Background Endometrial carcinoma (EC) is the most common gynecological malignant tumors which poses a serious threat to women health. This study aimed to screen the candidate genes differentially expressed in EC by bioinformatics analysis. Methods GEO database and GEO2R online tool were applied to screen the differentially expressed genes (DEGs) of EC from the microarray datasets. Protein-protein interaction (PPI) network for the DEGs was constructed to further explore the relationships among these genes and identify hub DEGs. Gene ontology and KEGG enrichment analyses were performed to investigate the biological role of DEGs. Besides, correlation analysis, genetic alteration, expression profile, and survival analysis of these hub DEGs were also investigated to further explore the roles of these hub gene in mechanism of EC tumorigenesis. qRT-PCR analysis was also performed to verify the expression of identified hub DEGs. Results A total of 40 DEGs were screened out as the DEGs with 3 upregulated and 37 downregulated in EC. The gene ontology analysis showed that these genes were significantly enriched in cell adhesion, response to estradiol, and growth factor activity, etc. The KEGG pathway analysis showed that DEGs were enriched in focal adhesion, leukocyte transendothelial migration, PI3K-Akt signaling pathway, and ECM-receptor interaction pathway. More importantly, COL1A1, IGF1, COL5A1, CXCL12, PTEN, and SPP1 were identified as the hub genes of EC. The genetic alteration analysis showed that hub genes were mainly altered in mutation and deep deletion. Expression validation by bioinformatic analysis and qRT-PCR also proved the expression of these six hub genes were differentially expressed in EC. Additionally, significantly better overall survival and disease-free survival were observed with six hub genes altered, and survival outcome in high expression of COL1A1, IGF1, and PTEN patients was also significantly better than low expression patients. Conclusions COL1A1, IGF1, COL5A1, CXCL12, PTEN, and SPP1 involved in the pathogenesis of EC and might be candidate genes for diagnosis of EC.

INTRODUCTION:Gap junctions (GJs) represent the best known intercellular communication (IC) system and are membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules to pass from cell to cell. In this study, we constructed an amino terminus of human Cx43 (Cx43NT-GFP), verified the overexpression of Cx43-NT in HUVEC cells and explored the impact of gap junctions (GJs) on multiple myeloma (MM).MATERIAL AND METHODS:The levels of phosphorylated Cx43(s368) and the change of MAPK pathway associated molecules (ERK1/2, JNK, p38, NFκB) were also investigated in our cell models. Cx43 mRNA and proteins were detected in both MM cell lines and mesenchymal stem cells (MSCs). Dye transfer assays demonstrated that gap junction intercellular communication (GJIC) occurring via Cx43 situated between MM and MSCs or MM and HUVECCx43NT is functional.RESULTS:Our results present evidence for a channel-dependent modulator action of connexin 43 on the migratory activity of MM cells toward MSCs or HUVECCx43-N was higher than those of spontaneous migration (p < 0.05) and protection them from apoptosis in the presence of dexamethasone via cytokines secretion. In the meantime, the migration of MM cells involves an augmented response of p38 and JNK signaling pathway of carboxyl tail of the protein.CONCLUSIONS:Our data suggest that GJIC between MM and MSCs is one of the essential factors in tumor cell proliferation and drug sensitivity, and is implicated in MM pathogenesis.

背景:多发性骨髓瘤骨病的发病机制目前尚未完全明确,骨髓间充质干细胞向成骨细胞分化障碍参与其中,而Notch1信号通路在间充质干细胞的增殖分化中起重要作用.目的:探讨Notch1信号通路在多发性骨髓瘤骨病中的作用.方法:分离培养多发性骨髓瘤患者和正常人骨髓间充质干细胞,Real-time PCR和Western blot检测成骨诱导分化前后Notch1和成骨基因Runx2的表达,以及Von Kossa染色鉴定钙质沉积程度.在多发性骨髓瘤患者间充质干细胞成骨诱导分化过程中,加入Notch1信号通路抑制剂DAPT和安慰剂,48 h后real-time PCR和western blot鉴定Notch1信号通路下游分子Hes1和成骨指标Runx2表达,2周后Von Kossa染色鉴定钙质沉积程度.结果与结论:成骨诱导48 h后,间充质干细胞的Notch1表达减低,但是骨髓瘤患者间充质干细胞的降低幅度小于正常对照间充质干细胞;48h后Runx2的表达在骨髓瘤患者间充质干细胞的表达明显弱于正常对照间充质干细胞;2周后,Von Kossa染色鉴定钙质沉积程度,骨髓瘤患者间充质干细胞明显弱于正常对照间充质干细胞;48h后Hes1表达在DAPT组明显低于安慰剂组;而Runx2的表达在DAPT组明显高于安慰剂组.2周后DAPT组钙质沉积明显强于安慰剂组.实验说明多发性骨髓瘤患者的间充质干细胞中,Notch1信号通路失活缺陷可能抑制其向成骨细胞分化.

Background: Proteasome inhibition with bortezomib has revolutionized the treatment of multiple myeloma, but the vast majority of patients eventually develop clinical bortezomib resistance through poorly understood mechanisms. Long-lived plasma cells, which are supported by niche environments, are postulated to be an important mediator of resistance to chemotherapy and disease relapse. CXCR4/CXCL12 (SDF-1) axis plays an important role in the interaction of long-lived plasma cells and the microenvironment. Granulocyte-Colony Stimulating Factor (G-CSF) is reported to possess the potential to inhibit the expression of SDF-1, thus to suppress the CXCR4/SDF-1 axis and mobilize long-lived plasma cells from the niche environments. Bortezomib is reported to be able to eliminate both short- and long-lived plasma cells. In this trial we hypothesized that inhibition of the CXCR4/SDF-1 axis by G-CSF would disrupt the interaction of plasma cells with the environment and increase the sensitivity of myeloma cells to bortezomib.

Objective To deplore the immunoregulatory function changes of mesenchymal stem cells(MSCs)from multiple myeloma(MM)patients and its effects on the pathogenesis of myeloma bone disease.Methods MSCs from MM patients and normal controls were isolated and the immunophenotype was detected.Real-time PCR was performed to detect the expressions of TGF-β1,TGF-β2,TGF-β3,IL-6,IL3,TNF-α,FasL and RANKL of MSCs.Transwell coculture systems were performed between MSCs and T cells.Lymphocyte proliferative assay was employed to detect the effect of MSCs on T cell proliferation.The effect of MSCs on T cell cycle and T cell activation markers CD25 and CD69 expression were analyzed by flow cytometry.Cleaved caspase 3 protein by western blot and hoechst 33258 staining were employed to detect the apotosis of T cells.Influence of T cells on the osteogenesis potential of MSCs were detected by Von kossa stain,real-time PCR and Western blot.Results MSCs from both MM patients and normal controls possessed similar morphology and immunophenotypes.MM derived MSCs exhibited increased expressions of TGF-β1,IL-6,IL-3,TNF-α and RANKL and decreased expression of TGF-β2,TGF-β3 and FasL.The inhibitory effect of MM derived MSCs on T cell proliferative ability was attenuated compared to control MSCs.MSCs from normal controls silence more T cells in Go/G1 phase than those from MM patients.The daupening effect of MM derived MSCs on activation-induced T apoptosis seemed to be enhanced.Expression of T cell activation markers were significantly inhibited by MSCs from normal controls.Both T cells cocultured with MM deprived MSCs and T cells directly from MM patients inhibited osteogenesis potential of MSCs from normal controls.Conclusion MSCs from MM patients showed impaired immunoregulatory capability on T cells.The activated T cells,in turn,inhibited the osteogenesis potential of MSCs.This may participate in the pathogenesis of myeloma bone disease.

Background and Aims. Abnormality of immune regulation exists in multiple myeloma (MM). Mesenchymal stem cells (MSCs), a key regulator for immunomodulatory function, have decreased osteogenic potential in MM patients. Here we investigated the immunomodulatory function of MSCs from MM patients (MM-MSCs) and its relationship with decreased osteogenic potential.Methods. Real-time PCR was performed to detect the cytokines expressed in MM-MSCs (n = 22) and MSCs from normal donors (ND-MSCs, n = 11). Lymphocyte proliferative assay was used to detect the effect of MSCs on T cell proliferation. The effect of MSCs on T-cell cycle and T-cell activation markers expression were analyzed by flow cytometry. Flow cytometry and Western blot were used to detect apoptosis of T cells. Influence of T cells on osteogenic potential of MSCs was detected.Results. MM-MSCs exhibited increased expression of TGF-beta 1, IL-6, IL-3, TNF-alpha, and RANKL and decreased expression of TGF-beta 2, TGF-beta 3 and FasL. The inhibitory effect of MM-MSCs on T.cell proliferative ability was attenuated. ND-MSCs silence more T cells in G0/G1 phase than MM-MSCs. The apoptosis-promoting effect of MM-MSCs on T cells seemed to be dampened. Expression of T-cell activation markers was significantly inhibited by ND-MSCs. T cells from normal donors possessed the ability to promote osteoblastic differentiation of ND-MSCs, but this ability of T cells both directly from MM patients and co-cultured with MM-MSCs was impaired.Conclusions. MSCs from MM patients showed impaired immunoinhibitory capability on T cells, which in turn lose the ability to stimulate osteogenesis of MSCs. (C) 2010 IMSS. Published by Elsevier Inc.

Engineered single chain antibodies have become a powerful source of immunotherapy against a wide range of diseases. Here, we present the generation of human CD28 single-chain antibody gene (CD28-ScFv), which contained variable fragments of heavy chain and light chain (VH and VL) of the anti-CD28 antibody, and a linking peptide (Gly4Ser)3 inserted in the middle of VH and VL. The fused gene CD28-ScFv was successfully expressed in BL21 (DE3) cells and confirmed by western blotting assay. The molecular weight of CD28-ScFv was 43 kDa and the major fraction was expressed as an insoluble body. By dissolving the insoluble bodies, renaturing in vitro and purifying with a Ni-NTA affinity column, highly purified expression products of CD28-ScFv were obtained. This product could recognize and bind to CD28+ positive T cells. The proliferation capacity of peripheral blood T cells was increased by purified CD28-ScFv. In this study, we improved orthodox renaturing techniques by combining the dilution renaturation with phase gradient dialysis. With this new method, highly purified CD28-ScFv products were developed and biological activity of the products was similar to that of the mouse monoclonal anti-human CD28 antibody.