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Objective(s): Colorectal cancer is one of the deadliest cancers worldwide, which can be prevented and even cured by early diagnosis and more efficient treatment modalities. Comprehensive transcriptional analysis has highlighted the importance of lncRNAs in CRC tumorigenesis. In this study, we identified co-expressed lncRNA networks based on public RNA sequencing data for biomarker prediction in CRC and then verified the best candidate experimentally. Materials and Methods: Publicly available RNA-sequencing data (BioProject PRJEB27536) of CRC samples and normal adjacent tissues were reanalyzed using the DESeq2 package in R to find differentially expressed lncRNAs. Pathway enrichment and gene network analysis were accomplished using GSEA and WGCNA to identify potential functions of lncRNAs with possible roles in tumorigenesis pathways. Subsequently, the expression of RP11-109D20.2 (lnc-Duox2-1:1) was assessed in fresh/frozen tissues obtained from 46 CRC patients by quantitative RT-PCR. Results: A total of 17939 DElncRNAs were identified between CRC and normal tissues via bioinformatics analyses. A significant up-regulation of RP11-109D20.2 (48%) was observed in CRC samples. Functional enrichment analysis showed that RP11-109D20.2 was mainly related to pathways like phosphoric ester hydrolase, oxidoreductase, phosphoric diester hydrolase, and cyclicnucleotide phosphodiester activities. Moreover, elevated expression of DUOX2 in tumors with high levels of RP11-109D20.2 suggests a link between these genes. Conclusion: Our data revealed that RP11-109D20.2 may have a considerable role in CRC progression. However, further functional analyses are essential to evaluate the probable role of RP11-109D20.2 as a potential diagnostic marker and its potential role in the dysregulation of cyclic nucleotide phosphodiesterase genes in CRC. [ABSTRACT FROM AUTHOR]

Objective: The immunoregulatory properties of mesenchymal stromal/stem cells (MSCs) bring a promise for the treatment of inflammatory diseases. However, their ability to suppress the immune system is unstable. To enhance their effectiveness against immune responses, it may be necessary to manipulate MSCs. Although some dsRNA transcripts come from invading viruses, the majority of dsRNA has an endogenous origin and is known as endo-siRNA. DICER1 is a ribonuclease protein that can generate small RNAs to modulate gene expression at the post-transcriptional level. We aimed to evaluate the expression of several immune-related genes at mRNA and protein levels in MSCs overexpressing DICER1 exogenously. Materials and Methods: In this comparative transcriptomic experimental study, the adipose-derived MSCs (Ad-MSCs) were transfected using the pCAGGS-Flag-hsDicer vector for the DICER1 overexpression. Following the RNA extraction, mRNA expression level of DICER1 and several inflammatory cytokines were examined. We performed a relative real-time polymerase chain reaction (PCR) assay and transcriptome analysis between two groups including DICER1- transfected MSCs and control MSCs. Moreover, media from the transfected MSCs were evaluated for various interferon response factors by ELISA. Results: The overexpression of DICER1 is associated with a significant increase in the mRNA expression level of COX-2, DDX-58, IFIH1, MYD88, RNase L, TLR3/4, and TDO2 genes and a downregulation of the TSG-6 gene in MSCs. Moreover, the expression levels of IL-1, 6, 8, 17, 18, CCL2, INF-γ, TGF-β, and TNF-α were higher in the DICER1-transfected MSCs group. Conclusion: It seems that the ectopic expression of DICER1 in Ad-MSCs is linked to alterations in the expression level of immune-related genes. It is suggested that the manipulation of immune-related pathways in MSCs via the Dicer1 overexpression could facilitate the development of MSCs with distinct immunoregulatory phenotypes. [ABSTRACT FROM AUTHOR]

Objective(s): The B18R protein encoded by the Vaccinia virus decoys Type 1 interferons and inhibits the activity of several type I IFN members. In vitro transcription protocols benefit from this molecule's involvement in enhancing cell viability by inhibiting interferon signal transduction. As a result of their immunomodulatory properties and potential to regenerate, mesenchymal stromal cells (MSCs) are increasingly considered an alternative treatment for a wide range of immune disorders. In this study, we investigated the modification of expression of several genes involved in immune-related pathways after preconditioning MSCs with two immune stimuli, including poly(I:C) and LPS. Materials and Methods: ASCs were isolated and primed with B18R, and after exposure to poly(I:C) and LPS, the expression of the same sets of genes as in the previous experiment was evaluated. Following total RNA isolation from primed cells and cDNA preparation, real-time quantitative PCR was performed for several immunomodulatory and immune-related genes, including IDO1, TDO2, COX-2, TGF-β1, TNF-α, IL-1β, IL-6, TLR3, TLR4, and MCP-1. Results: Pretreatment of MSCs with poly(I:C) and LPS significantly increased the expression of all mentioned genes, while upon the B18R challenge followed by poly(I:C) and LPS treatment, they were down-regulated. Finally, it was observed that the relative expression level of IFN-β has significantly decreased in MSCs+B18R+poly(I:C) and LPS in comparison with these groups without B18R. Conclusion: The data indicated that the presence of B18R prevents the overexpression of several immune-related genes, which are overexpressed in the in vitro inflammatory environment. [ABSTRACT FROM AUTHOR]

Abstract Background Dendritic Cells (DC) are potent antigen presenting cells with the ability to prime naïve T cells and convert them to cytotoxic T-lymphocytes (CTL). We evaluated the capability of autologous DCs transfected with total tumor and normal RNA to induce cytotoxic CTL as the preliminary step to design a DC-based vaccine in the esophageal squamous cell carcinoma (ESCC). Methods Monocytes-derived DCs were electroporated with either total tumor RNA or normal RNA. T cells were then primed with tumor RNA transfected DCs and lytic effects of the generated CTL were measured with Cytotoxicity assay and IFN-γ Release Elispot assay. Results Cytotoxicity was induced against DCs loaded with tumoral RNA (%24.8 ± 5.2 SEM) while in normal RNA-loaded DCs, it was minimal (%6.1 ± 2.4 SEM) and significantly lower (p < 0.05). INF-γ secretion was more than 2-folds higher in tumoral RNA-loaded DCs when compared with normal RNA-loaded DCs (p < 0.05). Conclusion Electroporating DCs with tumor RNA generated tumor antigen presenting cells which in turn enhanced cytotoxic effects of the T cells against ESCC. This may be a useful autologous ex vivo screening tool for confirming the lytic effects of primed T cells on tumors and evaluate probable further adverse effects on noncancerous tissues. These data provide crucial preliminary information to establish a total tumor RNA-pulsed DC vaccine therapy of ESCC.

Objective(s): Cell-based therapeutic approaches have witnessed significant developments during the last decade especially after approval of MSCs based treatment of graft versus host disease. Several cell-based approaches have shown immunomodulatory behavior during regeneration following the unknown cascade of events but the exact mechanisms are yet to be defined. Clinical applications of cell-based drugs are hampered all over the world because of incomplete understanding of molecular mechanisms requiring the application of mechanistic approaches to solving the mystery. Current work has given us the idea that Nanos2 enhances the cellular pluripotency characteristics while downregulating the innate immunity genes, simultaneously. Materials and Methods: The immunomodulatory behavior of cells was studied against cells carrying the ectopic expression of Nanos2 in comparison with Stella and Oct4 individually and simultaneously using SON vector (Stella, Nanos2 and Oct4). Results: It was observed that overexpression of Nanos2 leads to down-regulation of Interferon- Stimulated Genes (ISGs)-mRNAs such as Ifitm1, lsg15, Oas2, and Oas12. Nanos2 overexpressing MEF cells have shown restrictive inflammatory effects when cells were treated with inflammatory stimuli such as LPS and Poly (I:C). Conclusion: From our recent findings in line with many others, it can be concluded that Nanos2 acts as a coin with two sides, regulating pluripotency and immunity together which enhances resistance against inflammatory stimuli. Nanos2 could be a potential candidate as a molecular drug for management of inflammation and immunomodulation but it requires a comprehensive comparative expression analysis of innate immunity genes in vitro and in vivo. [ABSTRACT FROM AUTHOR]

C-X-C chemokine receptor type 4 (CXCR4), initially recognized as a co-receptor for HIV, contributes to several disorders, including the WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome. CXCR4 binds to its ligand SDF-1 to make an axis involved in the homing property of stem cells. This study aimed to employ WHIM syndrome pathogenesis as an inspirational approach to reinforce cell therapies. Wild type and WHIM-type variants of the CXCR4 gene were chemically synthesized and cloned in the pCDH-513B-1 lentiviral vector. Molecular cloning of the synthetic genes was confirmed by DNA sequencing, and expression of both types of CXCR4 at the protein level was confirmed by western blotting in HEK293T cells. Human adipose-derived mesenchymal stem cells (Ad-MSCs) were isolated, characterized, and subjected to lentiviral transduction with Wild type and WHIM-type variants of CXCR4. The presence of copGFP-positive MSCs confirmed the high efficiency of transduction. The migration ability of both groups of transduced cells was then assessed by transwell migration assay in the presence or absence of a CXCR4-blocking agent. Our qRT-PCR results showed overexpression of CXCR4 at mRNA level in both groups of transduced MSCs, and expression of WHIM-type CXCR4 was significantly higher than Wild type CXCR4 (P <0.05). Our results indicated that the migration of genetically modified MSCs expressing WHIM-type CXCR4 had significantly enhanced towards SDF1 in comparison with Wild type CXCR4 (P <0.05), while it was reduced after treatment with CXCR4 antagonist. These data suggest that overexpression of WHIM-type CXCR4 could lead to enhanced and sustained expression of CXCR4 on human MSCs, which would increase their homing capability; hence it might be an appropriate strategy to improve the efficiency of cell-based therapies. [ABSTRACT FROM AUTHOR]

Mesenchymal Stem/Stromal Cells (MSCs) have been applied to modulate various immune-mediated conditions. Prolonged culture of MSCs in vitro reduces their therapeutic efficacy. Pretreatment of the cells with some chemical agents during in vitro expansion could overcome this limitation. This study intended to determine whether pretreatment of adipose-derived MSCs (ASCs) with Human chorionic gonadotrophin (hCG), a glycoprotein hormone, and Vitamin E, an antioxidant, will improve their immunomodulatory ability. In this regard, ASCs were harvested from human processed lipoaspirate. LPS-induced ASCs were preconditioned with 1 mg of hCG and 600 µM of vitamin E for 24h. TSG-6, COX-2, IL-1β, and IL-6 were assessed at the mRNA level in preconditioned and control groups. ASCs were also co-cultured with peripheral blood mononuclear cells (PBMCs) in vitro to determine the functionality of these cells. Results showed that hCG and vitamin E significantly downregulate the pro-inflammatory COX-2, IL-1β, and IL-6 gene expression, while they did not significantly increase TSG-6 expression. Besides, the co-culturing of pretreated ASCs with PBMCs demonstrated that the amount of PBMCs in treated groups (with hCG and vitamin E) was significantly lower than in control groups. These findings revealed that the preconditioning of ASCs with hCG and vitamin E might enhance their immunoregulatory capacity. [ABSTRACT FROM AUTHOR]

The three amino acid loop extension ( TALE) class myeloid ecotropic viral integration site 1 ( MEIS1) homeobox gene is known to play a crucial role in normal and tumor development. In contrast with its well-described cancer stemness properties in hematopoietic cancers, little is known about its role in solid tumors like esophageal squamous cell carcinoma (ESCC). Here, we analyzed MEIS1 expression and its clinical relevance in ESCC patients and also investigated its correlation with the SOX2 self-renewal master transcription factor in the ESCC samples and in the KYSE-30 ESCC cell line. MEIS1 mRNA and protein expression were significantly decreased in ESCC disease ( P < 0.05). The inverse correlation between MEIS1 mRNA expression and tumor cell metastasis to the lymph nodes ( P = 0.004) was significant. Also, MEIS1 protein levels inversely correlated to lymph node involvement ( P = 0.048) and high tumor stage (stages III/IV, P = 0.030). The low levels of DNA methylation in the MEIS1 promoter showed that this suppression does not depend on methylation. We showed that downregulation of EZH2 restored MEIS1 expression significantly. Also, we investigated that MEIS1 downregulation is concomitant with increased SOX2 expression. To the best of our knowledge, this is the first report on the MEIS1 gene in ESCC. The inverse correlation of MEIS1 with metastasis, tumor staging, and the role of EZH2 in methylation, together with its correlation with stemness factor SOX2 expression, led us to predict cancer stemness properties for MEIS1 in ESCC. [ABSTRACT FROM AUTHOR]

Objective(s): The limited homing potential of bone-marrow-derived mesenchymal stem cells (BM-MSC) is the key obstacle in MSC-based therapy. It is believed that chemokines and chemokine receptor interactions play key roles in cellular processes associated with migration. Meanwhile, MSCs express a low level of distinct chemokine receptors and they even lose these receptors on their surface after a few passages which influence their therapeutic applications negatively. This study investigated whether treatment of BM-MSCs with hypoxia-mimicking agents would increase expression of some chemokine receptors and cell migration. Materials and Methods: BM-MSCs were treated at passage 2 for our gene expression profiling. All qPCR experiments were performed by SYBR Green method in CFX-96 Bio-Rad Real-Time PCR. The Boyden chamber assay was utilized to investigate BM-MSC homing. Results: Possible approaches to increasing the expression level of chemokine receptors by different hypoxia-mimicking agents such as valproic acid (VPA), CoCl2, and desferrioxamine (DFX) are described. Results show DFX efficiently up-regulate the CXCR7 and CXCR4 gene expression while VPA increase only the CXCR7 gene expression and no significant change in expression level of CXCR4 and the CXCR7 gene was detectable by CoCl2 treatment. Chemotaxis assay results show that pre-treatment with DFX, VPA, and CoCl2 enhances significantly the migration ability of BM-MSCs compared with the untreated control group and DFX treatment accelerates MSCs homing significantly with a higher rate than VPA and CoCl2 treatments. Conclusion: Our data supports the notion that pretreatment of MSC with VPA and DFX improves the efficiency of MSC therapy by triggering homing regulatory signaling pathways. [ABSTRACT FROM AUTHOR]

Objective(s): Epigenetic regulation of gene expression can be carried out through chromatin remodeling enzymes such as SWI/SNF. Brg1 also known as SMARCA4 is a catalytic subunit of SWI/SNF, which is necessary for MMPs expression. Matrix metalloproteinases (MMPs) are known as important player enzymes during tumor progression and metastasis. Aberrant epigenetic modification of chromatin should be precisely clarified to reveal probable unknown pathways in ESCC progression. Probable role of Brg1 in ESCC tumorigenesis and metastasis was studied through the assessment of Brg1 mRNA expression in KYSE30, and further evaluation about the biology of Brg1 was performed through the Brg1 silencing. Materials and Methods: Level of Brg1 mRNA expression in KYSE30 was compared to normal tissues using the real time polymerase chain reaction (PCR). Moreover, KYSE30 cells were transfected with Brg1-siRNA to silence the Brg1. Results: Our results showed for the first time that Brg1 mRNA expression was increased in KYSE30 cell line (ESCC cell line) compared with normal esophageal tissue of ESCC patients. Rate of transfection in KYSE30 was also between 40 to 50%, using the pSilencer-Brg1shRNA (1:1 ratio). Conclusion: Our data indicated that chromatin remodeling machinery is a novel aspect in tumor biology of ESCC, and overexpression of Brg1 as an important member of SWI/SNF might be involved in the migration and invasion of ESCC tumoral cells. [ABSTRACT FROM AUTHOR]