Objective: To explore the pathogenic mechanism of the miR-340/high mobility group box 1 (HMGB1) axis in the formation of liver fibrosis. Methods: A rat liver fibrosis model was established by injecting CCl(4) intraperitoneally. miRNAs targeting and validating HMGB1 were selected with gene microarrays after screening the differentially expressed miRNAs in rats with normal and hepatic fibrosis. The effect of miRNA expressional changes on HMGB1 levels was detected by qPCR. Dual luciferase gene reporter assays (LUC) was used to verify the targeting relationship between miR-340 and HMGB1. The proliferative activity of the hepatic stellate cell line HSC-T6 was detected by thiazolyl blue tetrazolium bromide (MTT) assay after co-transfection of miRNA mimics and HMGB1 overexpression vector, and the expression of extracellular matrix (ECM) proteins type I collagen and α-smooth muscle actin (SMA) was detected by western blot. Statistical analysis was performed by analysis of variance and the LSD-t test. Results: Hematoxylin-eosin and Masson staining results showed that the rat model of liver fibrosis was successfully established. Gene microarray analysis and bioinformatics prediction had detected eight miRNAs possibly targeting HMGB1, and animal model validation had detected miR-340. qPCR detection results showed that miR-340 had inhibited the expression of HMGB1, and a luciferase complementation assay suggested that miR-340 had targeted HMGB1. Functional experiments results showed that HMGB1 overexpression had enhanced cell proliferation activity and the expression of type I collagen and α-SMA, while miR-340 mimics had not only inhibited cell proliferation activity and the expression of HMGB1, type I collagen, and α-SMA, but also partially reversed the promoting effect of HMGB1 on cell proliferation and ECM synthesis. Conclusion: miR-340 targets HMGB1 to inhibit the proliferation and ECM deposition in hepatic stellate cells and plays a protective role during the process of liver fibrosis.
Animals ; Rats ; Cell Proliferation ; Collagen Type I/metabolism* ; Fibrosis ; Hepatic Stellate Cells ; HMGB1 Protein/genetics* ; Liver Cirrhosis/pathology* ; MicroRNAs/metabolism*

OBJECTIVE: To investigate the role of long non-coding RNA ZEB1-AS1 in cerebral ischemia/reperfusion injury (CI/RI). METHODS: We detected the temporal changes of ZEB1-AS1 and HMGB1 expression using qPCR and Western blotting in SD rats following CI/RI induced by middle cerebral artery occlusion (MCAO). The rat models of CI/RI were subjected to injections of vectors for ZEB1-AS1 overexpression or knockdown into the lateral ventricle, and the changes in cognitive function, brain water content, blood-brain barrier integrity, and IL-1β and TNF-α levels in the cerebrospinal fluid (CSF) and serum were observed. Neuronal loss and cell apoptosis in the cortex of the rat models were detected by FJC and TUNEL methods, and HMGB1 and TLR-4 expressions were analyzed with Western blotting. We also examined the effects of ZEB1-AS1 knockdown on apoptosis and expressions of HMGB1 and TLR-4 in SH-SY5Y cells with oxygen-glucose deprivation/reoxygenation (OGD/R). RESULTS: In CI/RI rats, the expressions of ZEB1-AS1 and HMGB1 in the brain tissue increased progressively with the extension of reperfusion time, reaching the peak levels at 24 h followed by a gradual decline. ZEB1-AS1 overexpression significantly aggravated icognitive impairment and increased brain water content, albumin content in the CSF, and IL-1β and TNF-α levels in the CSF and serum in CI/RI rats (P < 0.05), while ZEB1-AS1 knockdown produced the opposite effects (P < 0.05 or 0.01). ZEB1-AS1 overexpression obviously increased the number of FJC-positive neurons in the cortex and enhanced the expressions of HMGB1 and TLR-4 in the rat models (P < 0.01); ZEB1-AS1 knockdown significantly reduced the number of FJC-positive neurons and lowered HMGB1 and TLR-4 expressions (P < 0.01). In SH-SY5Y cells with OGD/R, ZEB1-AS1 knockdown significantly suppressed cell apoptosis and lowered the expressions of HMGB1 and TLR-4 (P < 0.01). CONCLUSION ZEB1-AS1 overexpression aggravates CI/RI in rats through the HMGB1/TLR-4 signaling axis.
Animals ; Cell Line, Tumor ; Cell Proliferation/genetics* ; HMGB1 Protein/metabolism* ; Humans ; Infarction, Middle Cerebral Artery ; Neuroblastoma ; RNA, Long Noncoding/metabolism* ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; Toll-Like Receptor 4/metabolism* ; Tumor Necrosis Factor-alpha ; Water

The study aimed to explore the mechanism and targets of Shenfu Injection in the regulation of inflammatory injury in chronic heart failure rats based on the high mobility group box-1/Toll like receptor 4/nuclear factor kappa-B(HMGB1/TLR4/NF-κB) signaling pathway. The rat model of chronic heart failure was established using isoproterenol. The modeled rats were divided into three groups by random number table: the model group, Shenfu group and glycopyrrolate group, and the normal group was also set. The rats were administrated for 15 consecutive days, and on the following day after the last administration, they were sacrificed for sample collection. The cardiac mass index and left ventricular mass index of the rats in each group were measured, and the echocardiogram was used to analyze the cardiac function indices, and ELISA to test the inflammatory indices in rat serum. The pathological morphology and fibrosis status of rat heart tissues were observed by HE staining and Masson staining, respectively. The content of HMGB1 was determined by immunofluorescence staining. The protein and mRNA expression of HMGB1/TLR4/TLR4 signaling pathway was detected by Western blot and RT-qPCR, respectively. The results showed that the chronic heart failure rat model was successfully prepared. The rats in the model group had reduced cardiac function, increased levels of HMGB1 and inflammatory factors(P<0.05), and elevated protein and mRNA expression of HMGB1, TLR4, MyD88, and NF-κB P65 in myocardial tissue(P<0.05), with fibrous connective tissue hyperplasia, inflammatory cell infiltration and severe fibrosis. Shenfu Injection improved cardiac function, decreased the levels of HMGB1 and inflammatory factors(P<0.05) and the protein and mRNA expression of HMGB1, TLR4, MyD88, and NF-κB P65 in myocardial tissue(P<0.05), ameliorated interstitial fibrous connective tissue hyperplasia and inflammatory cell infiltration, and reduced fibrosis. In conclusion, Shenfu Injection can reduce inflammatory damage and improve cardiac function in chronic heart failure rats by regulating the HMGB1/TLR4/NF-κB signaling pathway.
Rats ; Animals ; NF-kappa B/metabolism* ; HMGB1 Protein/pharmacology* ; Toll-Like Receptor 4/metabolism* ; Myeloid Differentiation Factor 88/metabolism* ; Hyperplasia ; Rats, Sprague-Dawley ; Signal Transduction ; RNA, Messenger ; Heart Failure/genetics* ; Fibrosis

To study the molecular mechanism for DNA hypomethylation of STAT3 promoter in CD4+ T cells from acute graft-versus-host disease (aGVHD) patients.
 Methods: We collected CD4+ T cells from peripheral blood of 42 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) from HLA-identical sibling donors. GADD45A expression level in CD4+ T cells was measured by real-time PCR and Western blot. The binding level between HMGB1 and GADD45A in CD4+ T cells was analyzed by co-immunoprecipitation, while the binding levels of HMGB1/GADD45A with STAT3 promoter were detected by chromatin immunoprecipitation-quantitative real-time PCR (ChIP-qPCR). After overexpression of HMGB1 and knockdown of GADD45A in normal CD4+ T cells, STAT3 expression and DNA methylation were measured by Western blot and bisulfite sequencing PCR, respectively.
 Results: GADD45A expression was significantly up-regulated in patients with aGVHD compared with that in the patients without aGVHD. More HMGB1-GADD45A complexes were found in CD4+ T cells from patients with aGVHD compared with that in patients without aGVHD. The bindings of HMGB1/GADD45A with STAT3 promoter were significantly increased, and the binding levels of HMGB1/GADD45A were negatively correlated with STAT3 promoter DNA methylation. The expression of STAT3 was significantly reduced and the DNA methylation of STAT3 promoter was significantly increased in CD4+ T cells with overexpression of HMGB1 and knockdown of GADD45A compared with CD4+ T cells only with overexpression of HMGB1.
 Conclusion: The increased expression of HMGB1/GADD45A plays an importent role in STAT3 promoter DNA hypomethylation, thereby promoting STAT3 expression in CD4+ T cells from aGVHD patients.
CD4-Positive T-Lymphocytes ; Cell Cycle Proteins ; metabolism ; DNA Demethylation ; Gene Expression Regulation ; genetics ; Graft vs Host Disease ; genetics ; HMGB1 Protein ; metabolism ; Hematopoietic Stem Cell Transplantation ; Humans ; Nuclear Proteins ; metabolism ; Promoter Regions, Genetic ; genetics ; STAT3 Transcription Factor ; genetics ; metabolism

PURPOSE: High mobility group box 1 (HMGB1) plays a central role in the pathogenesis of sepsis and multiple organ dysfunction syndromes. We investigated the associations of a single nucleotide polymorphism (SNP; rs1045411) in HMGB1 with various clinical parameters, severity, and prognosis in patients with sepsis, severe sepsis, or septic shock. MATERIALS AND METHODS: We enrolled 212 adult patients followed for 28 days. All patients were genotyped for rs1045411, and the serum levels of HMGB1 and several cytokines were measured. RESULTS: The proportions of patients according to genotype were GG (71.2%), GA (26.4%), and AA (2.4%). Among patients with chronic lung disease comorbidity, patients with a variant A allele had higher positive blood culture rates and higher levels of various cytokines [interleukin (IL)-1beta, IL-6, IL-10, IL-17, and tumor necrosis factor-alpha] than those with the GG genotype. In the analysis of those with diabetes as a comorbidity, patients with a variant A allele had higher blood culture and Gram-negative culture rates than those with GG genotypes; these patients also had a higher levels of IL-17. In the analysis of those with sepsis caused by a respiratory tract infection, patients with a variant A allele had higher levels of IL-10 and IL-17 (all p<0.05). This polymorphism had no significant impact on patient survival. CONCLUSION: The variant A allele of rs1045411 appears to be associated with a more severe inflammatory response than the GG genotype under specific conditions.
Adult ; Aged ; Alleles ; Asian Continental Ancestry Group/genetics ; China/epidemiology ; Cytokines/*blood/*genetics ; Female ; Genotype ; HMGB1 Protein/blood/*genetics ; Humans ; Interleukin-10/genetics ; Interleukin-17/genetics ; Interleukin-6/blood ; Male ; Middle Aged ; Polymorphism, Genetic/*genetics ; Polymorphism, Single Nucleotide/*genetics ; Prognosis ; Republic of Korea ; Sepsis/immunology/*metabolism/mortality ; Shock, Septic/immunology/*metabolism/mortality ; Survival ; Tumor Necrosis Factor-alpha/genetics

OBJECTIVETo study the effects of 1,25-(OH)(2)D(3) on airway remodeling and expression of high mobility group box 1 (HMGB1) and IL-17 in asthmatic mice.

METHODSFifty female mice were randomly divided into 5 groups: control, asthma, low-dose, middle-dose, and high-dose intervention groups (n=10 each). Asthma was induced by intraperitoneal injections of ovalbumin (OVA) and aerosol inhalation of OVA solution. The low-dose, middle-dose, and high-dose intervention groups were administered with 1,25-(OH)(2)D(3) solution at the dosage of 1, 4 and 10 μg/kg respectively by intraperitoneal injections before asthma challenge. The airway structural changes were assessed by hematoxylin and eosin staining. mRNA expression levels of HMGB1 and IL-17 in the lung tissues were evaluated by RT-PCR. The protein levels of HMGB1 and IL-17 in the lung tissues were observed by immunohistochemistry.

RESULTSThe airway wall thickness, protein and mRNA expression levels of HMGB1 and IL-17 were higher in the untreated asthma group than in the control group (P<0.05). The airway wall thickness, protein and mRNA expression levels of HMGB1 and IL-17 were lower in the middle-dose and low-dose intervention groups than in the untreated asthma group, and the middle-dose intervention group demonstrated lower airway wall thickness, protein and mRNA expression levels of HMGB1 and IL-17 than in the low-dose intervention group (P<0.05). However, the airway wall thickness, protein and mRNA expression levels of HMGB1 and IL-17 in the high-dose intervention group were higher than in the untreated asthma group (P<0.05).

CONCLUSIONSHMGB1 and IL-17 may be involved in the airway remodeling process in asthmatic mice. A moderate amount of HMGB1 and IL-17 may be involved in the airway remodeling process in asthmatic mice. A moderate amount of 1,25-(OH)(2)D(3) can improve the airway remodeling, but a higher dose of 1,25-(OH)(2)D(3) may affect adversely the airway remodeling process.

Airway Remodeling ; drug effects ; Animals ; Asthma ; drug therapy ; metabolism ; pathology ; Calcitriol ; pharmacology ; Dose-Response Relationship, Drug ; Female ; HMGB1 Protein ; analysis ; genetics ; physiology ; Interleukin-17 ; analysis ; genetics ; physiology ; Lung ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C

OBJECTIVETo observe the effect of high-mobility group box-1 protein (HMGB1) on the proliferation of human nasopharyngeal carcinoma cell line C666-1 and explore the possible underlying mechanisms.

METHODSCultured C666-1 cells were treated with a siRNA targeting HMGB1 gene. The changes in the cell proliferation were detected by CCK8 analysis, the cell cycle distribution was assayed with flow cytometry, and the expressions of cyclin D1, CDK6 and related pathway proteins were detected with Western blotting. The effect of a HMGB1 plasmid carrying the reporter gene GFP on the proliferation of C666-1 cells was tested with CCK8 and EDU analysis.

RESULTSCompared with the control cells, the cells transfected with the siRNA targeting HMGB1 showed obviously suppressed cell proliferation (P<0.001), cell cycle arrest in G1 phase (P<0.001), and down-regulated expressions of cyclin D1, CDK6, STAT3 and P-STAT3. Overexpression of HMGB1 in cells transfected with the HMGB1 plasmid showed a significantly increased ratio of S phase cells (P<0.05) and obviously enhanced cell proliferation (P<0.001).

CONCLUSIONHMGB1 can promote the proliferation of human nasopharyngeal carcinoma cell line C666-1 by up- regulating cyclin D1 and CDK6 via the STAT3 signaling pathway.

Carcinoma ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Division ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 6 ; metabolism ; Down-Regulation ; HMGB1 Protein ; genetics ; Humans ; Nasopharyngeal Neoplasms ; pathology ; RNA, Small Interfering ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Transfection ; Up-Regulation

OBJECTIVETo inquire into the influence of silencing HMGB1 expression by small interfering RNA (siRNA) on cell growth, proliferation, invasion and metastasis of colorectal cancer LoVo cells both in vitro and in vivo.

METHODSLentivirus-mediated HMGB1 siRNA was transfected into LoVo cells to silence the HMGB1 expression. The HMGB1 mRNA and protein expression after siRNA transfection was detected by RT-PCR and Western blot. MTT assay was used to observe the cell proliferation and to draw a growth curve. Cell cycle was measured by flow cytometry. The ability of invasion and speed of cell migration were evaluated by transwell chamber invasion and cell scratch assay. The influence of HMGB1 silencing on the proliferation of LoVo cells in vivo was observed in LoVo tumor-bearing nude mice.

RESULTSLentivirus-mediated siRNA was successfully transfected into colorectal cancer cell line LoVo. The expression of HMGB1 mRNA and protein in the HMGB1-siRNA group were 0.24±0.04 and 0.21±0.03, respectively. Compared with the HMGB1-siRNA-Neg group (0.82±0.13, 1.15±0.18) and control group (0.93±0.15, 1.21±0.20), the difference was significant (P<0.05). MTT assay showed that the cell proliferation in the HMGB1-siRNA group was significantly inhibited when compared with that in the HMGB1-siRNA-Neg group and control group (P<0.05). Flow cytometry showed that the proliferation index (PI) of HMGB1-siRNA group was 38.27±1.32, significantly lower than 54.66±1.74 in the HMGB1-siRNA-Neg group and 57.43±1.29 in the control group (P<0.05). The transwell assay showed that the number of penetrated cells in the HMGB1-siRNA group was 14.0±3.5, significantly lower than 51.0±6.7 in the HMGB1-siRNA-Neg group and 68.0±5.3 in the control group (P<0.05). Similarly, the scrape wound recovered significantly slower in the HMGB1-siRNA group (83.61±23.21) µm than that in the other two groups (202.86±46.46) µm and (214.58±57.38) µm(P<0.05). The nude mouse xenograft tumor experiment showed that the final tumor volume was (521±34) mm3 in the HMGB1-siRNA group, significantly smaller than that in the HMGB1-siRNA-Neg group of (763±46) mm3 and control group of (802±51) mm3 (P<0.05).

CONCLUSIONSLentivirus-mediated HMGBl-siRNA can effectively inhibit the HMGB1 expression in colorectal cancer LoVo cells both in vitro and in vivo. HMGB1 gene silencing can slow the growth of colorectal cancer cells, extend the cell proliferation cycle, decrease their invasion and migration, and significantly inhibit the growth of xenograft tumor in nude mice.

Animals ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Colorectal Neoplasms ; pathology ; therapy ; Gene Expression ; HMGB1 Protein ; genetics ; metabolism ; Humans ; In Vitro Techniques ; Lentivirus ; Mice ; Mice, Nude ; Neoplasm Invasiveness ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; therapeutic use ; Transfection ; Tumor Burden

miR-200c has been shown to regulate the epithelial-mesenchymal transition (EMT) by inhibiting ZEB1 and ZEB2 expression in breast cancer cells. This study further examined the role of miR-200c in the invasion and metastasis of breast cancer that goes beyond the regulation on ZEB1 and ZEB2 expression. In this study, the bioinformatics software (miRanda) was used to predict the target gene of miR-200c and Renilla luciferase assay to verify the result. The metastatic breast cancer cells MDA-MB-231 were cultured and transfected with the miR-200c mimic or inhibitor. The expressions of miR-200c and HMGB1 were detected by RT-PCR and Western blotting, respectively. Transwell assay and wound healing assay were employed to examine the invasive and migrating ability of transfected cells. Target prediction and Renilla luciferase analysis revealed that HMGB1 was a putative target gene of miR-200c. After transfection of MDA-MB-231 cells with the miR-200c mimic or inhibitor, the expression of miR-200c was significantly increased or decreased when compared with cells transfected with the miR-200c mimic NC or inhibitor NC. Moreover, the expression of HMGB1 was reversely correlated with that of miR-200c in transfected cells. Tranwell assay showed that the number of invasive cells was significantly reduced in miR-200c mimic group when compared with miR-200c inhibitor group. It was also found that the migrating ability of cells transfected with miR-200c mimics was much lower than that of cells transfected with miR-200c inhibitors. It was suggested that miR-200c can suppress the invasion and migration of breast cancer cells by regulating the expression of HMGB1. miR-200c and HMGB1 may become useful biomarkers for progression of breast cancer and targets of gene therapy.
Biomarkers, Tumor ; Breast Neoplasms ; genetics ; metabolism ; Cell Movement ; genetics ; Epithelial-Mesenchymal Transition ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; HEK293 Cells ; HMGB1 Protein ; genetics ; Homeodomain Proteins ; biosynthesis ; Humans ; MicroRNAs ; genetics ; Neoplasm Invasiveness ; genetics ; Neoplasm Metastasis ; genetics ; pathology ; Repressor Proteins ; biosynthesis ; Transcription Factors ; biosynthesis ; Zinc Finger E-box Binding Homeobox 2 ; Zinc Finger E-box-Binding Homeobox 1

OBJECTIVETo investigate the effects of 1,25-(OH)(2)D(3) on the airway remodeling and expression of high-mobility group box 1 (HMGB1) and Toll-like receptor 4 (TLR4) in the lungs among asthmatic mice.

METHODSThirty female mice (BALB/c strain) were randomly divided into control, asthma and 1,25-(OH)(2)D(3) intervention groups. An asthmatic mouse model was established by intraperitoneal injection and aerosol inhalation of ovalbumin. The intervention group was given 1,25-(OH)(2)D(3) by intraperitoneal injection 0.5 hour before each aerosol inhalation, while the control group used normal saline instead. The hematoxylin-eosin staining was used to observe the mouse airway structural changes. The mRNA and protein expression of HMGB1 and TLR4 was measured by RT-PCR and immunohistochemistry, respectively. Pearson correlation analysis was performed.

RESULTSThe asthma group had a significantly increased airway wall thickness compared with the control group (P<0.05); the intervention group had a significantly lower increase in airway wall thickness than the asthma group (P<0.05). The mRNA and protein expression of HMGB1 and TLR4 was significantly higher in the asthma group than in the control group (P<0.05); the mRNA and protein expression of HMGB1 and TLR4 in the intervention group was significantly lower than that in the asthma group, but still higher than that in the control group (P<0.05). A positive correlation was found between the protein expression of HMGB1 and TLR4 (P<0.01), and so was their mRNA expression (P<0.01).

CONCLUSIONSHMGB1 and TLR4 may be involved in asthmatic airway remodeling. 1,25-(OH)(2)D(3) can reduce the airway remodeling in asthmatic mice, which may be related to the downregulation of HMGB1 and TLR4 expression in the lungs of asthmatic mice.

Airway Remodeling ; drug effects ; Animals ; Asthma ; drug therapy ; metabolism ; Calcitriol ; pharmacology ; therapeutic use ; Female ; HMGB1 Protein ; genetics ; Lung ; metabolism ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; analysis ; Toll-Like Receptor 4 ; genetics