miR‐548d‐3p inhibits the invasion and migration of gastric cancer cells by targeting GKN1

BackgroundThe aim of this study was to explore the function and mechanism of GKN1 in gastric cancer (GC) progression.MethodsFirstly, we used GEO2R to perform differential gene analysis on {"type":"entrez-geo","attrs":{"text":"GSE26942","term_id":"26942"}}GSE26942 and {"type":"entrez-geo","attrs":{"text":"GSE79973","term_id":"79973"}}GSE79973 and constructed the protein–protein interaction network of differential genes by STRING. Next, the cytoHubba, Mcode plugins, and GEPIA were used to obtain our follow‐up research object GKN1. Then, the function of GKN1 in GC was verified by scratch and transwell assay in GC cells. We further analyzed the genes related to GKN1 through LinkedOmics, and exported top 100 genes positively or negatively correlated with GKN1. Meanwhile, Metascape was performed on these genes. Finally, we analyzed the miRNAs that bind to GKN1 through the miRDB and verified the correlation between miR‐548d‐3p and GKN1 using dual‐fluorescence and quantitative PCR experiments.ResultsBioinformatics analysis showed that there were 52 differential genes on {"type":"entrez-geo","attrs":{"text":"GSE26942","term_id":"26942"}}GSE26942 and {"type":"entrez-geo","attrs":{"text":"GSE79973","term_id":"79973"}}GSE79973. In addition, the results of functional assays indicated that overexpressed GKN1 can inhibit GC cell migration and invasion, while GKN1 knockdown demonstrated the opposite effect. Additionally, Metascape analysis results showed that the 3′‐UTR region of mRNA is rich in AU sequences, based on which we infer that mRNA may be regulated by miRNA. Dual‐fluorescence and quantitative PCR assays clarified that miR‐548d‐3p may be one of the target miRNAs of GKN1, which was up‐regulated in GC tissues.ConclusionsIn summary, we clarified that miR‐548d‐3p regulates GKN1 to participate in GC cell migration and invasion, and provides a possible target for the prognostic diagnosis and treatment of GC.     

A human immunodeficiency caused by mutations in the PIK3R1 gene

Recently, patient mutations that activate PI3K signaling have been linked to a primaryantibody deficiency. Here, we used whole-exome sequencing and characterized the moleculardefects in 4 patients from 3 unrelated families diagnosed with hypogammaglobulinemia andrecurrent infections. We identified 2 different heterozygous splice site mutations thataffect the same splice site in PIK3R1, which encodes the p85αsubunit of PI3K. The resulting deletion of exon 10 produced a shortened p85α proteinthat lacks part of the PI3K p110-binding domain. The hypothetical loss ofp85α-mediated inhibition of p110 activity was supported by elevated phosphorylationof the known downstream signaling kinase AKT in patient T cell blasts. Analysis of patientblood revealed that naive T and memory B cell counts were low, and T cell blasts displayedenhanced activation-induced cell death, which was corrected by addition of the PI3Kδinhibitor IC87114. Furthermore, B lymphocytes proliferated weakly in response toactivation via the B cell receptor and TLR9, indicating a B cell defect. The phenotypeexhibited by patients carrying the PIK3R1 splice site mutation is similarto that of patients carrying gain-of-function mutations in PIK3CD. Ourresults suggest that PI3K activity is tightly regulated in T and B lymphocytes and thatvarious defects in the PI3K-triggered pathway can cause primary immunodeficiencies.     

In silico functional and tumor suppressor role of hypothetical protein PCNXL2 with regulation of the Notch signaling pathway

Since the last decade, various genome sequencing projects have led to the accumulation of an enormous set of genomic data; however, numerous protein-coding genes still need to be functionally characterized. These gene products are called “hypothetical proteins”. The hypothetical protein pecanex-like protein 2 Homo sapiens (PCNXL2) is found to be mutated in colorectal carcinoma with microsatellite instability; therefore, annotation of the function of PCNXL2 in tumorigenesis is very important. In the present study, bioinformatics analysis of PCNXL2 was performed at the molecular level to assess its role in the progression of cancer for designing new anti-cancer drugs. The retrieved sequence of PCNXL2 was functionally and structurally characterized through the web tools Pfam, Batch CD (conserved domain) search, ExPASy, COACH and I-TASSER directed for pathway analysis and design to explore the intercellular interactions of PCNXL2 involved in cancer development. The present study has shown that PCNXL2 encodes multi-pass transmembrane proteins whose tumor suppressor function may involve regulating Notch signaling by transporting protons across the membrane to provide suitable membrane potential for γ secretase function, which may liberate the Notch intracellular domain NICD from the receptor to inside the cell. Furthermore, domain A of PCNXL2 may exhibit nuclear transport activity of NICD from the cytoplasm to the nucleus through interaction with a nuclear localization signal that may act as an activator for Notch signaling in the nucleus. Conclusively, the tumor suppressor role of PCNXL2 by regulation of the Notch signaling pathway and its functional and structural characteristics are important findings. However, further studies are required to validate the putative role of PCNXL2 as a cancer biomarker in cancer development.

Since the last decade, various genome sequencing projects have led to the accumulation of an enormous set of genomic data; however, numerous protein-coding genes still need to be functionally characterized.     

OGG1 contributes to hepatocellular carcinoma by promoting cell cycle‐related protein expression and enhancing DNA oxidative damage repair in tumor cells

BackgroundThis study aimed to analyze the expression of 8‐oxoguanine DNA glycosylase (OGG1) in patients with hepatocellular carcinoma (HCC) and its effect on prognosis by bioinformatics techniques and to determine its possible carcinogenic mechanism through data mining.MethodsThe difference in OGG1 expression between healthy people and HCC patients was searched and analyzed by TCGA and GEO databases, and the effect of OGG1 on prognosis was judged by survival analysis. Meanwhile, the possible molecular mechanism of OGG1 in the tumorigenesis and development of HCC was explored by GO analysis, KEGG analysis, immune infiltration analysis, protein–protein interaction network, promoter methylation analysis, and so forth. Quantitative polymerase chain reaction (qPCR) was used to examine the gene expression in 36 pairs of HCC tissues and adjacent tissues.ResultsThe expression of OGG1 in HCC patients was higher than that in healthy people, and the overexpression of OGG1 might stimulate cell proliferation by increasing the activity of cell cycle‐related proteins.ConclusionThe alteration of OGG1 was significantly correlated with the tumorigenesis and development of HCC. OGG1 is expected to be a new biomarker for evaluating the prognosis of HCC and a new target for the treatment of HCC.     

Inhibition of MAP kinases and down regulation of TNF-α, IL-β and COX-2 genes by the crude extracts from marine bacteria

Crude ethyl acetate extracts from marine bacterial isolates Staphylococcus arlettae KP2 (GenBank accession No. EU594442) and Planococcus maritimus KP8 (GenBank accession No. EU594443) isolated from Andaman seas were studied for their anti-inflammatory effect by lymphocyte proliferation assay (LPA) employing peripheral blood mononuclear cells (PBMCs). The crude extracts from both the bacteria down regulated the synthesis of inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2), besides markedly inhibiting p38 mitogen activated protein (MAP) kinase. These results suggest that the crude ethyl acetate extracts from both the isolates do contain compounds capable of inhibiting inflammation in mitogen induced PBMC and efforts to score potential bioactive molecules from these extracts may prove to be a promising preposition.     

人脐静脉内皮细胞损伤过程中核转录因子κB及单核细胞趋化蛋白1mRNA的变化

背景:既往实验表明,炎性因子与内皮细胞的功能有密切的关系,观察保护和损伤因素对体外培养人血管内皮细胞炎性因子作用的报道较少.目的:实验拟观察姜黄素和肿瘤坏死因子α对人脐静脉内皮细胞分泌核因子κB以及单核细胞趋化蛋白1mRNA的影响.设计、时间及地点:细胞观察实验,于2006-12/2007-08在解放军第四军医大学组织工程实验室完成.材料:健康人脐带由解放车第四军医大学唐都医院提供.内皮细胞培养采用M199完全培养基.方法:实验分为3组,正常对照组为无血清M199培养基;肿瘤坏死因子α组:在正常细胞培养液中加入浓度为10μg/L的肿瘤坏死因子α诱导损伤1.5h;姜黄素组:先在培养液中加入10μg/L的肿瘤坏死因子α30min诱导损伤,再分别加入终浓度为6.25,12.5,25,50,100mg/L的姜黄素孵育1h.主要观察指标:①四甲基偶氮唑盐法检测不同浓度姜黄素对人脐静脉内皮细胞增殖的影响.②免疫细胞化学染色法观察胞核内核转录因子κB的表达.③反转录-聚合酶链反应检测单核细胞趋化蛋白1mRNA的表达.结果:姜黄素可以提高肿瘤坏死因子α诱导损伤的人脐静脉内皮细胞活性(P＜0.01),并呈一定剂量依赖性.肿瘤坏死因子α可刺激核因子κBp65表达的强度,姜黄素可以减少核因子κBp65的表达.与正常对照组相比,肿瘤坏死因子α作用30min后,单核细胞趋化蛋白1mRNA相对表达量明显增高(p＜0.01);加入不同浓度的姜黄素作用1h后,单核细胞化蛋白1mRNA相对表达量明显减低(P＜0.05, P＜0.01).结论:姜黄素对损伤的内皮细胞具有保护作用,其作用途径可能通过抑制核转录因子κB活性,并下调单核细胞趋化蛋白1mRNA的表达,从而阻滞了单核细胞的聚集.     

Fibroblast growth factor improves the motility of human mesenchymal stem cells expanded in a human plasma‐derived xeno‐free medium through αVβ3 integrin

Human mesenchymal stem cells (MSC) are being explored for cell therapies targeting varied human diseases. For that, cells are being expanded in vitro, many times with fetal bovine serum (FBS) as the main source of growth factors. However, animal‐derived components should not be used, to avoid immune rejection from the patient that receives the MSC. To solve this issue, different xeno‐free media are being developed, and an industrial‐grade human plasma fraction (SCC) is a promising candidate to substitute FBS. Indeed, we have previously shown that MSC expanded in SCC‐medium maintain their phenotype and genetic stability. However, a reduction on MSC motility was observed when comparing with MSC motility on FBS‐medium. Thus, in this present study, we have tested different factors to improve the motility of MSC in SCC‐medium. Time lapse assays and experiments with transwells revealed that supplementation of the xeno‐free medium with FGF or PDGF, but not TNF‐α or SDF‐1, increased MSC motility. Interestingly, FGF and PDGF supplementation also led to alterations on MSC morphology to a shape similar to the one observed when using FBS. The mechanism behind the effect of FGF on MSC motility involved the increased expression of αVβ3 integrin. Furthermore, assays with small molecule inhibitors revealed that the signalling molecule p38 MAPK is important for MSC motility and that MEK/ERK and PI3K/AKT also have a role on FGF‐supplemented expanded MSC. Thus, it was found that FGF supplementation can improve the motility of xeno‐free‐expanded MSC and that the cells motility is regulated by αVβ3 integrin.     

Cytokine mRNA expression during an in vitro response of human B lymphocytes: kinetics of B cell tumor necrosis factor alpha, interleukin (IL)6, IL-10, and transforming growth factor beta 1 mRNAs

Expression of mRNA for eight cytokines was analyzed in an in vitro response-proliferation and Ig-secretion--of normal human B lymphocytes. This was made possible by the use of murine thymoma cells as helper cells in conjunction with human T cell supernatant, and the design of human DNA sequence-specific primers for RT-polymerase chain reaction. mRNAs for interleukin (IL)2 and IL-4, but also for IL-1 alpha and IL-1 beta remained undetectable during the whole culture period in highly purified B cells prepared by a three-step purification protocol. However, tumor necrosis factor alpha and IL-6 mRNAs peaked during days 1-3 after culture start and became undetectable after 5-6 d, shortly before bulk B cell proliferation started to decline. In contrast, transforming growth factor beta 1 mRNA, after a progressive increase during the first few days, and IL-10 mRNA, after a peak on days 1-3, remained detectable in immunoglobulin (Ig)-secreting cultures throughout the observation period of 22 d. Clonal analysis on 8-d cultures that had been seeded with single B cells by autocloning with the cell sorter, revealed that 85% of 77 B cell clones studied, expressed TGF-beta 1 mRNA, and only 19% IL-10 mRNA. These findings show a differentiation stage-related cytokine program during a B cell response, whereby (a) B cells can become activated without IL-1 alpha or IL-1 beta expression; (b) mRNA for positive (IL-10) and negative (TGF-beta 1) autoregulatory factors coexists in cell populations during the later phase of the response, although not necessarily in all B cell clones; and (c) normal Ig-secreting cells cease IL-6 expression in contrast to their malignant counterparts, myeloma cells.     

Temperature-dependent phosphorylation state of the H5R protein synthesised at the early stage of infection in cells infected with vaccinia virus ts mutants of the B1R and F10L protein kinases

The phosphorylation state of H5R protein was investigated by two-dimensional gel electrophoresis of proteins of BSC-40 cells infected at 32 degrees or 39.5 degrees with vaccinia virus ts mutants of the viral B1R or F10L protein kinase genes. A temperature-dependent increase of underphosphorylated H5R protein (pI 6.8) was demonstrated in the case of the B1R, but not of the F10L gene. The temperature-dependent cytoplasmic location of underphosphorylated H5R protein after infection with the ts mutants of the B1R gene was the consequence of the associated viral DNA replication block. These results show that the B1R protein kinase controls the phosphorylation state of the H5R protein synthesised at the early stage in vaccinia-virus-infected cells.     

The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin α2β1

BACKGROUND: Activation of the platelet integrin alpha 2 beta 1 is closely regulated due to the high thrombogenicity of its ligand. As a beta 1 interacting kinase, ILK represents a candidate intracellular regulator of alpha 2 beta 1 in human platelets. OBJECTIVES: We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha 2 beta 1 activation in HEL cells, a megakaryocytic cell line. METHODS: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta 1-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha 2 beta 1 function assessed by overexpression studies in HEL cells. RESULTS: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta 1-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha 2 beta 1-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha 2 beta 1. CONCLUSIONS: Our findings that ILK regulates alpha 2 beta 1 in HEL cells, is activated in platelets and associates with beta 1-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.     

Tensile strain and magnetic particle force application do not induce MAP3K8 and IL‐1B differential gene expression in a similar manner to fluid shear stress in human mesenchymal stem cells

Mechanical forces, important in a variety of cellular processes, including proliferation, differentiation and gene expression, are also key in the development, remodelling and maintenance of load‐bearing tissues such as cartilage and bone. Thus, there is great interest in using in vitro mechanical conditioning of mesenchymal stem cells (MSCs), multipotent adult stem cells, for tissue engineering of these tissues. In a previous gene expression study, we reported a potentially important role for mitogen‐activated protein kinase kinase kinase 8 (MAP3K8) and interleukin‐1β (IL‐1B) in MAPK signalling in MSCs exposed to fluid shear stress. In this follow‐up study, we examined the expression of these genes in MSCs exposed to other types of mechanical force: uniaxial tensile strain (3% cell elongation) and forces generated through the exposure of magnetic particle‐labelled MSCs to an oscillating magnetic field (maximum field strength 90 mT). Exposure to both types of mechanical force for 1 h did not significantly alter the gene expression of MAP3K8 or IL‐1B over the 24 h period subsequent to force exposure. These data demonstrate that uniaxial tensile strain and magnetic particle‐based forces do not induce MAP3K8‐related MAPK signalling in the same manner as does fluid flow‐induced shear stress. This illustrates divergence in the process of mechanotransduction in mechanically stimulated MSCs. Copyright © 2010 John Wiley & Sons, Ltd.     

The complement component C5a induces the expression of plasminogen activator inhibitor-1 in human macrophages via NF-κB activation

BACKGROUND: Atherosclerosis is considered to be a chronic inflammatory disorder. Activation of the complement cascade is a major aspect of chronic inflammatory diseases. Complement components were identified in atherosclerotic plaques, and a correlation between adverse events and C5a plasma levels was found. These findings support the notion that complement activation contributes to development and progression of atherosclerotic lesions. OBJECTIVES: We investigated whether complement components C3a and C5a regulate plasminogen activator inhibitor (PAI-1) in human macrophages. METHODS: Human monocyte-derived macrophages (MDM) and human plaque macrophages were cultured and incubated with the complement component C5a. RESULTS: C5a increased PAI-1 up to 11-fold in human MDM and up to 2.7-fold in human plaque macrophages. These results were confirmed at the mRNA level using real time-polymerase chain reaction. Pertussis toxin or anti-C5aR/CD88 antibody completely abolished the effect of recombinant human C5a on PAI-1 production, suggesting a role of the C5a receptor. Experiments with antitumor necrosis factor (TNF)-alpha antibodies and tiron showed that the effect of C5a was not mediated by TNF-alpha or oxidative burst. Furthermore C5a induced NF-kappaB binding to the cis element in human macrophages and the C5a-induced increase in PAI-1 was completely abolished by an NF-kappaB inhibitor. CONCLUSIONS: We conclude that C5a upregulates PAI-1 in macrophages via NF-kappaB activation. We hypothesize that - if operative in vivo- this effect could favor thrombus development and thrombus stabilization in the lesion area. On the other hand one could speculate that C5a-induced upregulation of PAI-1 in plaque macrophages could act as a defense mechanism against plaque destabilization and rupture.     

The phe-124-Cys and A-161T variants of the human 5-HT1B receptor gene are not major determinants of the clinical response to sumatriptan

BACKGROUND: The 5-HT(1B/1D) receptor agonist sumatriptan is highly effective in the treatment of migraine. However, some patients do not respond to sumatriptan or experience recurrence of the headache after initial relief. In addition, some patients report chest symptoms after the use of sumatriptan. OBJECTIVE: To assess whether 2 genetic variants (F124C changing a phenylalanine for a cysteine and polymorphism A/T at nucleotide position -161 in the 5' regulatory region) of the 5-HT(1B) receptor play a major role in the therapeutic response to sumatriptan. The 5-HT(1B) receptor most likely mediates the therapeutic action and coronary side effects of sumatriptan, and both F124C and A-161T have relevant functional consequences on either the affinity of sumatriptan to bind to the 5-HT(1B) receptor or on receptor expression level itself, respectively. METHOD: Genomic DNA of a relatively small but very well-characterized set of migraine patients with consistently good response to sumatriptan (n = 14), with no response (n = 12), with recurrence of the headache (n = 12), with chest symptoms (n = 13), and patients without chest symptoms (n = 27) was available for the genetic analyses and screened for the F124C variant and the A-161T polymorphism in the human 5-HT(1B) receptor gene. RESULTS: F124C was not detected in any of the patients studied. In addition, we did not observe drastic changes in allele frequencies of the A-161T polymorphism that might hint to a causal relation with the therapeutic effect of sumatriptan. CONCLUSION: We have not obtained any evidence that variants F124C and A-161T of the 5-HT(1B) receptor are major determinants in the clinical response to sumatriptan.     

Retracted Article: Down-regulation of Rab10 inhibits hypoxia-induced invasion and EMT in thyroid cancer cells by targeting HIF-1α through the PI3K/Akt pathway

Rab10, a member of the Rab family, is localized to endocytic compartments and serves as a regulator of intracellular vesicle trafficking. Previous studies mainly paid attention to the role of Rab10 in transport. Recently, Rab10 has been reported to be involved in the progression of various cancers. However, the biological functions of Rab10 in thyroid cancer remain unknown. In this study, we demonstrated that Rab10 was highly expressed in thyroid cancer tissues and cell lines. Down-regulation of Rab10 inhibited hypoxia-induced migration, invasion and epithelial–mesenchymal transition (EMT) of thyroid cancer cells. Moreover, HIF-1α and the PI3K/Akt pathway were involved in the inhibitory effect of Rab10 down-regulation on thyroid cancer cell invasion and EMT induced by hypoxia. Taken together, our study provided further evidence to support the role of Rab10 as a therapeutic target for thyroid cancer.

Rab10, a member of the Rab family, is localized to endocytic compartments and serves as a regulator of intracellular vesicle trafficking.