OBJECTIVE: To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD). METHODS: Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild-type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E-cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins. RESULTS: In the coculture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P < 0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS-treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa. CONCLUSION Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.
Humans ; Mice ; Animals ; Caco-2 Cells ; beta Catenin/metabolism* ; Culture Media, Conditioned/pharmacology* ; Tight Junctions/metabolism* ; Intestinal Mucosa ; Inflammatory Bowel Diseases ; Tight Junction Proteins/metabolism* ; Inflammation/metabolism* ; Fibroblasts/metabolism* ; Mice, Inbred C57BL ; Glycoproteins/metabolism* ; Wnt Proteins/pharmacology* ; Frizzled Receptors/metabolism*

Objective: To investigate the expression of glycoprotein non metastatic melanoma protein B (GPNMB) in renal eosinophilic tumors and to compare the value of GPNMB with CK20, CK7 and CD117 in the differential diagnosis of renal eosinophilic tumors. Methods: Traditional renal tumor eosinophil subtypes, including 22 cases of renal clear cell carcinoma eosinophil subtype (e-ccRCC), 19 cases of renal papillary cell carcinoma eosinophil subtype (e-papRCC), 17 cases of renal chromophobe cell carcinoma eosinophil subtype (e-chRCC), 12 cases of renal oncocytoma (RO) and emerging renal tumor types with eosinophil characteristics [3 cases of eosinophilic solid cystic renal cell carcinoma (ESC RCC), 3 cases of renal low-grade eosinophil tumor (LOT), 4 cases of fumarate hydratase-deficient renal cell carcinoma (FH-dRCC) and 5 cases of renal epithelioid angiomyolipoma (E-AML)], were collected at the Affiliated Drum Tower Hospital of Nanjing University Medical School from January 2017 to March 2022. The expression of GPNMB, CK20, CK7 and CD117 was detected by immunohistochemistry and statistically analyzed. Results: GPNMB was expressed in all emerging renal tumor types with eosinophil characteristics (ESC RCC, LOT, FH-dRCC) and E-AML, while the expression rates in traditional renal eosinophil subtypes e-papRCC, e-chRCC, e-ccRCC and RO were very low or zero (1/19, 1/17, 0/22 and 0/12, respectively); the expression rate of CK7 in LOT (3/3), e-chRCC (15/17), e-ccRCC (4/22), e-papRCC (2/19), ESC RCC (0/3), RO (4/12), E-AML(1/5), and FH-dRCC (2/4) variedly; the expression of CK20 was different in ESC RCC (3/3), LOT(3/3), e-chRCC(1/17), RO(9/12), e-papRCC(4/19), FH-dRCC(1/4), e-ccRCC(0/22) and E-AML(0/5), and so did that of CD117 in e-ccRCC(2/22), e-papRCC(1/19), e-chRCC(16/17), RO(10/12), ESC RCC(0/3), LOT(1/3), E-AML(2/5) and FH-dRCC(1/4). GPNMB had 100% sensitivity and 97.1% specificity in distinguishing E-AML and emerging renal tumor types (such as ESC RCC, LOT, FH-dRCC) from traditional renal tumor types (such as e-ccRCC, e-papRCC, e-chRCC, RO),respectively. Compared with CK7, CK20 and CD117 antibodies, GPNMB was more effective in the differential diagnosis (P<0.05). Conclusion: As a new renal tumor marker, GPNMB can effectively distinguish E-AML and emerging renal tumor types with eosinophil characteristics such as ESC RCC, LOT, FH-dRCC from traditional renal tumor eosinophil subtypes such as e-ccRCC, e-papRCC, e-chRCC and RO, which is helpful for the differential diagnosis of renal eosinophilic tumors.
Humans ; Kidney Neoplasms/pathology* ; Carcinoma, Renal Cell/pathology* ; Diagnosis, Differential ; Angiomyolipoma/diagnosis* ; Biomarkers, Tumor/metabolism* ; Leukemia, Myeloid, Acute/diagnosis* ; Membrane Glycoproteins

Objective: To study using isotope-labeled relative and absolute quantitative proteomics methodologies to screen for salivary biological markers as a simple, non-invasive tool for identifying hepatitis B-related HCC at an early stage. Methods: Saliva samples were collected to extract salivary proteins. Isotope-labeled relative and absolute quantitative proteomics were used to analyze the differentially expressed proteins between the hepatocellular carcinoma (HCC) and non-HCC groups. Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assays were used to verify differential proteins and identify markers in liver cancer tissues and saliva. Statistical analysis was used to analyze the diagnostic efficiency of salivary biomarkers. Results: 152 differentially expressed salivary proteins were screened out between the HCC and non-HCC groups. Western blot, immunohistochemistry, and enzyme-linked immunosorbent assays validated that the expressions of α-1-acid glycoprotein 1 (ORM1) and alpha-fetoprotein (AFP) were significantly increased in HCC (P < 0.05). There was a significant correlation between salivary AFP and serum AFP (P < 0.05). HCC was diagnosed when salivary α-1-acid glycoprotein 1 combined with AFP. The area under the receiver operating characteristic curve was 0.8726 (95% confidence interval: 0.8104 ~ 0.9347), the sensitivity was 78.3%, and the specificity was 88%. Conclusion: Salivary AFP and α-1-acid glycoprotein 1 can serve as potential biomarkers for hepatitis B-related hepatocellular carcinoma.
Humans ; Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/pathology* ; alpha-Fetoproteins/metabolism* ; Biomarkers ; Hepatitis B ; ROC Curve ; Glycoproteins ; Biomarkers, Tumor

OBJECTIVE: To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout. METHODS: The Sidt2 knockout (Sidt2^-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed. RESULTS: Sidt2^-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 μmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2^-/- HL7702 cells. CONCLUSION Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.
Humans ; Lysosome-Associated Membrane Glycoproteins/metabolism* ; Autophagy ; Apoptosis ; Hepatocytes ; Lysosomes/metabolism* ; Chloroquine/pharmacology* ; Nucleotide Transport Proteins/metabolism*

The aim of this study was to clone the chicken zp1 gene encoding zona pellucida 1 (Zp1) and investigate its tissues expression profile and its effect on osteoblast mineralization. The expression level of zp1 was quantified in various tissues of laying hens and in the tibia of the pre- and post-sexual maturity by RT-qPCR. Zp1 overexpressed vector was transfected into chicken calvarial osteoblasts which were induced differentiation for 8 days, and the extracellular mineral and the expression of mineralization-related genes were detected. The full-length chicken zp1 gene is 3 045 bp, encoding 958 amino acids residuals, and has two N-glycosylation sites. The highest expression level of the zp1 gene was found in the liver, followed by the tibia and yolk membrane, while no expression was detected in the heart and eggshell gland. Compared with the pre-sexual maturity hens, the concentration of estrogen (E2) in plasma, the content of glycosaminoglycan (GAG) and the expression level of the zp1 gene in the tibia with post-sexual maturity were higher. The extracellular matrix and the level of osteoblast mineralization-related genes showed a significantly upregulated expression in chicken calvarial osteoblasts with Zp1 overexpressed and addition of estrogen. The expression of the zp1 gene is tissue-specific and positively regulated osteoblast mineralization under the action of estrogen, laying the foundation for elucidating the functional properties of Zp1 in chicken bones during the egg production period.
Female ; Animals ; Zona Pellucida Glycoproteins ; Membrane Glycoproteins/metabolism* ; Chickens/genetics* ; Egg Proteins/metabolism* ; Receptors, Cell Surface ; Estrogens

Cancer stem cell-like cells (CSCs) play an integral role in the heterogeneity, metastasis, and treatment resistance of head and neck squamous cell carcinoma (HNSCC) due to their high tumor initiation capacity and plasticity. Here, we identified a candidate gene named LIMP-2 as a novel therapeutic target regulating HNSCC progression and CSC properties. The high expression of LIMP-2 in HNSCC patients suggested a poor prognosis and potential immunotherapy resistance. Functionally, LIMP-2 can facilitate autolysosome formation to promote autophagic flux. LIMP-2 knockdown inhibits autophagic flux and reduces the tumorigenic ability of HNSCC. Further mechanistic studies suggest that enhanced autophagy helps HNSCC maintain stemness and promotes degradation of GSK3β, which in turn facilitates nuclear translocation of β-catenin and transcription of downstream target genes. In conclusion, this study reveals LIMP-2 as a novel prospective therapeutic target for HNSCC and provides evidence for a link between autophagy, CSC, and immunotherapy resistance.
Humans ; Autophagy ; Carcinoma, Squamous Cell/pathology* ; Cell Line, Tumor ; Glycogen Synthase Kinase 3 beta/metabolism* ; Head and Neck Neoplasms/pathology* ; Neoplastic Stem Cells/pathology* ; Squamous Cell Carcinoma of Head and Neck/pathology* ; Lysosome-Associated Membrane Glycoproteins

OBJECTIVES: To investigate the effects of injury time, postmortem interval (PMI) and postmortem storage temperature on mRNA expression of glycoprotein non-metastatic melanoma protein B (Gpnmb), and to establish a linear regression model between Gpnmb mRNA expression and injury time, to provide aimed at providing potential indexes for injury time estimation. METHODS: Test group SD rats were anesthetized and subjected to blunt contusion and randomly divided into 0 h, 4 h, 8 h, 12 h, 16 h, 20 h and 24 h groups after injury, with 18 rats in each group. After cervical dislocation, 6 rats in each group were collected and stored at 0 ℃, 16 ℃ and 26 ℃, respectively. The muscle tissue samples of quadriceps femoris injury were collected at 0 h, 12 h and 24 h postmortem at the same temperature. The grouping method and treatment method of the rats in the validation group were the same as above. The expression of Gpnmb mRNA in rat skeletal muscle was detected by RT-qPCR. The Pearson correlation coefficient was used to evaluate the correlation between Gpnmb mRNA expression and injury time, PMI, and postmortem storage temperature. SPSS 25.0 software was used to construct a linear regression model, and the validation group data was used for the back-substitution test. RESULTS: The expression of Gpnmb mRNA continued to increase with the prolongation of injury time, and the expression level was highly correlated with injury time (P<0.05), but had little correlation with PMI and postmortem storage temperature (P>0.05). The linear regression equation between injury time (y) and Gpnmb mRNA relative expression (x) was y=0.611 x+4.489. The back-substitution test proved that the prediction of the model was accurate. CONCLUSIONS The expression of Gpnmb mRNA is almost not affected by the PMI and postmortem storage temperature, but is mainly related to the time of injury. Therefore, a linear regression model can be established to infer the time of injury.
Animals ; Rats ; Glycoproteins ; Linear Models ; Melanoma ; Membrane Glycoproteins/genetics* ; Postmortem Changes ; Rats, Sprague-Dawley ; RNA, Messenger/metabolism* ; Time Factors

Influenza B virus is one of the causes for seasonal influenza, which can account for serious illness or even death in some cases. We tested the expression of extracellular domain of hemagglutinin (HA-ecto) of influenza B viruses in mammalian cells, and then determined the immunogenicity of HA-ecto in mice. The gene sequence encoding influenza B virus HA-ecto, foldon sequence, and HIS tag was optimized and inserted into pCAGGS vector. The opening reading frame (ORF) of neuraminidase was also cloned into pCAGGS. The pCAGGS-HA-ecto and pCAGGS-NA were co-transfected into 293T cells using linear polyethylenimine. Cell supernatant after transfection was collected after 96 h, and the secreted trimmeric HA-ecto protein was purified by nickel ion affinity chromatography and size exclusion chromatography. Subsequently, the mice were immunized with HA-ecto protein, and the corresponding antibody titers were detected by ELISA and hemagglutination inhibition (HAI) assays. The results showed that soluble trimeric HA-ecto protein could be obtained using mammalian cell expression system. Moreover, trimeric HA-ecto protein, in combination with the adjuvant, induced high levels of ELISA and HAI antibodies against homogenous and heterologous antigens in mice. Thus, the soluble HA-ecto protein expressed in mammalian cells could be used as a recombinant subunit vaccine candidate for influenza B virus.
Animals ; Hemagglutinin Glycoproteins, Influenza Virus/genetics* ; Hemagglutinins/genetics* ; Influenza B virus/metabolism* ; Influenza Vaccines/genetics* ; Mammals/metabolism* ; Mice ; Mice, Inbred BALB C

OBJECTIVE: To analysis the specific protein markers of essential thrombocythemia (ET) based on proteomics technology, to explore and verify the differential protein related to platelet activation. METHODS: Blood samples were obtained from ET patients and healthy people and a certain protein mass spectrometry was detected using label-free quantitative technology. The proteins relative abundance increased or down-regulated by 1.3 times in the disease group compared with the control group, and the protein abundance in the two groups t test P＜0.05 were defined as differential proteins. Bioinformatics analysis of the differential proteins was performed using GO and KEGG. The difference in the average protein abundance between the two groups was analyzed by t test and P＜0.05 was considered statistically significant. Differential proteins were selected for verification by parallel reaction monitoring (PRM) technology. RESULTS: A total of 140 differential proteins were found, of which 72 were up-regulated and 68 were down-regulated. KEGG enrichment showed that the differential protein expression was related to the platelet activation pathway. The differential proteins related to platelet activation were GPV, COL1A2, GP1bα, COL1A1 and GPVI. Among them, the expressions of GPV, GP1bα and GPVI were up-regulated, and the expressions of COL1A2 and COL1A1 were down-regulated. PRM verification of COL1A1, GP1bα, GPVI and GPV was consistent with LFP proteomics testing. CONCLUSION Differential proteins in ET patients are related to platelet activation pathway activation.Differential proteins such as GPV, GPVI, COL1A1 and GP1bα can be used as new targets related to ET platelet activation.
Blood Platelets/metabolism* ; Humans ; Platelet Activation ; Platelet Membrane Glycoproteins/metabolism* ; Technology ; Thrombocythemia, Essential

OBJECTIVES: The biomarkers targeting colorectal cancer (CRC) prognosis are short of high accuracy and sensitivity in clinic. Through bioinformatics analysis, we aim to identify and confirm a series of key genes referred to the diagnosis and prognosis of CRC. METHODS: GSE31905, GSE35279, and GSE41657 were selected as complete RNA sequencing data sets of CRC and colorectal mucosa (CRM) tissues from the NCBI-GEO database, and the differentially expressed genes (DEGs) were analyzed. The common DEGs in these 3 data sets were obtained by Venn map, and enriched by STRING network system and Cytoscape software. The Kaplan-Meier plotter website was used to verify the correlation between the enriched genes and the prognosis of CRC. RESULTS: For the whole RNA sequencing data sets of CRC and normal intestinal mucosa samples, the DEGs of CRC and CRM in the 3 data sets (|log CONCLUSIONS The above 11 genes verified by bioinformatics retrieval and analysis can predict the poor prognosis of CRC to a certain extent, and they provide a possible target for the diagnosis and treatment of CRC.
Biomarkers, Tumor/metabolism* ; Colorectal Neoplasms/genetics* ; Computational Biology ; Formins ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Glycoproteins ; Humans ; Intercellular Signaling Peptides and Proteins ; Oncogenes ; Prognosis ; Protein Interaction Maps