scholarly journals Protective Role of Coxsackie-Adenovirus Receptor in the Pathogenesis of Inflammatory Bowel Diseases

2018 ◽  
Vol 2018 ◽  
pp. 1-11
Author(s):  
Xiong Chen ◽  
Rui Liu ◽  
Xiaoming Liu ◽  
Canxia Xu ◽  
Xiaoyan Wang
Keyword(s):  
Ulcerative Colitis ◽  
Western Blot ◽  
Rat Model ◽  
Coxsackie Adenovirus Receptor ◽  
Qrt Pcr ◽  
Inflammatory Bowel ◽  
Adenovirus Receptor ◽  
Permeability Assay

Aim. To investigate the role of Coxsackie-adenovirus receptor (CAR) in inflammatory bowel disease (IBD). Background. CAR, a type I transmembrane protein with functions in virus attachment, has been shown to be associated with epithelial tight junctions (TJs) and mediates cell adhesion, implying its potential roles in the pathogenesis of IBD. Methods and Materials. To determine the effect of CAR in IBD using QPCR and Western blotting to determine the expression of CAD in TNF-α induced NCM460 and SW480 cells and IBD tissues compared to control groups. Furthermore, TJs dysregulation, FITC-Dextran permeability assay, qRT-PCR, Western blot, and IF assessed the permeability in CAR overexpressed cells treated with TNF-α. HE, qRT-PCR, Western blot, and IHC assay were used to assess the CAR overexpressed cells whether they have the effect to cure DSS induced ulcerative colitis rat model in vivo. Result. We found CAR levels in human colon cell lines are significantly downregulated under the treatment of tumor necrosis factor-alpha (TNF-α). Furthermore, overexpression of CAR markedly prevented TNF-α induced inflammatory response, TJs dysregulation, and permeability disruption (FITC-Dextran permeability assay) in cells. Consistent with these findings in vitro, we found that CAR overexpression could suppress gut inflammation, attenuate the downregulation of TJ protein ZO-1 and Occludin, and limit the induction of barrier permeability in a DSS induced ulcerative colitis rat model in vivo. Together, our findings strongly suggest that CAR could protect tight junctions and has an anti-inflammatory effect during the pathogenesis of IBD. Thus CAR may serve as a therapeutic target for the diagnosis and treatment of IBD.

Requirement of CREB in Normal and Malignant Hematopoiesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1168-1168
Author(s):  
Jerry C. Cheng ◽  
Deepa Shankar ◽  
Stanley F. Nelson ◽  
Kathleen M. Sakamoto
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Western Blot ◽  
Blot Analysis ◽  
Hematopoietic Cells ◽  
Wild Type ◽  
Qrt Pcr ◽  
T315i Mutation

Abstract CREB is a nuclear transcription factor that plays an important role in regulating cellular proliferation, memory, and glucose homeostasis. We previously demonstrated that CREB is overexpressed in bone marrow cells from a subset of patients with acute leukemia at diagnosis. Furthermore, CREB overexpression is associated with an increased risk of relapse and decreased event-free survival in adult AML patients. Transgenic mice that overexpress CREB in myeloid cells developed myeloproliferative/myelodysplastic syndrome after one year. To further understand the role of CREB in leukemogenesis and in normal hematopoiesis, we employed RNA interference methods to inhibit CREB expression. To achieve sustained, CREB-specific gene knockdown in leukemia and normal hematopoietic cells, a lentiviral-based small hairpin (shRNA) approach was taken. Three CREB specific shRNAs were generated and tested for efficiency of gene knockdown in 293T cells. Knockdown efficiency approached 90 percent by Western blot analysis compared to vector alone and luciferase controls. Human myeloid leukemia cell lines, K562, TF1, and MV411, were then infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed using quantitative real time (qRT)-PCR, Western blot analysis, and growth and viability assays. Lentiviral CREB-shRNA achieved between 50 to 90 percent knockdown of CREB compared to control shRNAs at the protein and mRNA levels. To control for non-specific effects, we performed qRT-PCR analysis of the interferon response gene, OAS1, which was not upregulated in cells transduced with CREB shRNA constructs. Within 72 hours, cells transduced with CREB shRNA had decreased proliferation and survival. Similar results were obtained with murine leukemia cells (NFS60 and BA/F3 bcr-abl).To study the role of CREB in normal hematopoiesis, both primary murine and human hematopoietic cells were transduced with our shRNA constructs, and methylcellulose-based colony assays were performed. Primary hematopoietic cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in colony number compared to control virus-infected cells (p<0.05). Bone marrow colonies consisted of myeloid progenitor cells that were mostly Mac-1+ by FACs analysis. Interestingly, there were fewer differentiated cells in the CREB shRNA transduced cells compared to vector control or wild type cells, suggesting that CREB is critical for both myeloid cell proliferation and differentiation. To study the in vivo effects of CREB knockdown on leukemia progression, we studied mice injected with BA/F3 cells that express both bcr/abl with the T315I mutation and a luciferase reporter gene. BA/F3 cells expressing the T315I mutation have a 2-fold increase in CREB overexpression compared to wild-type cells. Disease progression was monitored using bioluminescence imaging with luciferin. CREB knockdown was 90 percent after transduction and prior to injection into SCID mice. We observed improved survival of mice injected with CREB shRNA transduced BA/F3 bcr-abl (T315I) compared to vector control cells. To understand the mechanism of growth suppression resulting from CREB downregulation, we performed microarray analysis with RNA from CREB shRNA transduced K562 and TF1 cells. Several genes were downregulated using a Human Affymetrix chip. Most notable was Beclin1, a tumor suppressor gene often deleted in prostate and breast cancer that has been implicated in autophagy. Our results demonstrate that CREB is required for normal and leukemic cell proliferation both in vitro and in vivo.

scholarly journals HIF-1α and HIF-2α Mediated PARVB Expression Promotes Tumor Growth and Metastasis in Malignantmelanoma

2020 ◽  
Author(s):  
Ting Wang ◽  
Zhiqiang Wu ◽  
Yifeng Bi ◽  
Haitao Sun ◽  
Zhipeng Wu ◽  
...  
Keyword(s):  
Malignant Melanoma ◽  
Tumor Growth ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Human Malignant Melanoma ◽  
Qrt Pcr ◽  
Tumor Growth And Metastasis

Abstract BackgroundMalignant melanoma is the leading cause of skin cancer-related death. The role of PARVB in malignant melanoma remains unclear. Hypoxia is a hallmark of solid tumors including melanoma. But the regulation role of hypoxia in PARVB expression has not been reported.MethodsHuman malignant melanoma tissues, cell lines and their controls were collected. IHC staining, qRT-PCR and Western blot were performed to reveal the differential PARVB expression. The role of PARVB in tumor growth and metastasis of malignant melanoma was evaluated in vitro and in vivo. The regulation role and mechanism of hypoxia and HIFs in PARVB expression was validated by qRT-PCR, Western blot, ChIP-PCR and Luciferase reporter assays.ResultsPARVB was upregulated in malignant melanoma and correlated with patient survival. OverexpressionofPARVB promoted tumor growth and metastasis of malignant melanoma. Furthermore, hypoxia induced HIF-1α and HIF-2α expression activated PARVB transcription and expression through binding to the specific hypoxia-responsive element (HRE) in the promoter region of PARVB.ConclusionsIn malignant melanoma, Hypoxia induced HIF-1α and HIF-2α expression could directly activate PARVB expression, which further promoted tumor growth and metastasis, inducing poor prognosis. These results indicated that PARVB might be a potential therapeutic target for malignant melanoma.

scholarly journals Knockdown of lncRNA X inactive specific transcript (XIST) radiosensitizes non-small cell lung cancer (NSCLC) cells through regulation of miR-16-5p/WEE1 G2 checkpoint kinase (WEE1) axis

2021 ◽  
Vol 35 ◽  
pp. 205873842096608
Author(s):  
Ran Du ◽  
Feng Jiang ◽  
Yanhua Yin ◽  
Jinfen Xu ◽  
Xia Li ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Western Blot ◽  
Small Cell ◽  
Luciferase Reporter ◽  
Small Cell Lung ◽  
Luciferase Reporter Gene ◽  
G2 Checkpoint ◽  
Qrt Pcr ◽  
Specific Transcript

Long non-coding RNA (lncRNA) X inactive specific transcript (XIST) is reported to play an oncogenic role in non-small cell lung cancer (NSCLC). However, the role of XIST in regulating the radiosensitivity of NSCLC cells remains unclear. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expressions of XIST and miR-16-5p in NSCLC in tissues and cells, and Western blot was used to assess the expression of WEE1 G2 checkpoint kinase (WEE1). Cell counting kit-8 (CCK-8), colony formation and flow cytometry assays were used to determine cell viability and apoptosis after NSCLC cells were exposed to different doses of X-rays. The interaction between XIST and miR-16-5p was confirmed by StarBase database, qRT-PCR and dual-luciferase reporter gene assays. TargetScan database was used to predict WEE1 as a target of miR-16-5p, and their targeting relationship was further validated by Western blot, qRT-PCR and dual-luciferase reporter gene assays. XIST was highly expressed in both NSCLC tissue and cell lines, and knockdown of XIST repressed NSCLC cell viability and cell survival, and facilitated apoptosis under the irradiation. MiR-16-5p was a target of XIST, and rescue experiments demonstrated that miR-16-5p inhibitors could reverse the role of XIST knockdown on radiosensitivity in NSCLC cells. WEE1 was validated as a target gene of miR-16-5p, and WEE1 could be negatively regulated by XIST. XIST promotes the radioresistance of NSCLC cells by regulating the expressions of miR-16-5p and WEE1, which can be a novel target for NSCLC therapy.

scholarly journals Role of Probiotics and Their Metabolites in Inflammatory Bowel Diseases (IBDs)

2021 ◽  
Vol 12 (1) ◽  
pp. 56-66
Author(s):  
Toumi Ryma ◽  
Arezki Samer ◽  
Imene Soufli ◽  
Hayet Rafa ◽  
Chafia Touil-Boukoffa
Keyword(s):  
Ulcerative Colitis ◽  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Clinical Symptoms ◽  
Therapeutic Potential ◽  
Short Chain Fatty Acids ◽  
Active Metabolites ◽  
Complex Disorders ◽  
Inflammatory Bowel

Inflammatory Bowel Disease (IBD) is a term used to describe a group of complex disorders of the gastrointestinal (GI) tract. IBDs include two main forms: Crohn’s Disease (CD) and Ulcerative Colitis (UC), which share similar clinical symptoms but differ in the anatomical distribution of the inflammatory lesions. The etiology of IBDs is undetermined. Several hypotheses suggest that Crohn’s Disease and Ulcerative Colitis result from an abnormal immune response against endogenous flora and luminal antigens in genetically susceptible individuals. While there is no cure for IBDs, most common treatments (medication and surgery) aim to reduce inflammation and help patients to achieve remission. There is growing evidence and focus on the prophylactic and therapeutic potential of probiotics in IBDs. Probiotics are live microorganisms that regulate the mucosal immune system, the gut microbiota and the production of active metabolites such as Short-Chain Fatty Acids (SCFAs). This review will focus on the role of intestinal dysbiosis in the immunopathogenesis of IBDs and understanding the health-promoting effects of probiotics and their metabolites.

scholarly journals Knockdown of Long Non-Coding RNA XIST Inhibited Doxorubicin Resistance in Colorectal Cancer by Upregulation of miR-124 and Downregulation of SGK1

2018 ◽  
Vol 51 (1) ◽  
pp. 113-128 ◽  
Author(s):  
Jia Zhu ◽  
Rui Zhang ◽  
Dongxiang Yang ◽  
Jibin Li ◽  
Xiaofei Yan ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Western Blot ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Glutathione S Transferase ◽  
Protein Levels ◽  
Qrt Pcr ◽  
Ic50 Value

Background/Aims: Doxorubicin (DOX) is a widely used chemotherapeutic agent for colorectal cancer (CRC). However, the acquirement of DOX resistance limits its clinical application for cancer therapy. Mounting evidence has suggested that aberrantly expressed lncRNAs contribute to drug resistance of various tumors. Our study aimed to explore the role and molecular mechanisms of lncRNA X-inactive specific transcript (XIST) in chemoresistance of CRC to DOX. Methods: The expressions of XIST, miR-124, serum and glucocorticoid-inducible kinase 1 (SGK1) mRNA in DOX-resistant CRC tissues and cells were detected by qRT-PCR or western blot analysis. DOX sensitivity was assessed by detecting IC50 value of DOX, the protein levels of P-glycoprotein (P-gp) and glutathione S-transferase-π (GST-π) and apoptosis. The interactions between XIST, miR-124 and SGK1 were confirmed by luciferase reporter assay, qRT-PCR and western blot. Xenograft tumor assay was used to verify the role of XIST in DOX resistance in CRC in vivo. Results: XIST expression was upregulated and miR-124 expression was downregulated in DOX-resistant CRC tissues and cells. Knockdown of XIST inhibited DOX resistance of CRC cells, as evidenced by the reduced IC50 value of DOX, decreased P-gp and GST-π levels and enhanced apoptosis in XIST-silenced DOX-resistant CRC cells. Additionally, XIST positively regulated SGK1 expression by interacting with miR-124 in DOX-resistant CRC cells. miR-124 suppression strikingly reversed XIST-knockdown-mediated repression on DOX resistance in DOX-resistant CRC cells. Moreover, SGK1-depletion-elicited decrease of DOX resistance was greatly restored by XIST overexpression or miR-124 inhibition in DOX-resistant CRC cells. Furthermore, XIST knockdown enhanced the anti-tumor effect of DOX in CRC in vivo. Conclusion: XIST exerted regulatory function in resistance of DOX possibly through miR-124/SGK1 axis, shedding new light on developing promising therapeutic strategy to overcome chemoresistance in CRC patients.

scholarly journals Metformin Sensitizes Osteosarcoma Cells to Chemotherapy Through IGF-1R/miR-610/FEN1 Pathway

2021 ◽  
Author(s):  
Suwei Dong ◽  
Yanbin Xiao ◽  
Ziqiang Zhu ◽  
Xiang Ma ◽  
Zhuohui Peng ◽  
...  
Keyword(s):  
Underlying Mechanism ◽  
Cytotoxic Agents ◽  
Luciferase Reporter ◽  
Osteosarcoma Cell ◽  
Metformin Treatment ◽  
Normal Tissues ◽  
Qrt Pcr ◽  
In Vivo Experiments

Abstract Background: Due to constitutive or acquired non-sensitive to cytotoxic agents, the prognosis of osteosarcoma remains unfavorable. It’s has been proved that metformin could enhance the chemosensitivity of cancer cells to anticancer drugs. A novel finding states that IGF-1R involves in cancer chemoresistance, However, whether IGF-1R play a role in metformin-induced osteosarcoma chemosensitivity is incompletely understood. Hence, the current study aimed to elucidate the role of metformin in OS cell chemosensitivity modulation to identify the underlying mechanism of metformin regulating the IGF-1R/miR-610/FEN1 signaling.Methods: Immunohistochemistry and qRT-PCR were used to evaluate the expression pattern of IGF-1R, miR-610 and FEN1 in osteosarcoma and paired normal tissues. Western blot and qRT-PCR were performed to determine changes in expression of key molecules in the IGF-1R/miR-610/FEN1 signaling pathway after various treatments. The direct modulation between miR-610 and FEN1 was monitored by luciferase reporter assay. Osteosarcoma cell sensitivity to chemotherapy was detected by MTS assay. In vivo experiments were conducted to further verify the role of the metformin in the chemosensitivity modulation of OS cells to ADM.Results: We found that IGF-1R, miR-610 and FEN1 were abberently expressed in osteosarcoma, and participated in apoptosis modulation (p < 0.05). We found that this effect was abated by metformin treatment. Luciferase reporter assays confirmed that FEN1 is a direct target of miR-610. Moreover, we observed that metformin treatment decreased IGF-1R and FEN1, but elevated miR-610 expression. Metformin sensitized OS cells to cytotoxic agents, while overexpression of FEN1 compromised the sensitizing effects of metformin partly. Furthermore, metformin was observed to enforce the ADM treatment effect in nude mice xenograft models.Conclusions: Overall, metformin enhanced the sensitivity of OS cells to cytotoxic agents via the IGF-1R/miR-610/FEN1 signaling axis, highlighting the capacity of metformin as an adjunct to the chemotherapy of OS.

scholarly journals Virulence of methicillin-resistant Staphylococcus aureus modulated by the YycFG two-component pathway in a rat model of osteomyelitis

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Shizhou Wu ◽  
Yunjie Liu ◽  
Lei Lei ◽  
Hui Zhang
Keyword(s):  
Staphylococcus Aureus ◽  
Western Blot ◽  
Rat Model ◽  
Biofilm Formation ◽  
Bacterial Biofilm ◽  
Invasive Ability ◽  
Methicillin Resistant ◽  
Two Component ◽  
Mrsa Strains

Abstract Objectives Methicillin-resistant Staphylococcus aureus (MRSA) strains present an urgent medical problem in osteomyelitis cases. Our previous study indicated that the YycFG two-component regulatory pathway is associated with the bacterial biofilm organization of MRSA strains. The aim of this study was to investigate the regulatory roles of ASyycG in the bacterial biofilm formation and the pathogenicity of MRSA strains using an antisense RNA strategy. Methods An ASyycG-overexpressing MRSA clinical isolate was constructed. The bacterial growth was monitored, and the biofilm biomass on bone specimens was examined using scanning electron microscopy and confocal laser scanning microscopy. Furthermore, quantitative RT-PCR (QRT-PCR) analysis was used to measure the expression of yycF/G/H and icaA/D in the MRSA and ASyycG strains. The expression of the YycG protein was quantified by Western blot assays. We validated the role of ASyycG in the invasive ability and pathogenicity of the strains in vivo using histology and peptide nucleic acid fluorescent in situ hybridization. Results The results showed that overexpression of ASyycG lead to a reduction in biofilm formation and exopolysaccharide (EPS) synthesis compared to the control MRSA strains. The ASyycG strains exhibited decreased expression of the yycF/G/H and icaA/D genes. Furthermore, Western blot data showed that the production of the YycG protein was inhibited in the ASyycG strains. In addition, we demonstrated that ASyycG suppressed the invasive ability and pathogenicity of the strain in vivo using an SPF (specific pathogen free) rat model. Conclusion In summary, the overexpression of ASyycG leads to a reduction in biofilm formation and bacterial pathogenicity in vivo, which provides a potential target for the management of MRSA-induced osteomyelitis.

The Multifaceted Role of Natural Agents in Colitis-Associated Cancer Prevention and Therapy

2021 ◽  
pp. 220-239
Author(s):  
Ashok Kumar Kumar Pandurangan ◽  
Suresh Kumar Anandasadagopan ◽  
Neesar Ahmed
Keyword(s):  
Oxidative Stress ◽  
Ulcerative Colitis ◽  
Inflammatory Bowel Disease ◽  
Colon Cancer ◽  
The Other ◽  
Preclinical Model ◽  
Natural Agents ◽  
Inflammatory Bowel ◽  
Prevention And Therapy

Inflammatory bowel disease (IBD) is comprised of ulcerative colitis (UC) and Crohn's disease (CD) that was recognized by the inflammation in the colon. There are no proper medications are available to control the IBD in patients. NASIDs such as Aspirin, diclofenac, and ibuprofen are widely used to control the inflammation. On the other hand, the untreated prolonged inflammation leads to the development of cancer in the colon termed as colitis-associated cancer or inflammation-driven colon cancer. Oxidative stress and inflammation play key roles in the pathogenesis of colitis-associated cancer. Single dose of azozymethane (AOM) and three cycles of 2% dextran sodium sulfate (DSS) induces colitis-associated cancer (CAC) in mouse. Hence, many natural products were tested in the preclinical model of colitis-associated cancer. Each of these natural agents modulate important signaling pathway to control the colitis-associated cancer (CAC). In this review, the authors tabulated all the natural agents that culminate the colitis-associated cancer in the preclinical models.

scholarly journals The Anti-Inflammatory Effects of Angiogenin in an Endotoxin Induced Uveitis in Rats

2020 ◽  
Vol 21 (2) ◽  
pp. 413
Author(s):  
Jihae Park ◽  
Jee Taek Kim ◽  
Soo Jin Lee ◽  
Jae Chan Kim
Keyword(s):  
Western Blot ◽  
Inflammatory Cytokines ◽  
Aqueous Humor ◽  
Rat Model ◽  
Inflammatory Responses ◽  
Inflammatory Cells ◽  
Ocular Tissue ◽  
Tnf Α ◽  
Anti Inflammatory

Angiogenin (ANG) is involved in the innate immune system and inflammatory disease. The aim of this study is to evaluate the anti-inflammatory effects of ANG in an endotoxin induced uveitis (EIU) rat model and the pathways involved. EIU rats were treated with balanced salt solution (BSS), a non-functional mutant ANG (mANG), or wild-type ANG (ANG). The integrity of the blood-aqueous barrier was evaluated by the infiltrating cell and protein concentrations in aqueous humor. Histopathology, Western blot, and real-time qRT-PCR of aqueous humor and ocular tissue were performed to analyze inflammatory cytokines and transcription factors. EIU treated with ANG had decreased inflammatory cells and protein concentrations in the anterior chamber. Compared to BSS and mANG, ANG treatment showed reduced expression of IL-1β, IL-8, TNF-α, and Myd88, while the expression of IL-4 and IL-10 was increased. Western blot of ANG treatment showed decreased expression of IL-6, inducible nitric oxide synthase (iNOS), IL-1β, TNF-α, and phosphorylated NF-κB and increased expression of IL-10. In conclusion, ANG seems to reduce effectively immune mediated inflammation in the EIU rat model by reducing the expression of proinflammatory cytokines, while increasing the expression of anti-inflammatory cytokines through pathways related to NF-κB. Therefore, ANG shows potential for effectively suppressing immune-inflammatory responses in vivo.

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