Piao, Yongwei;Yun, So Yoon;Kim, Hee Soo;Park, Bo Kyung;Ha, Hae Chan;Fu, Zhicheng;Jang, Ji Min;Back, Moon Jung;Shin, In Chul;Won, Jong Hoon;Kim, Dae Kyong
Biomolecules & Therapeutics
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v.30
no.6
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pp.529-539
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2022
Rheumatoid arthritis (RA) is a multifactorial immune-mediated disease, the pathogenesis of which involves different cell types. T-cell activation plays an important role in RA. Therefore, inhibiting T-cell activation is one of the current therapeutic strategies. Cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig), also known as abatacept, reduces cytokine secretion by inhibiting T-cell activation. To achieve a homeostatic therapeutic effect, CTLA4-Ig has to be administered repeatedly over several weeks, which limits its applicability in RA treatment. To overcome this limitation, we increased the number of sialic acid-capped antennas by genetically engineering the CTLA4 region to increase the therapeutic effect of CTLA4-Ig. N-acetylglucosaminyltransferase (GnT) and α2,6-sialyltransferase (α2,6-ST) were co-overexpressed in Chinese hamster ovary (CHO) cells to generate a highly sialylated CTLA4-Ig fusion protein, named ST6. The therapeutic and immunogenic effects of ST6 and CTLA4-Ig were compared. ST6 dose-dependently decreased paw edema in a mouse model of collagen-induced arthritis and reduced cytokine levels in a co-culture cell assay in a similar manner to CTLA4-Ig. ST6- and CTLA4-Ig-induced T cell-derived cytokines were examined in CD4 T cells isolated from peripheral blood mononuclear cells after cell killing through irradiation followed by flow- and magnetic-bead-assisted separation. Interestingly, compared to CTLA4-Ig, ST6 was substantially less immunogenic and more stable and durable. Our data suggest that ST6 can serve as a novel, less immunogenic therapeutic strategy for patients with RA.
Background: Coinfection with avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) is common in chickens, and the molecular mechanism of the synergistic pathogenic effects of the coinfection is not clear. Exosomes have been identified as new players in the pathogenesis of retroviruses. The different functions of exosomes depend on their cargo components. Objectives: The aim of this study was to investigate the function of co-regulation differentially expressed proteins in exosomes on coinfection of ALV-J and REV. Methods: Here, viral replication in CEF cells infected with ALV-J, REV or both was detected by immunofluorescence microscopy. Then, we analyzed the exosomes isolated from supernatants of chicken embryo fibroblast (CEF) cells single infected and coinfected with ALV-J and REV by mass spectrometry. KEGG pathway enrichment analyzed the co-regulation differentially expressed proteins in exosomes. Next, we silenced and overexpressed tripartite motif containing 62 (TRIM62) to evaluate the effects of TRIM62 on viral replication and the expression levels of NCK-association proteins 1 (NCKAP1) and actin-related 2/3 complex subunit 5 (ARPC5) determined by quantitative reverse transcription polymerase chain reaction. Results: The results showed that coinfection of ALV-J and REV promoted the replication of each other. Thirty proteins, including TRIM62, NCK-association proteins 1 (NCKAP1, also known as Nap125), and Arp2/3-5, ARPC5, were identified. NCKAP1 and ARPC5 were involved in the actin cytoskeleton pathway. TRIM62 negatively regulated viral replication and that the inhibition of REV was more significant than that on ALV-J in CEF cells coinfected with TRIM62. In addition, TRIM62 decreased the expression of NCKAP1 and increased the expression of ARPC5 in coinfected CEF cells. Conclusions: Collectively, our results indicated that coinfection with ALV-J and REV competitively promoted each other's replication, the actin cytoskeleton played an important role in the coinfection mechanism, and TRIM62 regulated the actin cytoskeleton.
Jiawei, Du;Hui, Zhao;Guibing, Song;Yuan, Pang;Lei, Jiang;Linsen, Zan;Hongbao, Wang
Animal Bioscience
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v.36
no.2
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pp.200-208
/
2023
Objective: Muscle acetylcholine receptors have five alpha subunits (α, β, δ, ε, or γ), and cholinergic receptor nicotinic gamma subunit (CHRNG) is the γ subunit. It may also play an essential role in biological processes, including cell differentiation, growth, and survival, while the role of CHRNG has not been studied in the literature. Therefore, the purpose of this study is to clarify the effect of CHRNG on the proliferation and differentiation of bovine preadipocytes. Methods: We constructed a CHRNG overexpression adenovirus vector and successfully overexpressed it on bovine preadipocytes. The effects of CHRNG on bovine preadipocyte proliferation were detected by Edu assay, cell counting Kit-8 (CCK-8), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), Western blot and other techniques. We also performed oil red O, RT-qPCR, Western blot to explore its effect on the differentiation of preadipocytes. Results: The results of Edu proliferation experiments showed that the number of EDU-positive cells in the overexpression group was significantly less. CCK-8 experiments found that the optical density values of the cells in the overexpression group were lower than those of the control group, the mRNA levels of proliferating cell nuclear antigen (PCNA), cyclin A2 (CCNA2), cyclin B1 (CCNB1), cyclin D2 (CCND2) decreased significantly after CHRNG gene overexpression, the mRNA levels of cyclin dependent kinase inhibitor 1A (CDKN1A) increased significantly, and the protein levels of PCNA, CCNB1, CCND2 decreased significantly. Overexpression of CHRNG inhibited the differentiation of bovine preadipocytes. The results of oil red O and triglyceride determination showed that the size and speed of lipid droplets accumulation in the overexpression group were significantly lower. The mRNA and protein levels of peroxisome proliferator activated receptor gamma (PPAR class="checkNonKBPoint">γ), CCAAT enhancer binding protein alpha (CEBPα), fatty acid binding protein 4 (FABP4), fatty acid synthase (FASN) decreased significantly. Conclusion: Overexpression of CHRNG in bovine preadipocytes inhibits the proliferation and differentiation of bovine preadipocytes.
Yawut, Natpaphan;Kaowinn, Sirichat;Cho, Il-Rae;Budluang, Phatcharaporn;Kim, Seonghye;Kim, Suhkmann;Youn, So Eun;Koh, Sang Seok;Chung, Young-Hwa
BMB Reports
/
v.55
no.2
/
pp.98-103
/
2022
Increased mRNA levels of cancer upregulated gene (CUG)2 have been detected in many different tumor tissues using Affymetrix microarray. Oncogenic capability of the CUG2 gene has been further reported. However, the mechanism by which CUG2 overexpression promotes cancer stem cell (CSC)-like phenotypes remains unknown. With recent studies showing that pyruvate kinase muscle 2 (PKM2) is overexpressed in clinical tissues from gastric, lung, and cervical cancer patients, we hypothesized that PKM2 might play an important role in CSC-like phenotypes caused by CUG2 overexpression. The present study revealed that PKM2 protein levels and translocation of PKM2 into the nucleus were enhanced in CUG2-overexpressing lung carcinoma A549 and immortalized bronchial BEAS-2B cells than in control cells. Expression levels of c-Myc, CyclinD1, and PKM2 were increased in CUG2-overexpressing cells than in control cells. Furthermore, EGFR and ERK inhibitors as well as suppression of Yap1 and NEK2 expression reduced PKM2 protein levels. Interestingly, knockdown of β-catenin expression failed to reduce PKM2 protein levels. Furthermore, reduction of PKM2 expression with its siRNA hindered CSC-like phenotypes such as faster wound healing, aggressive transwell migration, and increased size/number of sphere formation. The introduction of mutant S37A PKM2-green fluorescence protein (GFP) into cells without ability to move to the nucleus did not confer CSC-like phenotypes, whereas forced expression of wild-type PKM2 promoted such phenotypes. Overall, CUG2-induced increase in the expression of nuclear PKM2 contributes to CSC-like phenotypes by upregulating c-Myc and CyclinD1 as a co-activator.
Objective: The study was conducted to screen differentially expressed miRNAs in sows at early pregnancy by high-throughput sequencing and explore its mechanism of action on embryo implantation. Methods: The blood serum of pregnant and non-pregnant Landrace×Yorkshire sows were collected 14 days after artificial insemination, and exosomal miRNAs were purified for high throughput miRNA sequencing. The expression patterns of 10 differentially expressed (DE) miRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR quantified the abundance of serum exosomal miR-192 in pregnant and control sows, and the diagnostic power was assessed by receiver operating characteristic (ROC) analysis. The target genes of DE miRNAs were predicted with bioinformatics software, and the functional and pathway enrichment analysis was performed on gene ontology and the Kyoto encyclopedia of genes and genomes terms. Furthermore, a luciferase reporter system was used to identify the target relation between miR-192 and integrin alpha 4 (ITGA4), a gene influencing embryo implantation in pigs. Finally, the expression levels of miRNAs and the target gene ITGA4 were analyzed by qRT-PCR, and western blot, with the proliferation of BeWo cells detected by cell counting kit-8 (CCK-8). Results: A total of 221 known miRNAs were detected in the libraries of the pregnant and non-pregnant sows, of which 55 were up-regulated and 67 were down-regulated in the pregnant individuals compared with the non-pregnant controls. From these, the expression patterns of 10 DE miRNAs were validated. The qRT-PCR analysis further confirmed a significantly higher expression of miR-192 in the serum exosomes extracted from pregnant sows, when compared to controls. The ROC analysis revealed that miR-192 provided excellent diagnostic accuracy for pregnancy (area under the ROC curve [AUC]=0.843; p>0.001). The dual-luciferase reporter assay indicated that miR-192 directly targeted ITGA4. The protein expression of ITGA4 was reduced in cells that overexpressed miR-192. Overexpression of miR-192 resulted in the decreased proliferation of BeWo cells and regulated the expression of cell cycle-related genes. Conclusion: Serum exosomal miR-192 could serve as a potential biomarker for early pregnancy in pigs. miR-192 targeted ITGA4 gene directly, and miR-192 can regulate cellular proliferation.
Objective: The present study was executed to explore the molecular mechanism of fibroblast growth factor 10 (FGF10) gene in bovine adipogenesis. Methods: The bovine FGF10 gene was overexpressed through Ad-FGF10 or inhibited through siFGF10 and their negative control (NC) in bovine adipocytes, and the multiplicity of infection, transfection efficiency, interference efficiency were evaluated through quantitative real-time polymerase chain reaction, western blotting and fluorescence microscopy. The lipid droplets, triglycerides (TG) content and the expression levels of adipogenic marker genes were measured during preadipocytes differentiation. The differentially expressed genes were explored through deep RNA sequencing. Results: The highest mRNA level was found in omasum, subcutaneous fat, and intramuscular fat. Moreover, the highest mRNA level was found in adipocytes at day 4 of differentiation. The results of red-oil o staining showed that overexpression (Ad-FGF10) of the FGF10 gene significantly (p<0.05) reduced the lipid droplets and TG content, and their down-regulation (siFGF10) increased the measurement of lipid droplets and TG in differentiated bovine adipocytes. Furthermore, the overexpression of the FGF10 gene down regulated the mRNA levels of adipogenic marker genes such as CCAAT enhancer binding protein alpha (C/EBPα), fatty acid binding protein (FABP4), peroxisome proliferator-activated receptor-γ (PPARγ), lipoprotein lipase (LPL), and Fas cell surface death receptor (FAS), similarly, down-regulation of the FGF10 gene enriched the mRNA levels of C/EBPα, PPARγ, FABP4, and LPL genes (p<0.01). Additionally, the protein levels of PPARγ and FABP4 were reduced (p<0.05) in adipocytes infected with Ad-FGF10 gene and enriched in adipocytes transfected with siFGF10. Moreover, a total of 1,774 differentially expressed genes (DEGs) including 157 up regulated and 1,617 down regulated genes were explored in adipocytes infected with Ad-FGF10 or Ad-NC through deep RNA-sequencing. The top Kyoto encyclopedia of genes and genomes pathways regulated through DEGs were the PPAR signaling pathway, cell cycle, base excision repair, DNA replication, apoptosis, and regulation of lipolysis in adipocytes. Conclusion: Therefore, we can conclude that the FGF10 gene is a negative regulator of bovine adipogenesis and could be used as a candidate gene in marker-assisted selection.
Objectives: The pulp is the center of the tooth containing nerves and blood vessels. The condition in which the pulp becomes inflamed due to caries or periodontitis is called pulpitis. Pulpitis is a difficult-to-treat disease and causes peripheral nerve tissue changes and severe pain; however, the relationship between neuronal activity and voltage-gated sodium channel 1.7 (Nav1.7) expression in the trigeminal ganglion (TG) during pulpitis has not been well studied. In this study, we found that experimentally induced pulpitis activates Nav1.7 expression in the periphery, leading to neuronal overexpression in the TG. Thus, we sought to identify ways to regulate this process. Methods: Acute pulpitis was induced in rat maxillary molars by treating the pulp with allyl isothiocyanate (AITC). Three days later, in vivo optical imaging was used to record and compare neural activities in the TG. Western blotting was used to identify molecular changes in terms of the expression of extracellular signal-regulated kinase (ERK), c-Fos, transient receptor potential ankyrin 1 (TRPA1), and collapsin response mediator protein-2 (CRMP2) in the brain stem. Results: The results confirmed the neurological changes in the TGs of the pulpitis model, and histological and molecular biological evidence confirmed that increased Nav1.7 expression induced by pulpitis leads to pain. Furthermore, selective inhibition of Nav1.7 resulted in changes in neural activity, suggesting that pulpitis induces increased Nav1.7 expression, and that effective control of Nav1.7 could potentially reduce pain. Conclusions: The inhibition of overexpressed Nav1.7 channels may modulate nociceptive signal processing in the brain and effectively control pain associated with pulpitis.
[68Ga]Ga-FAPI-04 is a promising radiopharmaceutical that binds specifically to fibroblast activation protein, which is overexpressed in more than 90% of malignant epithelial tumors but not in normal healthy tissue. This study aimed to develop an efficient method for producing 68Ga-labelled FAPI-04 using a cassette-based automated synthesizer. [68Ga]GaCl3 was eluted from an Eckert & Ziegler Medical germanium-68/gallium-68 generator using 2.5 mL of 0.1 M HCl. The synthesis of the [68Ga]Ga-FAPI-04 was performed using different concentrations of HEPES (1~2.5 M; 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid) in 3~10 minutes; amounts of FAPI-04 precursor (5~50 ㎍) and reaction temperature (25℃~100℃) were optimized on the BIKBox® synthesizer. The labeling efficiency of [68Ga]Ga-FAPI-04 was greater than 96% (decay corrected) using 25 ㎍ FAPI-04 synthesized in 10 minutes at 100℃ in 2 M HEPES (pH 3.85), and its stability was greater than 99% at 6 hours. The total synthesis time of [68Ga]Ga-FAPI-04 was 32.4 minutes, and the product met all quality control criteria. In this study, we developed and optimized a labeling method using [68Ga]Ga-FAPI-04 using a cassette-based synthesizer. The devised method is expected to be useful for supplying [68Ga]Ga-FAPI-04 for diagnosis in clinical practice.
Kim, Woo Jin;Yoon, Jung Min;Lee, Kyoung Hee;Han, Seon Jin;Shin, Won Hyuk;Yim, Jae-Joon;Yoo, Chul-Gyu;Lee, Choon Taek;Han, Sung Koo;Shim, Young-Soo;Kim, Young Whan
Tuberculosis and Respiratory Diseases
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v.56
no.6
/
pp.638-645
/
2004
Background : Uteroglobin is a protein produced by the normal bronchial epithelium and its expression level is lower in non-small cell lung cancer tissues and cell lines. It mainly functions as an anti-inflammatory, and when it is overexpressed in cancer cells, the neoplastic phenotype is antagonized. cPLA2 and COX-2, which are also associated with inflammation, were reported to be related to cancer. The relationship between cPLA2, COX-2 and uteroglobin is unclear. The relationship between uteroglobin and ERK, which is related to cell growth, is also not unclear. This study investigated the changes in the cPLA2 and COX-2 expression levels and the ERK activities after the overexpression of uteroglobin in non-small cell lung cancer cell lines. Methods : The A549 and NCI-H460 cell lines were infected by adenovirus-null and adenovirusuteroglobin. The cChange in the cPLA2, COX-2 expression level and ERK activity after uteroglobin overexpression was measured by Western blot. The change in MMP activity was measured by zymography. Results : Western blot revealed decreased expression levels of cPLA2, and COX-2, and increased pERK levels in nonsmall cell lung cancer cells after uteroglobin overexpression. Zymography revealed no changes in the MMP-2 activity and lower MMP-9 activity. U0126, which is a specific inhibitor of ERK-activating kinase MEK-1/-2, prevented the decrease in the MMP-9 activity Conclusions : A decrease in cPLA2 expression, COX-2 expression, MMP-9 activity and a increase in ERK activity may be related to the anticancer effects of uteroglobin in nonsmall cell lung cancer cells.
Background : Immunotherapy for cancer has not been successful because of several obstacles in tumor and its environment. Inappropriate secretions of cytokines and growth factors by tumors cause substantial changes in the immune responses against tumors, affording the tumors some degree of protection from immune attack. Uteroglobin (UG, Clara cell secretory protein) has been known to have anti-inflammatory, immunomodulatory and anti-cancer activities. However, in lung cancer cells, UG expression is decreased. This study investigated the role of UG in the immunomodulation of lung cancer. Methods : The UG protein was overexpressed by Adenovirus(Ad)-UG transduction in non-small cell lung cancer cell lines. The concentration of Prostaglandin $E_2$ ($PGE_2$) was measured by Enzyme Immunoassay (EIA). Peripheral blood mononuclear cells (PBMC) from whole blood were prepared with Ficoll. PBMC were cultured in RPMI 1640, supernatant of A549, or A549 with UG or NS-398. Concentration of Th 1 type and Th 2 type cytokines from PBMC were measured by ELISA. Results : UG suppressed $PGE_2$, Cyclooxygenase-2 (COX-2) product. Both Th1 type such as Interleukin-2 (IL-2), Interferon-${\gamma}$ (IFN-${\gamma}$) and Tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) and Th2 type cytokines such as IL-10 and Tumor growth factor-${\beta}$ (TGF-${\beta}$) were increased when PBMC were cultured with supernatant of non small lung cancer cells. UG and COX-2 inhibitor, NS-398 induced normal immune response of PBMC. Although Th 1 type cytokines were increased, Th 2 type cytokines were reduced by UG. Conclusion : UG suppressed PGE2, COX-2 product. Supernatant of NSCLC induced imbalance of immune response of PBMC. However, UG reversed this imbalance. These results suggest that UG may be used in the development of immunotherapy for lung cancer.
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