR-21 and its target gene CCL20 are both highly overexpressed in the microenvironment of colorectal tumors: significance of their regulation.

Recently, we reported a functional interaction between miR-21 and its identified chemokine target CCL20 in colorectal cancer (CRC) cell lines. Here, we investigated whether such functional interactions are permitted at the cellular level which would require an inverse correlation of expression and also co-expression of miR-21 and CCL20 in the same cell. Expression profiling was performed using qPCR, and ELISA, in situ hybridization and immunohistochemistry were applied for the presentation of their cellular localization. We demonstrated that miR-21 as well as CCL20 were both significantly upregulated in CRC tissues; thus, showing no antidromic expression pattern. This provided an initial clue that miR-21 and CCL20 may not be expressed in the same cell. In addition, we located miR-21 expression at the cellular level predominantly in stromal cells such as tumor-associated fibroblasts and to a minor degree in immune cells such as macrophages and lymphocytes. Likewise, CCL20 expression was primarily detected in tumor-infiltrating immune cells. Thus, investigating the cellular localization of miR-21 and its target CCL20 revealed that both molecules are expressed predominantly in the microenvironment of CRC tumors.

Thoracotomy procedures effect cytokine levels after thoracoabdominal esophagectomy.

Pulmonary complications together with surgical complications are the most frequent causes for morbidity and mortality after thoracoabdominal esophagectomy. The con-tinuous improvement of surgical techniques has led to a decrease in surgical complications, whereas up to 30% of the patients develop postoperative pulmonary complications such as acute lung injury (ALI) or even the more severe acute respiratory distress syndrome (ARDS), which are characterized by an acute inflammation in the lung parenchyma and the airspace. Evidence from several studies indicates that a complex network of inflammatory cytokines and mediators play a key role in mediation, amplification, and perpetuation of the process of lung injury and that the thoracotomy itself is a risk factor for developing ALI or ARDS. In this trial, the cytokine levels of IL6, IL8 and IL10 were measured and compared in 30 patients who had undergone an extended radical thoracoabdominal esophagectomy for esophageal cancer, via anterolateral thoracotomy (n=17) or posterolateral thoracotomy (n=13). Patients of both groups were similar in terms of age, sex and preoperative pulmonary function as well as in the anesthetic procedures they have undergone. All patients displayed significantly increased serum levels of IL6 and IL8 after thoracoabdominal esophagectomy. However, patients who were subjected to an anterolateral thoracotomy were reported with significantly higher serum levels of IL6 and IL8 compared to patients who had received a posterolateral thoracotomy. Thus, the choice of the thoracotomy method during the thoracoabdominal esophagectomy and the resultant cytokine levels may contribute to the occurrence of postoperative pulmonary complications and may have an impact on the extent and severity of the surgical stress.

miR-21 functionally interacts with the 3'UTR of chemokine CCL20 and down-regulates CCL20 expression in miR-21 transfected colorectal cancer cells.

As deregulation of miRNAs and chemokine CCL20 was shown to play a role in colorectal cancer (CRC) pathogenesis, we analyzed the functional interactions of candidate miRNAs with CCL20 mRNA. After target prediction software programs indicated a role for miR-21 in CCL20 regulation, we applied the luciferase reporter assay system to demonstrate that miR-21 functionally interacts with the 3'UTR of CCL20 mRNA and down-regulates CCL20 in miR-21 mimic transfected CRC cell lines (Caco-2, SW480 and SW620). Thus, regulation of CCL20 expression by miR-21 might be a regulatory mechanism involved in progression of CRC.

Effect of preoperative FOLFOX chemotherapy on CCL20/CCR6 expression in colorectal liver metastases.

AIM: To evaluate the influence of preoperative FOLFOX chemotherapy on CCL20/CCR6 expression in liver metastases of stage IV colorectal cancer (CRC) patients. METHODS: Using Real Time-PCR, enzyme-linked immunosorbent assay, Western Blots and immunohistochemistry, we have analyzed the expression of CCL20, CCR6 and proliferation marker Ki-67 in colorectal liver metastasis (CRLM) specimens from stage IV CRC patients who received preoperative FOLFOX chemotherapy (n = 53) and in patients who did not receive FOLFOX chemotherapy prior to liver surgery (n = 29). RESULTS: Of the 53 patients who received FOLFOX, time to liver surgery was ≤ 1 mo in 14 patients, ≤ 1 year in 22 patients and > 1 year in 17 patients, respectively. In addition, we investigated the proliferation rate of CRC cells in liver metastases in the different patient groups. Both CCL20 and CCR6 mRNA and protein expression levels were significantly increased in patients who received preoperative FOLFOX chemotherapy ≤ 12 mo before liver surgery (P < 0.001) in comparison to patients who did not undergo FOLFOX treatment. Further, proliferation of CRLM cells as measured by Ki-67 was increased in patients who underwent FOLFOX treatment. CCL20 and CCR6 expression levels were significantly increased in CRLM patients who had undergone preoperative FOLFOX chemotherapy. CONCLUSION: This chemokine/receptor up-regulation could lead to increased proliferation/migration through an autocrine mechanism which might be used by surviving metastatic cells to escape cell death caused by FOLFOX.