Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.
Humans ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Colorectal Neoplasms/pathology* ; Lysine/metabolism* ; NF-kappa B/metabolism* ; TNF Receptor-Associated Factor 6/metabolism* ; Transcription Factor AP-1/metabolism* ; Ubiquitin/metabolism*

Chikusetsusaponin IVa (CsIVa) is a natural active monomer of triterpene saponins in the Chinese herbal medicine of Panax japonicus, which has anti-inflammatory, anti-tumor and other effects. However, its function and mechanism in triple negative breast cancer (TNBC) remain unclear. This study investigated the inhibitory effect and mechanisms of CsIVa on the proliferation of triple negative breast cancer cell line MDA-MB-231. In this study, we found that CsIVa could significantly inhibit the proliferation of MDA-MB-231 cells and eliminate its potential toxic effect on normal breast cells (MCF-10A). The transcriptome sequencing results showed that the inhibition of proliferation of MDA-MB-231 cells by CsIVa was closely related to cell cycle and the pathway regulating cell cycle. Further studies confirmed that CsIVa blocked the cell cycle in G2/M phase by down-regulating the expression of cyclin dependent kinase 1 (CDK1), cyclin B1 and up-regulating the expression of cyclin dependent kinase inhibitor 1A (p21). Moreover, CsIVa can block cell cycle through inhibiting PI3K/AKT signal pathway. In conclusion, CsIVa regulates the expression of cell cycle related proteins (p21, CDK1, cyclin B1) via inhibiting the activity of PI3K/AKT signaling pathway, blocks TNBC cell cycle, and thus exerts its anti-tumor activity.

OBJECTIVE: To investigate protective effect of Cordyceps sinensis (CS) through autophagy-associated adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in acute kidney injury (AKI)-induced acute lung injury (ALI). METHODS: Forty-eight male Sprague-Dawley rats were divided into 4 groups according to a random number table, including the normal saline (NS)-treated sham group (sham group), NS-treated ischemia reperfusion injury (IRI) group (IRI group), and low- (5 g/kg·d) and high-dose (10 g/kg·d) CS-treated IRI groups (CS1 and CS2 groups), 12 rats in each group. Nephrectomy of the right kidney was performed on the IRI rat model that was subjected to 60 min of left renal pedicle occlusion followed by 12, 24, 48, and 72 h of reperfusion. The wet-to-dry (W/D) ratio of lung, levels of serum creatinine (Scr), blood urea nitrogen (BUN), inflammatory cytokines such as interleukin- β and tumor necrosis factor- α, and biomarkers of oxidative stress such as superoxide dismutase, malonaldehyde (MDA) and myeloperoxidase (MPO), were assayed. Histological examinations were conducted to determine damage of tissues in the kidney and lung. The protein expressions of light chain 3 II/light chain 3 I (LC3-II/LC3-I), uncoordinated-51-like kinase 1 (ULK1), P62, AMPK and mTOR were measured by Western blot and immunohistochemistry, respectively. RESULTS: The renal IRI induced pulmonary injury following AKI, resulting in significant increases in W/D ratio of lung, and the levels of Scr, BUN, inflammatory cytokines, MDA and MPO (P<0.01); all of these were reduced in the CS groups (P<0.05 or P<0.01). Compared with the IRI groups, the expression levels of P62 and mTOR were significantly lower (P<0.05 or P<0.01), while those of LC3-II/LC3-I, ULK1, and AMPK were significantly higher in the CS2 group (P<0.05 or P<0.01). CONCLUSION CS had a potential in treating lung injury following renal IRI through activation of the autophagy-related AMPK/mTOR signaling pathway in AKI-induced ALI.
Rats ; Male ; Animals ; AMP-Activated Protein Kinases/metabolism* ; Cordyceps/metabolism* ; Rats, Sprague-Dawley ; Kidney/pathology* ; Acute Kidney Injury/metabolism* ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism* ; Reperfusion Injury/metabolism* ; Cytokines/metabolism* ; Acute Lung Injury/drug therapy* ; Mammals/metabolism*

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*

OBJECTIVES: To investigate the epidemiological characteristics of antisocial personality disorder (ASPD) in young men in Chengdu and explore the characteristics and risks of violence. METHODS: Stratified random sampling was used to conduct a self-assessment questionnaire survey in 4 108 males aged from 18 to 34 in Chengdu, including general demographic characteristics, structured clinical interview for the diagnostic and statistical manual of mental disorders-Ⅳ axis Ⅱ disorders (SCID-Ⅱ) personality disorder screen questionnaire, violence questionnaire, psychosis screening questionnaire (PSQ), Alcohol Use Disorders Identification Test (AUDIT) and drug use. χ² test, univariate and multivariate logistic regression were used for analysis and odds ratio (OR) was calculated. RESULTS: The positive rate of ASPD was 5.91%, which was associated with young age, unmarried, unemployment state, low educational level, violent behavior, psychotic symptoms, alcohol and drug use (P<0.05). Young men with ASPD also had a risk (P<0.05) of violence (OR was 8.51), multiple violence (OR was 16.57), injury (OR was 6.68), intentional violence (OR was 11.41), etc., the risk decreased after controlling for psychotic symptoms and substance abuse, but was still statistically significant. CONCLUSIONS The risk of violence, severe violence and intentional violence in young men in Chengdu is high, and psychotic symptoms and substance abuse increase the risk of ASPD violence and relate characteristics.
Alcoholism/epidemiology* ; Antisocial Personality Disorder/epidemiology* ; Comorbidity ; Humans ; Male ; Substance-Related Disorders/epidemiology* ; Surveys and Questionnaires

OBJECTIVE: To investigate the molecular mechanism of one patient with abnormal serological phenotype in RhD and discuss the transfusion strategy. METHODS: The RhD variant sample was screened from a patient with IgM type anti-D antibody and further determined by three different sources of anti-D antibodies. Ten exons and the adjacent introns of the RHD gene were amplified, purified and sequenced. RhCE phenotypes and RHCE genotypes were detected. RESULTS: The patient with Rh variant showed abnormal results of serological tests. The RHD gene sequence analysis showed that the RHD*01W.01 with a variation (c.809T>G, p.Val270Gly) in exon 6 of the RHD gene was found in the patient. The RhCE phenotype was CcEe. The genotyping results of RHCE were consistent with the serological typing results. CONCLUSION The Rh variant of the patient is RHD*01W.01, these findings indicate that RhD variants should be analyzed by molecular assays for the sake of safe transfusion.
Alleles ; Blood Transfusion ; Exons ; Genotype ; Humans ; Phenotype ; Rh-Hr Blood-Group System/genetics*

Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.
Amino Acid Sequence ; Cloning, Molecular ; Geranyltranstransferase/genetics* ; Pogostemon ; Transcription Factors/genetics*

Objective:To investigate the factors affecting phenotypes in the patients of methylmalonic acidemia combined with homocysteinemia cblC type with MMACHC c. 609G>A homologous variant. Methods:A retrospective study on the clinical manifestations, complications, treatment, and outcome in 164patients of cblC type with MMACHC c. 609G>A homologous variant was conducted.The patients were diagnosed by biochemical and genetic analysisfrom January 1998 to December 2020. Results:Among the 164 patients, 2 cases were prenatally diagnosed and began treatment after birth. They are 3 and 12 years old with normal physical and mental development. Twenty-one cases were diagnosed by newborn screening. Among them, 15 cases had with normal development. They were treated fromthe age of two weeks at the asymptomatic period. Six cases began treatment aged 1 to 3 months after onset. Their development was delayed. One hundred and forty-one cases were clinically diagnosed. Their onset age ranges from a few minutes after birth to 6 years old. 110 cases had early-onset (78.0%). 31 cases had late-onset (22.0%). Five of them died. 24 patients lost to follow-up. Of the 141 clinically diagnosed patients, 130 (92.2%) with psychomotor retardation, 69 (48.9%) with epilepsy, 39 (27.7%) with anemia, 30 (21.3%) had visual impairment, 27 (19.1%) had hydrocephalus, 26 (18.4%) had feeding difficulties, 7 (5.0%) with liver damage, and 5 (3.5%) with metabolic syndrome. The frequency of hydrocephalus and seizures was significantly higher in the early-onset group. The urinary methylmalonic acid increased significantly in the patients with epilepsy. During the long-term follow-up, the level of plasma total homocysteine in the seizure-uncontrolled group was significantly higher than that in the seizure-controlled group, the difference had a statistical significance ( P<0.05). Conclusion:Most of the patients with MMACHC c. 609G>A homozygous variant had early-onset disease, with a high mortality and disability rate. If not treated in time, it will lead to neurological damage, resulting in epilepsy, mental retardation, hydrocephalus, and multiple organ damage. Pre-symptomatic diagnosis and treatment are crucial to prevent irreversible neurological damage. Neonatal screening and prenatal diagnosis are important to improve the outcome of the patients.

Objective:To analyze the clinical features, genetic characteristics, treatment and follow-up results of patients with hydrocephalus caused by methylmalonic acidemia combined with homocysteinuria, and to discuss the optimal strategies for assessing and treating such patients.Methods:From January 1998 to December 2020, 76 patients with hydrocephalus due to methylmalonic acidemia combined with homocysteinuria in the Department of Pediatrics in 11 hospitals including Peking University First Hospital were diagnosed by biochemical, genetic analysis and brain imaging examination. The patients were divided into operation-group and non-operation-group according to whether they underwent ventriculoperitoneal shunt. The clinical features, laboratory examinations, genotype, and follow-up data were retrospectively analyzed. Data were compared between the two groups using rank sum test, and categorical data were compared using χ 2 test. Results:Among the 76 patients (51 male, 25 female), 5 were detected by newborn screening, while 71 were diagnosed after clinical onset, 68 cases (96%) had early-onset, 3 cases (4%) had late-onset. The most common clinical manifestations of 74 cases with complete data were psychomotor retardation in 74 cases (100%), visual impairment in 74 cases (100%), epilepsy in 44 cases (59%), anemia in 31 cases (42%), hypotonia or hypertonia in 21 cases (28%), feeding difficulties in 19 cases (26%) and disturbance of consciousness in 17 cases (23%). Genetic analysis was performed in 76 cases, all of whom had MMACHC gene variations, including 30 homozygous variations of MMACHC c.609G>A. The most common variations were c.609G>A (94, 62.7%), followed by c.658_660del (18, 12.0%), c.567dupT (9, 6.0%) and c.217C>T (8, 5.3%). Therapy including cobalamin intramuscular injection, L-carnitine and betaine were initiated immediately after diagnosis. A ventriculoperitoneal shunt operation was performed in 41 cases (operation group), and 31 patients improved after metabolic intervention (non-operation group). There was no significant difference in the age of onset, the age of diagnosis, the blood total homocysteine, methionine, and urinary methylmalonic acid concentration between the two groups (all P>0.05). The symptoms of psychomotor development, epilepsy, and visual impairments improved gradually after a long-term follow-up in the operation group. Conclusions:Hydrocephalus is a severe complication of methylmalonic acidemia combined with homocysteinuria. The most common clinical manifestations are psychomotor retardation, visual impairment, and epilepsy. It usually occurs in early-onset patients. Early diagnosis and etiological treatment are very important. Hydrocephalus may improve after metabolic intervention in some patients. For patients with severe ventricular dilatation, prompt surgical intervention can improve the prognosis.

OBJECTIVE: To analyze the different subtypes caused by c.721C>T substitution in the exon 7 of the ABO gene, and to investigate the related molecular mechanism of different antigens expression. METHODS: ABO subtypes in 7 samples were identified by standard serological methods. The exons 6, 7, and adjacent intron of ABO gene were amplified by Polymerase Chain Reaction (PCR), and the PCR products were analyzed by direct DNA sequencing and cloning sequencing. RESULTS: ABO subtypes phenotypes were A CONCLUSION c.721C>T substitution in the ABO gene causes p.Arg241Trp exchange resulting in the decreasing of GTA or GTB activities and weaker antigen expression. O.01.07 is a null allele which cannot form a functional catalytic enzyme has no effect on A
ABO Blood-Group System/genetics* ; Alleles ; Exons ; Genotype ; Mutation, Missense