[Identification of immune-related prognostic signature for colon adenocarcinoma based on weighted gene co-expression network analysis].

Objective To identify immune-related molecular markers in an attempt to predict prognosis of colon adenocarcinoma (COAD). Methods Immune related genes (IREGs) was analyzed based on the TCGA database. Weighted gene co-expression network analysis (WGCNA) and Cox regression analysis were used to establish risk models. According to the median risk score, COAD patients were divided into high risk and low risk groups. The prognostic difference were compared between the two groups. The function of the model was validated using GEO. Results A total of 1015 IREGs was obtained. The established model consisted of three genes: RAR related orphan receptor C (RORC), leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2) and lectin galactoside-binding soluble galectin 4 (LGALS4). The high-risk group had significantly poorer prognosis than low-risk group in the GEO database, and it was validated using a GEO database. Further analysis via univariate and multivariate Cox regression analyses revealed that risk model could function as independent prognostic factor for COAD patients. Conclusion The risk model based on IREGs can predict the prognosis of patients with COAD.

Simultaneous isolation of mesenchymal stem cells and endothelial progenitor cells derived from murine bone marrow.

Mesenchymal stem or stromal cells (MSCs) are identified as sources of pluripotent stem cells with varying degrees of plasticity. Endothelial progenitor cells (EPCs) originate from either bone marrow (BM) or peripheral blood and can mature into cells that line the lumen of blood vessels. MSC and EPC therapies exhibit promising results in a variety of diseases. The current study described the simultaneous isolation of EPCs and MSCs from murine BM using a straightforward approach. The method is based on differences in attachment time and trypsin sensitivity of MSCs and EPCs. The proposed method revealed characteristics of isolated cells. Isolated MSCs were positive for cell surface markers, cluster of differentiation (CD)29, CD44 and stem cell antigen-1 (Sca-1), and negative for hematopoietic surface markers, CD45 and CD11b. Isolated EPCs were positive for Sca-1 and vascular endothelial growth factor receptor 2 and CD133. The results indicate that the proposed method ensured simultaneous isolation of homogenous populations of MSCs and EPCs from murine BM.

miR-34a Regulates Multidrug Resistance via Positively Modulating OAZ2 Signaling in Colon Cancer Cells.

Although aberrant expression of miR-34a, an essential tumor suppressor miRNA, has been frequently observed in colon cancer (CCa), whether miR-34a can regulate CCa progression by modulating other facets of this malignancy (such as multidrug resistance, MDR) remains unknown. Here, we report for the first time that miR-34a expression was significantly downregulated in clinical CCa samples from oxaliplatin-resistant patients and in experimentally established multidrug-resistant CCa cells. By using histoculture drug response assay, we further confirmed that clinical CCa samples with lower miR-34a expression appeared to be more resistant to chemotherapy. Functionally, ectopic expression of exogenous miR-34a resensitized multidrug-resistant HCT-8/OR cells to oxaliplatin treatment, whereas miR-34a inhibition augmented the oxaliplatin resistance in chemosensitive HCT-8 cells. Mechanistically, miR-34a positively regulated the mRNA stability of the ornithine decarboxylase antizyme 2 (OAZ2) by directly targeting its three prime untranslated region (3'UTR). Consequently, suppression of the expression of miR-34a/OAZ2 signaling by chemotherapeutic agents significantly enhanced the activation of MDR-associated ATP-binding cassette (ABC) transporters and antiapoptosis pathways, thus leading to MDR development in CCa cells. Collectively, our combined analysis reveals a critical role of miR-34a/OAZ2 cascade in conferring a proper cellular response to CCa chemotherapy.

Interleukin-1ß induces intercellular adhesion molecule-1 expression, thus enhancing the adhesion between mesenchymal stem cells and endothelial progenitor cells via the p38 MAPK signaling pathway.

Endothelial progenitor cells (EPCs) are an important component of stem-cell niches, which are able to promote the self-renewal and pluripotency of mesenchymal stem cells (MSCs). The biological functions of these two cell types is dependent on adhesion, and the adhesion between MSCs and EPCs is important due to their critical role in neovascularization and bone regeneration in tissue engineering. Intercellular adhesion molecule-1 (ICAM-1, also known as cluster of differentiation 54), is a member of the immunoglobulin supergene family, which functions in cell-cell and cell-matrix adhesive interactions. Compared with other adhesion molecules, ICAM-1 is expressed in hematopoietic and nonhematopoietic cells, and can mediate adhesive interactions. The present study aimed to investigate the importance of ICAM-1 in the adhesion of MSCs and EPCs, and demonstrated that adhesion between these cells could be regulated by interleukin (IL)-1ß via the p38 mitogen­activated protein kinase pathway. In addition, the results confirmed that ICAM-1 served a critical role in regulation of adhesion between MSCs and EPCs. ELISA, cell immunofluorescence, western blot analysis and adhesion assay were used to confirm our theory from phenomenon to essence. The present study provided evidence to support and explain the adhesion between MSCs and EPCs. Furthermore, the present findings provide a theoretical basis for further stem­cell niche transplantation to increase understanding of the function of MSCs and the crosstalk between MSCs and EPCs in the stem-cell niche.

VEGF secreted by mesenchymal stem cells mediates the differentiation of endothelial progenitor cells into endothelial cells via paracrine mechanisms.

Stem cell therapy is a promising treatment strategy for ischemic diseases. Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) adhere to each other in the bone marrow cavity and in in vitro cultures. We have previously demonstrated that the adhesion between MSCs and EPCs is critical for MSC self­renewal and their multi­differentiation into osteoblasts and chondrocytes. In the present study, the influence of the indirect communication between EPCs and MSCs on the endothelial differentiation potential of EPCs was investigated, and the molecular mechanisms underlying MSC­mediated EPC differentiation were explored. The effects of vascular endothelial growth factor (VEGF), which is secreted by MSCs, on EPC differentiation via paracrine mechanisms were examined via co­culturing MSCs and EPCs. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to detect the expression of genes and proteins of interest. The present results demonstrated that co­culturing EPCs with MSCs enhanced the expression of cluster of differentiation 31 and von Willebrand factor, which are specific markers of an endothelial phenotype, thus indicating that MSCs may influence the endothelial differentiation of EPCs in vitro. VEGF appeared to be critical to this process. These findings are important for the understanding of the biological interactions between MSCs and EPCs, and for the development of applications of stem cell­based therapy in the treatment of ischemic diseases.

Mesenchymal stem cells promote endothelial progenitor cell proliferation by secreting insulin­like growth factor­1.

Bone marrow mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) interact with each other. EPCs are able to promote the self­renewal of MSCs as niche cells in murine bone marrow, and MSCs are able to promote EPC proliferation in vitro in a co­culture system. It has previously been reported that MSCs can secrete insulin­like growth factor­1 (IGF­1), which serves critical functions in EPC proliferation. However, the mechanism underlying the IGF­1­mediated proliferation of EPCs remains unclear. The aim of the present study was to reveal the molecular mechanisms regulating this process. The effects of IGF­1, which is secreted by MSCs, on EPC proliferation via the PI3K/Akt signaling pathway were examined by MTT assay, reverse transcription­quantitative polymerase chain reaction and western blot analysis. The present study treated EPCs with various concentrations of IGF­1. The results demonstrated that IGF­1 significantly induced the proliferation of cultured EPCs. However, this effect was offset by treatment with the phosphatidylinositol 3­kinase (PI3K) inhibitor LY294002. These results indicated that the pro­proliferative effects of IGF­1 are mediated in response to the PI3K/protein kinase B signaling pathway.

Construction of In Vivo Fluorescent Imaging of Echinococcus granulosus in a Mouse Model.

Human hydatid disease (cystic echinococcosis, CE) is a chronic parasitic infection caused by the larval stage of the cestode Echinococcus granulosus. As the disease mainly affects the liver, approximately 70% of all identified CE cases are detected in this organ. Optical molecular imaging (OMI), a noninvasive imaging technique, has never been used in vivo with the specific molecular markers of CE. Thus, we aimed to construct an in vivo fluorescent imaging mouse model of CE to locate and quantify the presence of the parasites within the liver noninvasively. Drug-treated protoscolices were monitored after marking by JC-1 dye in in vitro and in vivo studies. This work describes for the first time the successful construction of an in vivo model of E. granulosus in a small living experimental animal to achieve dynamic monitoring and observation of multiple time points of the infection course. Using this model, we quantified and analyzed labeled protoscolices based on the intensities of their red and green fluorescence. Interestingly, the ratio of red to green fluorescence intensity not only revealed the location of protoscolices but also determined the viability of the parasites in vivo and in vivo tests. The noninvasive imaging model proposed in this work will be further studied for long-term detection and observation and may potentially be widely utilized in susceptibility testing and therapeutic effect evaluation.

A Rapid and Convenient Method for in Vivo Fluorescent Imaging of Protoscolices of Echinococcus multilocularis.

Human and animal alveolar echinococcosis (AE) are important helminth infections endemic in wide areas of the Northern hemisphere. Monitoring Echinococcus multilocularis viability and spread using real-time fluorescent imaging in vivo provides a fast method to evaluate the load of parasite. Here, we generated a kind of fluorescent protoscolices in vivo imaging model and utilized this model to assess the activity against E. multilocularis protoscolices of metformin (Met). Results indicated that JC-1 tagged E. multilocularis can be reliably and confidently used to monitor protoscolices in vitro and in vivo. The availability of this transient in vivo fluorescent imaging of E. multilocularis protoscolices constitutes an important step toward the long term bio-imaging research of the AE-infected mouse models. In addition, this will be of great interest for further research on infection strategies and development of drugs and vaccines against E. multilocularis and other cestodes.

[Effect of Intercellular Adhesion Molecule-1 on Adherence Between Mesenchymal Stem Cells and Endothelial Progenitor Cells].

OBJECTIVE: To investigate the effects of intercellular adhesion molecule-1(ICAM-1) on the adherence between mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC). METHODS: MSC and EPC were isolated, cultured and expanded from the 6-8 weeks aged C57BL/6 murine bone marrow by in vitro. Immuno-fluorescence was used to detect the expression of ICAM-1 in MSC group, EPC group and co-cultured MSC and EPC group. The mRNA and protein levels of ICAM-1 were detected by RT-PCR and Western blot respectively, then, the ICAM-1 adherence between MSC and EPC was observed by adding different concentration of neutralizing antibody. RESULTS: The expression of ICAM-1 on surface of MSC and EPC could be detected by cell immunofluorescence method. According to results of the semiquantitative fluorescene detection, the fluorescence strength of MSC+EPC co-cultured group (89.02 ± 24.52) was higher than that of MSC group (31.25 ± 2.95) and EPC group (34.32 ± 5.02), and there was statistical difference between them (P < 0.01), but there was no obvious difference between MSC group and EPC group (P > 0.05). RT-PCR detection showed that the expression levels of ICAM-1 in MSC+EPC co-cultured group were higher than that in MSC group and that in EPC group (P < 0.01), and expression level of ICAM in EPC group was higher than that in MSC group (P < 0.01). Western blot detection showed that the expression level of ICAM-1 protein in MSC+EPC co-cultured group (0.33 ± 0.4) was higher than that in MSC group (0.11 ± 0.01) (P < 0.05) and than that in EPC group (0.19+0.02) (P < 0.05), However, the expression level of ICAM-1 protein in EPC group was higher than that in MSC group (P < 0.05). The test of different concentrations against neutralizing antibody showed that with the increasing of concentration of ICAM-1 neutralizing antibody, the adhesion capability of MSC and EPC was gradually decreasing. CONCLUSION: The ICAM-1 can mediate the adherence process between MSC and EPC.

E-cadherin-mediated contact of endothelial progenitor cells with mesenchymal stem cells through ß-catenin signaling.

Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) are attached to each other in the bone marrow (BM) cavity and in in vitro cultures, and this adhesion has important physiological significance. We demonstrated that cell proliferation could be promoted when MSCs were co-cultured with EPCs, which was beneficial to angiogenesis, tissue repair, and regeneration. The adhesion of MSCs and EPCs could promote the pluripotency of MSCs, particularly self-renewal and multi-differentiation to osteoblasts, chondrocytes, and adipocytes. This study focused on the mechanism of adhesion between EPCs and MSCs. The results showed that E-cadherin (E-cad) mediated the adhesion of MSCs and EPCs through the E-cad/beta-catenin signaling pathway. The E-cad of EPCs occupied a dominant position during this process, which activated and up-regulated the beta-catenin (ß-catenin) of MSCs to improve cohesion and exert their biological function.