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1.
Article in Chinese | WPRIM | ID: wpr-936371

ABSTRACT

OBJECTIVE@#To investigate the effect of metformin on the proliferation and apoptosis of HER-2-positive breast cancer cell line SKBR3 and explore the possible mechanism of its action.@*METHODS@#SKBR3 cells were treated with different concentrations (20-120 μmol/L) of metformin, and the changes in cell proliferation and colony formation ability were assessed using CCK-8 assay and crystal violet staining, respectively. Flow cytometry was performed to analyze cell apoptosis and cell cycle changes. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect mRNA expressions of YAP, TAZ, EGFR, CTGF, CYR61, E-cadherin, N-cadherin, vimentin and fibronectin in the treated cells, and the protein expressions of YAP and TAZ were detected using Western blotting; immunofluorescence assay was used to observe YAP/TAZ nuclear translocation in the cells.@*RESULTS@#Metformin treatment significantly inhibited the proliferation of SKBR3 cells (P < 0.05) in a concentration- and time-dependent manner. The results of flow cytometry showed that metformin significantly promoted apoptosis and caused cell cycle arrest at G1 phase in SKBR3 cells. Metformin treatment significantly down-regulated the mRNA expressions of YAP, TAZ, EGFR, CTGF and CYR61, N-cadherin, vimentin and fibronectin (P < 0.05) and up-regulated the expression of E-cadherin (P < 0.05); Western blotting results showed that YAP and TAZ protein expressions were significantly down-regulated in the cells after metformin treatment (P < 0.05). Immunofluorescence assay revealed that metformin treatment caused the concentration of YAP and TAZ in the cytoplasm, and significantly reduced their amount in the cell nucleus.@*CONCLUSION@#Metformin can inhibit proliferation and promote apoptosis and epithelal-mesenchymal transition of HER-2 positive breast cancer cells possibly by that inhibing YAP and TAZ expression and their nuclear localization.


Subject(s)
Apoptosis , Cadherins , Cell Proliferation , ErbB Receptors , Fibronectins , Metformin/pharmacology , Neoplasms , Protein Serine-Threonine Kinases , RNA, Messenger , Transcription Factors/metabolism , Vimentin
2.
Article in Chinese | WPRIM | ID: wpr-936334

ABSTRACT

OBJECTIVE@#To explore the mechanism by which estradiol modulates the immunophenotype of macrophages through the endoplasmic reticulum stress pathway.@*METHODS@#Peritoneal macrophages isolated from C57 mice were cultured in the presence of 60 ng/mL interferon-γ (IFN-γ) followed by treatment with estradiol (1.0 nmol/L) alone, estradiol with estrogen receptor antagonist (Acolbifene, 4 nmol/L), estradiol with IRE1α inhibitor (4 μ 8 C), or estradiol with IRE1α agonist. After the treatments, the expression levels of MHC-Ⅱ, iNOS and endoplasmic reticulum stress marker proteins IRE1α, eIF2α and ATF6 in the macrophages were detected with Western blotting, and the mRNA levels of TGF-β, IL-6, IL-10 and TNF-α were detected with RT-PCR.@*RESULTS@#Estrogen treatment of the macrophages significantly decreased the expressions of M1-related proteins MHC-Ⅱ (P=0.021) and iNOS (P < 0.001) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.004), increased the mRNA expression of TGF-β (P=0.002) and IL-10 (P=0.008), and up-regulated the protein expressions of IRE1α (P < 0.001) and its downstream transcription factor XBP-1 (P < 0.001). Addition of the estrogen inhibitor obviously blocked the effect of estrogen. Compared with estrogen treatment alone, combined treatment of the macrophages with estrogen and the IRE1α inhibitor 4 μ 8 C significantly up-regulated the protein expressions of MHC-Ⅱ (P=0.002) and iNOS (P=0.003) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.024), and obviously down-regulated the mRNA expression of TGF-β (P < 0.001) and IL-10 (P < 0.001); these changes were not observed in cells treated with estrogen and the IRE1α agonist.@*CONCLUSION@#Estrogen can inhibit the differentiation of murine macrophages into a pro-inflammatory phenotype by up-regulating the IRE1α-XBP-1 signaling axis, thereby producing an inhibitory effect on inflammatory response.


Subject(s)
Animals , Cell Differentiation/drug effects , Endoribonucleases/metabolism , Estradiol/pharmacology , Estrogens/metabolism , Interleukin-10 , Interleukin-6/metabolism , Macrophages, Peritoneal/metabolism , Mice , Phenotype , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/metabolism , Signal Transduction/drug effects , Transforming Growth Factor beta/metabolism , Tumor Necrosis Factor-alpha/metabolism , Up-Regulation/drug effects , X-Box Binding Protein 1/metabolism
3.
Article in Chinese | WPRIM | ID: wpr-928665

ABSTRACT

OBJECTIVE@#To investigate the effect of monoammonium glycyrrhizinate on the stem cell-like characteristics, oxidative stress and mitochondrial function of acute promyelocytic leukemia cells NB4.@*METHODS@#CCK-8 method was used to detect the viability of acute promyelocytic leukemia cells NB4, and the appropriate dose was screened; Cloning method was used to detect the proliferation rate of NB4 cell; Western blot was used to detect the expression of cell cycle-related protein; flow cytometry was used to detect cell apoptosis and sort NB4 stem cells positive (CD133+); Stem cell markers (Oct4, ABCG2, Dclk1) were detected by RT-PCR; ROS was detected by fluorescence; The kit was used to detect the level of oxidative stress markers (MDA); The flow cytometry was used to detect the change of mitochondrial membrane potential; Western blot was used to detect the expression of mitochondrial damage index-related proteins (Bax/BCL-2).@*RESULTS@#Compared with the control group, if the concentration of MAG was less than 5 μmol/L, the cell NB4 viability showed no significant difference; if the concentration was higher than 5 μmol/L, the inhibitory effect on the growth of cell NB4 increased and showed significant difference (P<0.05), according to the results of CCK-8 experiment, four groups were set based on the concentration of MAG 0 μmol/L, MAG 5 μmol/L, MAG 10 μmol/L, and MAG 20 μmol/L; compared with the control group (MAG 0 μmol/L), the cells in MAG 5 μmol/L group showed no significant difference, while the proliferation rate, cyclin expression, mitochondrial membrane potential, stem cell CD133+ ratio, and marker mRNA level ( Oct4, ABCG2, Dclk1) of NB4 cell were significantly reduced (P<0.05); the apoptosis rate, reactive oxygen species, MDA content and Bax/BCL-2 expression of NB4 cell significantly increased (P<0.05).@*CONCLUSION@#Monoammonium glycyrrhizinate has a significant inhibitory effect on acute promyelocytic leukemia cells NB4, which may be related to the regulation of stem cell-like characteristics, oxidative stress and mitochondrial function.


Subject(s)
Apoptosis , Cell Line, Tumor , Doublecortin-Like Kinases , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Leukemia, Promyelocytic, Acute , Mitochondria , Oxidative Stress , Protein Serine-Threonine Kinases , Stem Cells
4.
Article in Chinese | WPRIM | ID: wpr-941045

ABSTRACT

OBJECTIVE@#To explore the role of salt-inducible kinase 2 (SIK2) in myocardial ischemia-reperfusion (IR) injury in rats.@*METHODS@#Fifteen male SD rats were randomized equally into sham operation group, myocardial IR model group, and SIK2 inhibitor group (in which the rats were treated with intravenous injection of 10 mg/kg bosutinib via the left femoral vein 24 h before modeling). Ultrasound was used to detect the cardiac function of the rats, and myocardial pathologies were observed with HE staining. Transmission electron microscopy was used to observe autophagy of myocardial cells, and Western blotting was performed to detect the contents of the autophagy-related proteins SIK2, LC3B, Beclin-1, p62 and the expressions of p-mTOR, mTOR, p-ULK1, and ULK1 in myocardial tissue.@*RESULTS@#Myocardial IR injury significantly increased the number of autophagosomes (P < 0.05) and the expression of SIK2 protein (P < 0.01) in the myocardial tissues. Treatment with bosutinib before modeling obviously lowered the expression of SIK2 protein (P < 0.01), alleviated myocardial pathologies, and reduced the number of autophagosomes (P < 0.05) in the myocardial tissue. The rats with myocardial IR injury showed obviously lowered LVEF and FS values (P < 0.001), which were significantly improved by bosutinib treatment (P < 0.05); no significant difference was detected in IVSDd or LVPWDd among the 3 groups (P > 0.05). Myocardial IR injury obviously increased the expressions of LC3-II/LC3-I and Beclin-1 proteins and lowered the expression of p62 protein (P < 0.01), and these changes were significantly rescued by bosutinib treatment (P < 0.05). The rat models of myocardial IR injury showed significantly increased expression of p-ULK1 (Ser757) (P < 0.01) and lowered expression of p-mTOR protein (P < 0.0001) in the myocardium, and these changes were obviously reversed by bosutinib (P < 0.01 or 0.05); there was no significant difference in mTOR and ULK1 expressions among the 3 groups (P > 0.05).@*CONCLUSION@#SIK2 may promote autophagy through the mTOR/ULK1 signaling pathway, and inhibiting SIK2 can reduce abnormal autophagy and alleviate myocardial IR injury in rats.


Subject(s)
Animals , Autophagy , Autophagy-Related Protein-1 Homolog/metabolism , Beclin-1/metabolism , Down-Regulation , Male , Myocardial Reperfusion Injury , Protein Serine-Threonine Kinases , Rats , Rats, Sprague-Dawley , Signal Transduction , TOR Serine-Threonine Kinases/metabolism
5.
Article in Chinese | WPRIM | ID: wpr-941006

ABSTRACT

OBJECTIVE@#To explore the mechanism by which inositol-requiring enzyme-1α (IRE1α) regulates autophagy function of chondrocytes through calcium homeostasis endoplasmic reticulum protein (CHERP).@*METHODS@#Cultured human chondrocytes (C28/I2 cells) were treated with tunicamycin, 4μ8c, rapamycin, or both 4μ8c and rapamycin, and the expressions of endoplasmic reticulum (ER) stress- and autophagy-related proteins were detected with Western blotting. Primary chondrocytes from ERN1 knockout (ERN1 CKO) mice and wild-type mice were examined for ATG5 and ATG7 mRNA expressions, IRE1α and p-IRE1α protein expressions, and intracellular calcium ion content using qPCR, Western blotting and flow cytometry. The effect of bafilomycin A1 treatment on LC3 Ⅱ/LC3 Ⅰ ratio in the isolated chondrocytes was assessed with Western blotting. Changes in autophagic flux of the chondrocytes in response to rapamycin treatment were detected using autophagy dual fluorescent virus. The changes in autophagy level in C28/I2 cells overexpressing CHERP and IRE1α were detected using immunofluorescence assay.@*RESULTS@#Tunicamycin treatment significantly up-regulated ER stress-related proteins and LC3 Ⅱ/LC3 Ⅰ ratio and down-regulated the expression of p62 in C28/I2 cells (P < 0.05). Rapamycin obviously up-regulated LC3 Ⅱ/LC3 Ⅰ ratio (P < 0.001) in C28/I2 cells, but this effect was significantly attenuated by co-treatment with 4μ8c (P < 0.05). Compared with the cells from the wild-type mice, the primary chondrocytes from ERN1 knockout mice showed significantly down-regulated mRNA levels of ERN1 (P < 0.01), ATG5 (P < 0.001) and ATG7 (P < 0.001), lowered or even lost expressions of IRE1α and p-IRE1α proteins (PP < 0.01), and increased expression of CHERP (P < 0.05) and intracellular calcium ion content (P < 0.001). Bafilomycin A1 treatment obviously increased LC3 Ⅱ/ LC3 Ⅰ ratio in the chondrocytes from both wild-type and ERN1 knockout mice (P < 0.01 or 0.05), but the increment was more obvious in the wild-type chondrocytes (P < 0.05). Treatment with autophagy dual-fluorescence virus resulted in a significantly greater fluorescence intensity of LC3-GFP in rapamycin-treated ERN1 CKO chondrocytes than in wild-type chondrocytes (P < 0.05). In C28/I2 cells, overexpression of CHERP obviously decreased the fluorescence intensity of LC3, and overexpression of IRE1α enhanced the fluorescence intensity and partially rescued the fluorescence reduction of LC3 caused by CHERP.@*CONCLUSION@#IRE1α deficiency impairs autophagy in chondrocytes by upregulating CHERP and increasing intracellular calcium ion content.


Subject(s)
Animals , Autophagy , Calcium/metabolism , Chondrocytes , Endoplasmic Reticulum/metabolism , Endoribonucleases/pharmacology , Homeostasis , Inositol , Mice , Mice, Knockout , Protein Serine-Threonine Kinases , RNA, Messenger/metabolism , Sirolimus/pharmacology , Tunicamycin/pharmacology
6.
Article in English | WPRIM | ID: wpr-929069

ABSTRACT

Antibody-mediated rejection (AMR) is one of the major causes of graft loss after transplantation. Recently, the regulation of B cell differentiation and the prevention of donor-specific antibody (DSA) production have gained increased attention in transplant research. Herein, we established a secondary allogeneic in vivo skin transplant model to study the effects of romidepsin (FK228) on DSA. The survival of grafted skins was monitored daily. The serum levels of DSA and the number of relevant immunocytes in the recipient spleens were evaluated by flow cytometry. Then, we isolated and purified B cells from B6 mouse spleens in vitro by magnetic bead sorting. The B cells were cultured with interleukin-4 (IL-4) and anti-clusters of differentiation 40 (CD40) antibody with or without FK228 treatment. The immunoglobulin G1 (IgG1) and IgM levels in the supernatant were evaluated by enzyme-linked immunosorbent assay (ELISA). Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting were conducted to determine the corresponding levels of messenger RNA (mRNA) and protein expression in cultured cells and the recipient spleens. The results showed that FK228 significantly improved the survival of allogeneic skin grafts. Moreover, FK228 inhibited DSA production in the serum along with the suppression of histone deacetylase 1 (HADC1) and HDAC2 and the upregulation of the acetylation of histones H2A and H3. It also inhibited the differentiation of B cells to plasma cells, decreased the transcription of positive regulatory domain-containing 1 (Prdm1) and X-box-binding protein 1 (Xbp1), and decreased the expression of phosphorylated inositol-requiring enzyme 1 α (p-IRE1α), XBP1, and B lymphocyte-induced maturation protein-1 (Blimp-1). In conclusion, FK228 could decrease the production of antibodies by B cells via inhibition of the IRE1α-XBP1 signaling pathway. Thus, FK228 is considered as a promising therapeutic agent for the clinical treatment of AMR.


Subject(s)
Animals , Depsipeptides , Endoribonucleases , Hematopoietic Stem Cell Transplantation , Histone Deacetylase Inhibitors/pharmacology , Mice , Protein Serine-Threonine Kinases , Skin Transplantation
7.
Article in Chinese | WPRIM | ID: wpr-927695

ABSTRACT

Sucrose non-fermenting-1-related protein kinase 2 (SnRK2) is a specific Ser/Thr protein kinase in plants. SnRK2 can regulate the expression of downstream genes or transcription factors through phosphorylation of substrates to achieve stress resistance regulation in different tissue parts, and make plants adapt to adverse environment. SnRK2 has a small number of members and a molecular weight of about 40 kDa, and contains a conserved N-terminal kinase domain and a divergent C-terminal regulatory domain, which plays an important role in the expression of enzyme. This review summarized the recent research progresses on the discovery, structure, and classification of SnRK2, and its function in response to various stresses and in regulating growth and development, followed by prospecting the future research direction of SnRK2. This review may provide a reference for genetic improvement of crop stress resistance.


Subject(s)
Abscisic Acid , Arabidopsis Proteins/genetics , Gene Expression Regulation, Plant , Growth and Development , Plants/genetics , Protein Kinases , Protein Serine-Threonine Kinases/genetics , Stress, Physiological/genetics
8.
Acta Physiologica Sinica ; (6): 110-116, 2022.
Article in Chinese | WPRIM | ID: wpr-927586

ABSTRACT

Hypertension is one of the strongest risk factors for cardiovascular diseases, cerebral stroke, and kidney failure. Lifestyle and nutrition are important factors that modulate blood pressure. Hypertension can be controlled by increasing physical activity, decreasing alcohol and sodium intake, and stopping tobacco smoking. Chronic kidney disease patients often have increased blood pressure, which indicates that kidney is one of the major organs responsible for blood pressure homeostasis. The decrease of renal sodium reabsorption and increase of diuresis induced by high potassium intake is critical for the blood pressure reduction. The beneficial effect of a high potassium diet on hypertension could be explained by decreased salt reabsorption by sodium-chloride cotransporter (NCC) in the distal convoluted tubule (DCT). In DCT cells, NCC activity is controlled by with-no-lysine kinases (WNKs) and its down-stream target kinases, Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1). The kinase activity of WNKs is inhibited by intracellular chloride ([Cl-]i) and WNK4 is known to be the major WNK positively regulating NCC. Based on our previous studies, high potassium intake reduces the basolateral potassium conductance, decreases the negativity of DCT basolateral membrane (depolarization), and increases [Cl-]i. High [Cl-]i inhibits WNK4-SPAK/OSR1 pathway, and thereby decreases NCC phosphorylation. In this review, we discuss the role of DCT in the blood pressure regulation by dietary potassium intake, which is the mechanism that has been best dissected so far.


Subject(s)
Blood Pressure , Diet , Humans , Kidney/metabolism , Kidney Tubules, Distal/metabolism , Phosphorylation , Potassium/pharmacology , Protein Serine-Threonine Kinases , Solute Carrier Family 12, Member 3/metabolism
9.
Chinese Journal of Biotechnology ; (12): 1189-1204, 2021.
Article in Chinese | WPRIM | ID: wpr-878624

ABSTRACT

The innate immune system initiates innate immune responses by recognizing pathogen-related molecular patterns on the surface of pathogenic microorganisms through pattern recognition receptors. Through cascade signal transduction, it activates downstream transcription factors NF-κB and interferon regulatory factors (IRFs), and then leads to the production of inflammatory cytokines and type Ⅰ interferon, which resists the infection of pathogenic microorganism. TBK1 is a central adapter protein of innate immune signaling pathway and can activate both NF-κB and IRFs. It is a key protein kinase in the process of anti-infection. The finetuning regulation of TBK1 is essential to maintain immune homeostasis and resist pathogen invasion. This paper reviews the biological functions and ubiquitin modification of TBK1 in innate immunity, to provide theoretical basis for clinical treatment of pathogenic infections and autoimmune diseases.


Subject(s)
Immunity, Innate , Interferon Regulatory Factor-3/metabolism , Protein Serine-Threonine Kinases/genetics , Signal Transduction , Ubiquitin
10.
Acta Physiologica Sinica ; (6): 115-125, 2021.
Article in Chinese | WPRIM | ID: wpr-878241

ABSTRACT

In eukaryotic cells, the endoplasmic reticulum (ER) is the key quality control organelle for cellular protein synthesis and processing. It also serves as an important site for Ca


Subject(s)
Adipose Tissue , Diabetes Mellitus, Type 2 , Endoplasmic Reticulum Stress , Endoribonucleases , Humans , Protein Serine-Threonine Kinases , eIF-2 Kinase
11.
Article in Chinese | WPRIM | ID: wpr-922030

ABSTRACT

OBJECTIVE@#To study the genetic variants of a child with Autism Spectrum Disorder (ASD) combined with epilepsy, and explore its possible pathogenic mechanism.@*METHODS@#Clinical data of the child were collected and evaluated, whole-exome sequencing (WES) technology was used to explore the genetic variants sites of the child and his parents and candidate genes were filtered out. Sanger sequencing were performed to verify the variants identified by WES and PolyPhen2 was utilized to predict the function of these variants. qPCR was carry out to determine the expression of the variant gene.@*RESULTS@#The proband carried a compound heterozygous mutation in the SIK3 gene (Chr11 q23.3, NM_025164.6), which contains a missense mutation c.1295A>G (p.N432S) inherited from the father and a deletion [c.2389_2391del(p.797del)] inherited from the mother. Both mutation sites are highly conservative, and PolyPhen2 predicted (c.1295A>G [p.N432S]) to be harmful. Compared to the mother, expression of SIK3in mRNA level in the peripheral blood of the proband and his father were both significantly decreased; compared to normal child, SIK3 expression in the peripheral blood of the proband and two other children with ASD were all decreased significantly too. In addition, studies on mice found that Sik3 gene has a marked higher level of expression in the brain.@*CONCLUSION@#The SIK3 gene variants may probably be associated with ASD. The detailed mechanism needs to be studied further, which may involve lipid metabolism dysfunction in the brain.


Subject(s)
Animals , Autism Spectrum Disorder/genetics , Epilepsy/genetics , Male , Mice , Mutation , Mutation, Missense , Protein Kinases , Protein Serine-Threonine Kinases/genetics , Whole Exome Sequencing
12.
Article in Chinese | WPRIM | ID: wpr-921814

ABSTRACT

To explore the regulatory effects of Xinfeng Capsules(XFC) on the apoptosis of synovial fibroblasts(FLS) and inflammation in rheumatoid arthritis(RA) via lncRNA MAPKAPK5-AS1(MK5-AS1). Thirty healthy people and 30 patients with RA due to spleen deficiency and dampness exuberance were collected for extracting the peripheral blood mononuclear cells(PBMCs) before and after XFC treatment, which were used to observe the correlation between MK5-AS1 and clinical indicators as well as MK5-AS1 expression before and after XFC treatment. Following the establishment of RA-FLS cell line and the preparation of XFC-containing serum, MK5-AS1-overexpression plasmid was constructed and transfected into RA-FLS for investigating the efficacy of XFC-containing serum in regulating inflammation and apoptosis of RA-FLS via MK5-AS1. The expression of MK5-AS1 in PBMCs of patients with RA due to spleen deficiency and dampness exuberance was decreased(P<0.001). The ROC curve analysis revealed the AUC of 83.9%. Correlation analysis showed that MK5-AS1 was negatively correlated with ESR, CRP, RF, CCP, and spleen deficiency and dampness exuberance syndrome score. The expression of MK5-AS1 increased significantly after XFC treatment(P<0.001). As demonstrated by association analysis, XFC decreased MK5-AS1, ESR, CRP, RF, and spleen deficiency and dampness exuberance syndrome score, with the degree of support all greater than 83%, confidence greater than 80%, and lift greater than 1. The results of RT-qPCR showed that the MK5-AS1 RNA expression significantly decreased after TNF-α stimulation(P<0.01), which, however, increased significantly after the intervention with XFC-containing serum(P<0.05). Such expression rose again after the transfection of pcDNA3.1-MK5-AS1(P<0.01). ELISA results showed that TNF-α stimulation elevated the expression of pro-inflammatory factor IL-17 but lowered the expression of anti-inflammatory factor IL-4(P<0.01). After intervention with XFC-containing serum, the expression of IL-17 decreased while that of IL-4 increased(P<0.01). The transfection of pcDNA3.1-MK5-AS1 contributed to the reduction in IL-17 expression but the elevation in IL-4 expression(P<0.01). The immunofluorescence(IF) findings demonstrated that the expression of pro-apoptotic protein Bax was down-regulated, whereas that of the anti-apoptotic protein Bcl-2 was up-regulated after TNF-α stimulation(P<0.01). After the intervention with XFC-containing serum, the Bax expression was increased, while Bcl-2 expression was decreased(P<0.01), which were remarkably collaborated by the transfection of pcDNA3.1-MK5-AS1(P<0.05). The expression of MK5-AS1 is significantly decreased in both RA-PBMCs and RA-FLS, implying that XFC inhibits inflammatory reaction and promotes the apoptosis in RA by regulating the expression of MK5-AS1.


Subject(s)
Apoptosis , Arthritis, Rheumatoid/genetics , Capsules , Drugs, Chinese Herbal , Fibroblasts , Humans , Inflammation/genetics , Intracellular Signaling Peptides and Proteins , Leukocytes, Mononuclear , Protein Serine-Threonine Kinases , RNA, Long Noncoding/genetics
13.
Article in English | WPRIM | ID: wpr-888498

ABSTRACT

To investigate the effects of salt-inducible kinase 2 (SIK2) on energy metabolism in rats with cerebral ischemia-reperfusion. Adult SD male rats were divided into 5 groups: sham group, ischemia group, reperfusion group, adenovirus no-load group, and SIK2 overexpression group with 5 animals in each group. The middle cerebral artery occlusion (MCAO) was induced with the modified Zea-Longa line thrombus method to establish the cerebral ischemia reperfusion model. Eight days before the MCAO, SIK2 overexpression was induced by injecting 7 μL adenovirus in the right ventricle, then MCAO was performed for followed by reperfusion HE staining was used to observe the pathological changes of cerebral tissue in rats; TTC staining was used to observe the volume of cerebral infarct. The levels of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) in rat brain tissue were detected by ELISA; the levels of SIK2 and hypoxia-inducible factor 1α (HIF-1α) in the rat brain tissues were detected by RT-qPCR and Western blotting. Compared with the sham group, SIK2 level was decreased in the ischemia group, and it was further declined in the reperfusion group (<0.05). Compared with the sham group and ischemic group, the pathological injury in reperfusion group were more severe, and the infarct size was larger; compared with the reperfusion group and adenovirus no-load group, the pathological injury of the SIK2 overexpression group was milder, and the infarct size is less. Compared with the sharn group, HIF-1α was increased in both ischemia group and reperfusion group, especially in ischemia group (all <0.05); HIF-1α level in the SIK2 overexpression group was higher than that in the reperfusion group and adenovirus no-load group (all <0.05). ATP level in ischemia group and reperfusion group was lower than that in the sham group, and the reperfusion group decreased more significantly than the ischemia group (<0.05); ADP content was increased in the ischemia and reperfusion group, and the ADP content in reperfusion group was significantly higher than that in the ischemia group (<0.05). ATP level in the SIK2 overexpression group was higher than that in the reperfusion group and adenovirus no-load group (all <0.05), and ADP was decreased in the SIK2 overexpression group (all <0.05). SIK2 can up-regulate the ATP level and down-regulate the ADP level in rat brain tissue and alleviate cerebral ischemia-reperfusion injury by increase the level of HIF-1α.


Subject(s)
Animals , Brain Ischemia , Energy Metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Infarction, Middle Cerebral Artery , Male , Protein Serine-Threonine Kinases , Rats , Rats, Sprague-Dawley , Reperfusion , Reperfusion Injury
14.
Chinese Medical Journal ; (24): 2054-2065, 2021.
Article in English | WPRIM | ID: wpr-887637

ABSTRACT

BACKGROUND@#The Nuclear Dbf2-related (NDR1) kinase is a member of the NDR/LATS family, which was a supplementary of Hippo pathway. However, whether NDR1 could inhibit glioblastoma (GBM) growth by phosphorylating Yes-associated protein (YAP) remains unknown. Meanwhile, the role of NDR1 in GBM was not clear. This study aimed to investigate the role of NDR1-YAP pathway in GBM.@*METHODS@#Bioinformation analysis and immunohistochemistry (IHC) were performed to identify the expression of NDR1 in GBM. The effect of NDR1 on cell proliferation and cell cycle was analyzed utilizing CCK-8, clone formation, immunofluorescence and flow cytometry, respectively. In addition, the xenograft tumor model was established as well. Protein interaction was examined by Co-immunoprecipitation and immunofluorescence to observe co-localization.@*RESULTS@#Bioinformation analysis and IHC of our patients' tumor tissues showed that expression of NDR1 in tumor tissue was relatively lower than that in normal tissues and was positively related to a lower survival rate. NDR1 could markedly reduce the proliferation and colony formation of U87 and U251. Furthermore, the results of flow cytometry showed that NDR1 led to cell cycle arrest at the G1 phase. Tumor growth was also inhibited in xenograft nude mouse models in NDR1-overexpression group. Western blotting and immunofluorescence showed that NDR1 could integrate with and phosphorylate YAP at S127 site. Meanwhile, NDR1 could mediate apoptosis process.@*CONCLUSION@#In summary, our findings point out that NDR1 functions as a tumor suppressor in GBM. NDR1 is identified as a novel regulator of YAP, which gives us an in-depth comprehension of the Hippo signaling pathway.


Subject(s)
Animals , Cell Nucleus/metabolism , Cell Proliferation , Glioblastoma , Humans , Mice , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Signal Transduction
15.
Clin. biomed. res ; 41(3): 245-253, 20210000.
Article in Portuguese | LILACS | ID: biblio-1348794

ABSTRACT

Dentre os sistemas neurais responsáveis pela ingestão dos alimentos, destaca-se a via dopaminérgica mesolímbica que, através da liberação de dopamina nos núcleos de accumbens, desperta prazer e motivação para recompensas químicas e naturais. Esta via de recompensa age através dos receptores dopaminérgicos transmembranares, que variam de DRD1 a DRD5. Desta forma, considerando os efeitos prazerosos despertados pela ingestão alimentar, é plausível que variações genéticas em genes do sistema dopaminérgico possam ter um papel na arquitetura genética da obesidade. Este estudo tem como objetivo realizar uma revisão narrativa da literatura sobre a influência de variantes genéticas nos receptores dopaminérgicos em fenótipos relacionados com a obesidade. Em conjunto, os principais achados desta revisão indicaram que os genes codificadores dos receptores DRD2 e DRD4 possam ser os mais relevantes no contexto da obesidade e fenótipos relacionados. No entanto, a obesidade é uma doença complexa e multifatorial e novos estudos são ainda necessários para uma melhor compreensão do impacto da dopamina nos desfechos relacionado à obesidade. É importante também destacar que esses efeitos podem ser específicos para subgrupos de pacientes e que outros fatores, além das variantes genéticas, devem ser considerados. (AU)


Among the neural systems responsible for food ingestion, the mesolimbic dopaminergic pathway stands out by eliciting pleasure and motivation for chemical and natural rewards through the release of dopamine in the nucleus accumbens. This reward pathway is regulated by transmembrane dopaminergic receptors, which range from DRD1 to DRD5. Thus, considering the pleasurable effects aroused by food intake, it is plausible that genetic variations in genes of the dopaminergic system may have a role in the genetic architecture of obesity. This study aims to conduct a narrative review of the literature on the influence of genetic variants of dopaminergic receptors on obesity-related phenotypes. Taken together, the main findings of this review indicated that the genes encoding the DRD2 and DRD4 receptors may be the most relevant in the context of obesity and related phenotypes. However, obesity is a complex and multifactorial disease and new studies are still being conducted to better understand the impact of dopamine on obesity-related outcomes. It is also important to note that these effects can be specific to subgroups of patients and that other factors, in addition to genetic variants, must be considered. (AU)


Subject(s)
Dopamine , Receptors, Dopamine , Feeding Behavior , Obesity , Protein Serine-Threonine Kinases
16.
Braz. j. med. biol. res ; 54(8): e10062, 2021. tab, graf
Article in English | LILACS | ID: biblio-1249323

ABSTRACT

Long non-coding RNA (lncRNA) is an essential regulator of carcinogenesis and cancer progression. In the study, we explored the role of lncRNA DLGAP1-AS1 in gastric cancer (GC). qRT-PCR was carried out to detect DLGAP1-AS1 expression in GC tissues and cell lines. CCK-8 assay, EdU assay, and transwell experiments were employed to detect the malignant biological behaviors of GC cells with DLGAP1-AS1 knockdown or overexpression. Bioinformatics and dual-luciferase report assay were used to confirm the binding relationship between DLGAP1-AS1 and miR-515-5p. MARK4 expression was detected by western blot after DLGAP1-AS1/miR-515-5p was selectively regulated. DLGAP1-AS1 was up-regulated in GC tissues and cell lines, and its high expression was closely associated with larger tumor size, higher TNM stage, and lymph node metastasis. Furthermore, DLGAP1-AS1 overexpression enhanced cell proliferation, migration, and invasion, and miR-515-5p could reverse these effects. DLGAP1-AS1 participated in the regulation of the MARK4 signaling pathway by targeting miR-515-5p. DLGAP1-AS1 promoted GC progression through miR-515-5p/MARK4 signaling pathway.


Subject(s)
Humans , Stomach Neoplasms/genetics , MicroRNAs/genetics , RNA, Long Noncoding/genetics , Gene Expression Regulation, Neoplastic , Cell Movement/genetics , Protein Serine-Threonine Kinases , Cell Line, Tumor
17.
Chinese Journal of Biotechnology ; (12): 2298-2312, 2020.
Article in Chinese | WPRIM | ID: wpr-878487

ABSTRACT

Polo-like kinase 1 (Plk1) is widely regarded as one of the most promising targets for cancer therapy due to its essential role in cell division and tumor cell survival. At present, most Plk1 inhibitors have been developed based on kinase domain, some of which are in clinical trial. However, inhibitors targeting kinase domain face off-target effect and drug resistance owing to the conserved nature and the frequent mutations in the ATP-binding pocket. In addition to a highly conserved kinase domain, Plk1 also contains a unique Polo-Box domain (PBD), which is essential for Plk1's subcellular localization and mitotic functions. Inhibitors targeting Plk1 PBD show stronger selectivity and less drug resistance for cancer therapy. Therefore, Plk1 PBD is an attractive target for the development of anti-cancer agents. In this review, we will summarize the up-to date drug discovery for targeting Plk1 PBD, including the molecular structure and cellular functions of Plk1 PBD. Small-molecule inhibitors targeting Plk1 PBD not only provide an opportunity to specifically inhibit Plk1 activity for cancer treatment, but also unveil novel biological basis regarding the molecular recognition of Plk1 and its substrates.


Subject(s)
Cell Cycle Proteins/genetics , Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics
18.
Article in Chinese | WPRIM | ID: wpr-880765

ABSTRACT

OBJECTIVE@#To screen the key genes related to the prognosis of lung adenocarcinoma through big data analysis and explore their clinical value and potential mechanism.@*METHODS@#We analyzed GSE18842, GSE27262, and GSE33532 gene expression profile data obtained from the Gene Expression Omnibus (GEO). Bioinformatics methods were used to screen the differentially expressed genes in lung adenocarcinoma tissues and KEGG and GO enrichment analysis was performed, followed by PPI interaction network analysis, module analysis, differential expression analysis, and prognosis analysis. The expressions of MAD2L1 and TTK by immunohistochemistry were verified in 35 non-small cell lung cancer specimens and paired adjacent tissues.@*RESULTS@#We identified a total of 256 genes that showed significant differential expressions in lung adenocarcinoma, including 66 up-regulated and 190 down-regulated genes. Thirty-two up-regulated core genes were screened by functional analysis, and among them 29 were shown to significantly correlate with a poor prognosis of patients with lung adenocarcinoma. All the 29 genes were highly expressed in lung adenocarcinoma tissues compared with normal lung tissues and were mainly enriched in cell cycle pathways. Seven of these key genes were closely related to the spindle assembly checkpoint (SAC) complex and responsible for regulating cell behavior in G2/M phase. We selected SAC-related proteins TTK and MAD2L1 to test their expressions in clinical tumor samples, and detected their overexpression in lung adenocarcinoma tissues as compared with the adjacent tissues.@*CONCLUSIONS@#Seven SAC complex-related genes, including TTK and MAD2L1, are overexpressed in lung adenocarcinoma tissues with close correlation with the prognosis of the patients.


Subject(s)
Adenocarcinoma of Lung/genetics , Big Data , Cell Cycle Proteins/genetics , Computational Biology , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/genetics , M Phase Cell Cycle Checkpoints , Mad2 Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics
19.
Article in Chinese | WPRIM | ID: wpr-828367

ABSTRACT

This study aimed to investigate the effect and possible mechanism of Bidens pilosa decoction on non-alcoholic fatty liver disease(NAFLD) induced by high fat and high glucose in mice. Bald/c mice were randomly divided into normal group, model group, metformin(200 mg·kg~(-1)) treatment group, Bidens pilosa decoction(10 g·kg~(-1)) treatment group, metformin and B. pilosa decoction(100 mg·kg~(-1)+5 g·kg~(-1)) treatment group. Except for the normal group, mice in the other four groups were fed with high-fat and high-glucose diet for 8 weeks to establish the non-alcoholic fatty liver model. After 4 weeks of treatment, blood was collected from the eyeballs, the mice were sacrificed, and relevant indicators were detected. The results showed that compared with the model group, blood lipid and blood glucose levels of each treatment group were significantly lower(P<0.05); HE staining results showed that liver pathological damage in each treatment group was significantly improved; oil red O staining results showed fat distribution in each treatment group significantly reduced(P<0.01); immunohistochemical staining showed that glucose regulated the protein expression of protein 78(GRP78) in liver tissues of each treatment group was also significantly reduced(P<0.01); Western blot results showed that endoplasmic reticulum stress signal pathway-related factors GRP78, phosphorylated-protein kinase R-like ER kinase(p-PERK), eukaryotic translation-initiation factor 2α(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(Chop), inositol requiring 1α(IRE1α), and cleaved-cysteinyl aspartate specific proteinase 12(cleaved-caspase-12) were significantly reduced(P<0.01). The results of the combined drug treatment group were better than those of the single drug treatment group. These results showed that B. pilosa decoction had the effect in improving non-alcoholic fatty liver, and its mechanism may be related to the down-regulation of the expression of endoplasmic reticulum stress(ERS)-related factors, and the reduction of the apoptosis of hepatocytes caused by ERS and the down-regulation of blood lipid and blood glucose levels.


Subject(s)
Animals , Apoptosis , Bidens , Endoplasmic Reticulum Stress , Endoribonucleases , Glucose , Mice , Non-alcoholic Fatty Liver Disease , Protein Serine-Threonine Kinases
20.
Article in Chinese | WPRIM | ID: wpr-828861

ABSTRACT

OBJECTIVE@#To investigate the expression of BUB1 gene in gastric cancer.@*METHODS@#Oncomine, GEPIA, BioGPS and Kaplan-Meier Plotter databases were used to analyze the difference of BUB1 gene expression between gastric cancer tissue and normal gastric tissue. The association of BUB1 expression level with the prognosis of gastric cancer patients was also analyzed. The Cancer Cell Line Encyclopedia (CCLE) was explored to analyze the expression of BUB1 in T cells and B cells in gastric cancer patients, and the String database was used to generate the network map of BUB1-related proteins and functional annotation of gene ontology (GO). The related pathways of KEGG were analyzed. Tumor immune assessment resource (TIMER) database was used to analyze the expression of BUB1 in immune infiltration and its effect on prognosis of gastric cancer patients. To further verify the results of gene chip analysis in Oncomine database, we collected 30 pairs of surgical specimens of gastric adenocarcinoma and adjacent tissues from patients admitted to the First Affiliated Hospital of Chengdu Medical College from March, 2018 to July, 2019. The results of BUB1 gene expression in Oncomine database were verified by PCR and immunohistochemistry.@*RESULTS@#Oncomine, GEPIA and BioGPS analyses showed that BUB1 was highly expressed in gastric cancer compared with normal gastric tissue. Kaplan-Meier survival analysis showed that the progression-free survival time (HR=0.52, 95% :0.41-0.67, < 0.05) and the overall survival time (HR=0.67, 95% :0.55-0.82, < 0.05) were prolonged in gastric cancer patients with a high expression of BUB1. Through String data collection, BUB1-related proteins were mainly enriched in 13 cellular components, 4 molecular functions and 12 biological processes, involving 4 signal pathways. TIMER database analysis showed that CD4 T cells and macrophages with high expressions of BUB1 mRNA in the immune microenvironment were associated with a favorable 5-year survival outcome of patients with gastric cancer. In the surgical specimens, real-time quantitative PCR showed that the expression level of BUB1 mRNA was significantly higher in gastric cancer tissues than in the adjacent gastric mucosa tissues, and immunohistochemical results demonstrated positive BUB1 staining in the gastric cancer tissues.@*CONCLUSIONS@#BUB1 gene is highly expressed in gastric cancer. BUB1 may reduce tumor immunosuppression and helps to evaluate the prognosis of patients with gastric cancer.


Subject(s)
Computational Biology , Humans , Kaplan-Meier Estimate , Prognosis , Protein Serine-Threonine Kinases , Genetics , Stomach Neoplasms , Genetics , Tumor Microenvironment
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