Objective To investigate the regulatory mechanism of transforming growth factor-β1 (TGF-β1) in different types of mitral valvular disease (MVD) with atrial fibrillation (AF). Methods From August 2011 to August 2012, patients with moderate to severe MVD accompanied by AF who required mitral valve replacement at the Department of Cardiovascular Surgery, West China Hospital, Sichuan University, were included. Based on echocardiographic results, patients were divided into two groups: a mitral regurgitation (MR) with AF (MR-AF) group and a mitral stenosis (MS) with AF (MS-AF) group. Left atrial tissue samples were collected during surgery. Techniques such as enzyme-linked immunosorbent assay, real-time fluorescence quantitative polymerase chain reaction, immunohistochemistry, and Western blotting were used to detect key molecules in the TGF-β1/JNK pathway. Results Sixteen patients were enrolled. There were 8 patients in the MR-AF group, including 5 males and 3 females, with an average age of (41.38±11.19) years; and 8 patients in the MS-AF group, including 6 males and 2 females, with an average age of (43.12±5.30) years. The left atrial volume load was higher in MR-AF patients, while the left atrial pressure load was higher in MS-AF patients. In MS-AF patients, the relative expression levels of MAPK9, JUN, CASP3, BAX, and BCL2 mRNA in left atrial tissues were significantly upregulated. The serum TGF-β1 protein level and the relative expression levels of p-JNK, p-c-Jun, and Caspase-3 proteins in the left atrial tissues of the MR-AF group were higher. Myocardial cell damage was more severe in the MS-AF group, and the protein expression level of Bcl-2 was higher. Conclusion Different MVD have distinct hemodynamic characteristics. The myocardium of the left atrium in MR-AF patients is more prone to apoptosis, possibly through the activation of the TGF-β1/JNK signaling pathway.

OBJECTIVES: To investigate the mechanism by which transforming growth factor‑β (TGF‑β) regulates major histocompatibility complex class I (MHC-I) expression in hepatocellular carcinoma (HCC) cells and its role in immune evasion of HCC. METHODS: HCC cells treated with TGF‑β alone or in combination with SB-431542 (a TGF-β type I receptor inhibitor) were examined for changes in MHC-I expression using RT-qPCR and Western blotting. A RNA interference experiment was used to explore the role of miR-23a-3p/IRF1 signaling in TGF‑β‑mediated regulation of MHC-I. HCC cells with different treatments were co-cultured with human peripheral blood mononuclear cells (PBMCs), and the changes in HCC cell proliferation was assessed using CCK-8 and colony formation assays. T-cell cytotoxicity in the co-culture systems was assessed with lactate dehydrogenase (LDH) release and JC-1 mitochondrial membrane potential assays, and T-cell activation was evaluated by flow cytometric analysis of CD69 cells and ELISA for TNF-α secretion. RESULTS: TGF‑β treatment significantly suppressed MHC-I expression in HCC cells and reduced T-cell activation, leading to increased tumor cell proliferation and decreased HCC cell death in the co-culture systems. Mechanistically, TGF-β upregulated miR-23a-3p, which directly targeted IRF1 to inhibit MHC-I transcription. Overexpression of miR-23a-3p phenocopied TGF‑β‑induced suppression of IRF1 and MHC-I. CONCLUSIONS We reveal a novel immune escape mechanism of HCC, in which TGF‑β attenuates T cell-mediated antitumor immunity by suppressing MHC-I expression through the miR-23a-3p/IRF1 signaling axis.
Humans ; MicroRNAs/genetics* ; Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/metabolism* ; Interferon Regulatory Factor-1/metabolism* ; Transforming Growth Factor beta/metabolism* ; Signal Transduction ; Histocompatibility Antigens Class I/metabolism* ; Cell Line, Tumor ; Tumor Escape ; Coculture Techniques

Objective To reveal the molecular mechanism by which nuclear epidermal growth factor receptor(nEGFR)synergistically regulates the expression of cell migration-inducing protein(CEMIP)by forming a complex with the transcription factor Yin Yang 1(YY1),and to investigate the biological functions of the nEGFR-YY1-CEMIP signaling axis in invasion of hepatocellular carcinoma(HCC).Methods After HCC cells were serum-starved for 24 h,the cells were treated with 100 ng/mL EGF.Thus,the cells were divided into a control group and EGF-treated groups at different time points.Nuclear expression and localization changes of EGFR were detected by Western blotting and immunofluorescence(IF).To investigate the interaction between nEGFR and YY1,their nuclear colocalization and interaction were examined by IF and co-immunoprecipitation(Co-IP),respectively.Transcriptional profiling was performed using RNA sequencing(RNA-seq)to identify differentially expressed genes at the genome-wide level.Combined with Gene Ontology(GO)functional enrichment analysis and transcription factor binding profiles via using the JASPAR database,CEMIP was identified as a candidate target gene.To validate the regulatory mechanism,the following experimental groups were established,Control,EGF,siYY1,and siYY1+EGF.The expression of CEMIP at protein and mRNA levels was detected by Western blotting and RT-qPCR.To elucidate the molecular mechanism of nEGFR/YY1 binding to the CEMIP promoter,the control and EGF-treated groups were established.Chromatin immunoprecipitation followed by quantitative PCR(ChIP-qPCR)was performed to assess the enrichment of nEGFR/YY1 at the CEMIP promoter region.Luciferase reporter assay was conducted following transfection with either wild-type EGFR(EGFR-WT),nuclear localization-deficient mutant(EGFR-dNLS),YY1 overexpression plasmid(YY1-OE),or dominant-negative YY1 mutant(YY1-DN)to evaluate changes in promoter activity.Subsequently,cell migration and invasion capabilities were evaluated using scratch wound healing assay and Transwell assay,while hyaluronic acid(HA)level was quantified by ELISA.The expression of matrix metalloproteinases(MMP2/9)was analyzed via Western blotting to assess the regulatory role of the nEGFR/YY1-CEMIP axis in the migration and invasion of HCC cells.By analyzing the CEMIP expression profiles in HCC patients from National Center for Biotechnology Information(NCBI)public databases,its potential association with tumor metastasis risk was validated.Results Western blotting and IF demonstrated that EGF treatment significantly induced nuclear translocation of EGFR,peaking at 30 min(P<0.001).Co-IP and IF assays indicated both physical interaction and nuclear co-localization between nEGFR and YY1.RNA-seq analysis identified CEMIP as a significantly differentially expressed gene.GO enrichment analysis revealed that CEMIP was significantly enriched in biological processes related to cell invasion promotion.JASPAR prediction identified conserved YY1 potential binding region within the CEMIP promoter region.Western blot and RT-qPCR analyses confirmed that EGF treatment up-regulated CEMIP at both protein and mRNA levels(P<0.05).Notably,YY1 knockdown significantly suppressed CEMIP expression,while exogenous EGF supplementation restored CEMIP level in YY1-deficient cells(P<0.05).ChIP-qPCR analysis demonstrated specific enrichment of the nEGFR/YY1 complex at the CEMIP promoter region,with EGF stimulation significantly enhancing its binding affinity(P<0.001).Luciferase reporter assay confirmed that nEGFR/YY1 robustly enhanced CEMIP promoter activity(P<0.01),while either the EGFR-dNLS or the YY1-DN substantially attenuated this transcriptional activation.Functional phenotyping showed that the nEGFR/YY1-CEMIP axis significantly enhanced the migration and invasion of HCC cells by promoting HA catabolism and up-regulating MMP2/9 expression(P<0.05).Analysis of NCBI datasets revealed that CEMIP expression was significantly up-regulated in HCC tumor tissues than adjacent normal tissues(P<0.001).Moreover,HCC patients with elevated CEMIP expression exhibited higher risk of metastasis(P<0.001).Conclusion nEGFR promotes HCC invasion by forming a transcriptional complex with YY1 to cooperatively activate CEMIP expression.

Objective To reveal the mechanism by which the programmed death-ligand 1(PD-L1)-protein tyrosine phosphatase 1B(PTP1B)-focal adhesion kinase(FAK)signaling axis promotes the progression of hepatocellular carcinoma(HCC)and elucidate its effector functions in HCC.Methods GEPIA database was used to plot a 10-year survival curve for PD-L1 and FAK expression levels in HCC patients.Immunohistochemical(IHC)staining was utilized to analyze the relative expression levels of PD-L1 and FAK phosphorylated at the Y397 site[p-FAK(Y397)]in HCC tissues,and the results were compared to those in the adjacent non-tumor tissues.Subsequently,endogenous PD-L1 expression was detected with Western blotting in HCC cell lines with low(SNU-387)and high(Hep3B)PD-L1 expression levels.After lentivirus-transduced SNU-387PDL1+and Hep3BPDL1-cells were constructed,the effect of high and low expression of PD-L1 on the expression of p-FAK(Y397)with Western blotting.To elucidate the functional mechanism of FAK in HCC,functional rescue experiments were performed by administering a FAK inhibitor to SNU-387PDL1+cells and a FAK activator to Hep3BPDL1-cells,combined with wound healing scratch assay,Transwell invasion assay,EdU proliferation assay,and colony formation assay to evaluate tumor malignant effects.The GENEMANIA database predicted functional interactions between protein tyrosine phosphatase 1B(PTP1B),PD-L1,and FAK.IHC staining was performed to analyze the correlation among PD-L1,PTP1B,and p-FAK(Y397)expression.Co-immunoprecipitation(Co-IP)and indirect immunofluorescence(IF)were applied to validate the interaction between PD-L1 and PTP1B.Western blotting was utilized to confirm the regulatory relationship between PD-L1 and PTP1B.In vitro PTP1B phosphatase activity assay measured the changes in PTP1B activity.Subsequently,Western blotting was used to screen cell lines with high endogenous PTP1B expression(SNU-387)and low endogenous PTP1B expression(Hep3B).Furthermore,Hep3BPTP1B+and SNU-387PTP1B-cell lines were generated,and then p-FAK(Y397)levels were then detected in these modified cell lines,and the aforementioned functional effect assays(migration,invasion,proliferation and colony formation)and rescue experiments were repeated.Furthermore,Western blotting was employed to detect changes in downstream signaling pathways following enhancement or attenuation of p-FAK(Y397)in SNU-387 and Hep3B cells.Results IHC staining revealed a positive correlation between PD-L1 and p-FAK(Y397)expression in HCC tissues(95%CI:1.065～3.801,P<0.01).In SNU-387PDL1+cells,PD-L1 overexpression significantly enhanced phosphorylation at the FAK Y397 site(P<0.01)and increased cell migration,invasion,proliferation,and colony formation capabilities(P<0.01),and these effects could be reversed by FAK inhibitor treatment(P<0.05).Conversely,in Hep3BPDL1-cells,PD-L1 knockdown significantly reduced FAK Y397 phosphorylation(P<0.01)and decreased cell migration,invasion,proliferation,and colony formation abilities(P<0.01),and these effects were restored by FAK activator treatment(P<0.05).IHC staining further showed a negative correlation between PTP1B expression and both PD-L1 and p-FAK(Y397)in HCC tissues(95%CI:1.886～3.514,P<0.05).Co-IP and IF assays confirmed a direct interaction between PD-L1 and PTP1B,with PD-L1 suppressing PTP1B expression level and reducing its activity(P<0.01).In SNU-387PTP1B-cells,PTP1B knockdown significantly increased FAK Y397 phosphorylation(P<0.01)and enhanced cell migration,invasion,proliferation,and colony formation(P<0.01),and these effects were reversed by FAK inhibitor(P<0.05).While in Hep3BPTP1B+cells,PTP1B overexpression significantly decreased FAK Y397 phosphorylation(P<0.01)and reduced cell migration,invasion,proliferation,and colony formation(P<0.01),and those effects were restored by FAK activator treatment(P<0.05).Furthermore,enhanced phosphorylation at the FAK Y397 site in SNU-387 cells activated downstream PI3K/AKT and MEK/ERK signaling pathways(P<0.01),whereas inhibition of FAK(Y397)phosphorylation in Hep3B cells attenuated the activation of these signaling pathways(P<0.01).Conclusion PD-L1 activates FAK by suppressing PTP1B,thereby promoting migration,invasion,and proliferation in HCC.

Objective:To establish a nomogram prediction model for recurrence within 2 years after radical resection of hepatocellular carcinoma (HCC) based on clinical and ultrasonographic characteristics.Methods:Clinical data from 405 HCC patients (including 327 males and 78 females), aged 60 (53, 66) years old, who underwent radical hepatectomy in the Zhongshan Hospital, Fudan University, from January to December 2021, were retrospectively collected. The patients were divided into two groups: the training group ( n=283) and the validation group ( n=122). Based on recurrence within 2 years after surgery, the 283 patients in the training group were further categorized into the recurrence group ( n=73) and the non-recurrence group ( n=210). Among the 122 patients in the validation group, 33 had recurrence within 2 years, while 89 did not. Data on age, microvascular invasion, alpha-fetoprotein (AFP), AFP lentil lectin-reactive fraction (AFP-L3), protein induced by vitamin K absence or antagonist-II (PIVKA-II), tumor number, and enhancement homogeneity were collected. Logistic regression analysis was performed on the training group to identify risk factors associated with postoperative recurrence, and a nomogram model for predicting HCC recurrence was constructed based on these factors. Calibration curves were used to compare the consistency between predicted and actual outcomes in both the training and validation groups. Results:Multivariate logistic regression analysis revealed that younger age ( OR=0.976, 95% CI: 0.953-1.000, P=0.004), higher AFP-L3 ( OR=1.066, 95% CI: 1.014-1.120, P=0.012), higher PIVKA-II ( OR=1.000, 95% CI: 1.000-1.001, P=0.042), multiple tumors ( OR=0.399, 95% CI: 0.225-0.706, P=0.038), and heterogeneous enhancement ( OR=0.472, 95% CI: 0.243-0.916, P=0.045) were significant risk factors for recurrence after partial hepatectomy in HCC patients. The nomogram constructed based on these variables had a C-index of 0.87 (95% CI: 0.81-0.93) in the training group and 0.83 (95% CI: 0.77-0.89) in the validation group. The calibration curves for predicting recurrence within 2 years after partial hepatectomy in HCC patients showed a high degree of fit in both the training and validation groups, indicating a good agreement between predicted and actual outcomes. Conclusion:The nomogram model constructed based on preoperative clinical and ultrasonographic characteristics can effectively predict the risk of recurrence within 2 years after radical resection of HCC.

Background/Aims: Total proctocolectomy with ileal pouch-anal anastomosis (IPAA) is widely accepted as a radical surgery for refractory ulcerative colitis (UC). Definite results on the appropriate pouch length for an evaluation of the risk-to-benefit ratio regarding technical complications and long-term quality of life (QOL) are still scarce. Methods: Data on UC patients who underwent IPAA from 2008 to 2022 in four well-established pouch centers affiliated to China UC Pouch Center Union were collected. Results: A total of 208 patients with a median follow-up time of 6.0 years (interquartile range, 2.3 to 9.0 years) were enrolled. The median lengths of the patients’ short and long pouches were 14.0 cm (interquartile range, 14.0 to 15.0 cm) and 22.0 cm (interquartile range, 20.0 to 24.0 cm), respectively. Patients with a short J pouch configuration were less likely to achieve significantly improved long-term QOL (p=0.015) and were prone to develop late postoperative complications (p=0.042), such as increased defecation frequency (p=0.003) and pouchitis (p=0.035). A short ileal pouch was an independent risk factor for the development of late postoperative complications (odds ratio, 3.100; 95% confidence interval, 1.519 to 6.329; p=0.002) and impaired longterm QOL improvement (odds ratio, 2.221; 95% confidence interval, 1.218 to 4.050, p=0.009). Conclusions The length of the J pouch was associated with the improvement in long-term QOL and the development of late post-IPAA complications. A long J pouch configuration could be a considerable surgical option for pouch construction.

Objective To investigate the relationships of the subsets and activation factors of peripheral blood innate lymphocytes (ILCs) with postoperative intracranial infection in patients with hypertensive basal ganglia hemorrhage. Methods A total of 105 patients with postoperative intracranial infection (infection group) and 105 patients without infection (non-infection group) were selected as research subjects. The patients in the infection group were divided into mild infection group (n=33), moderate infection group (n=50) and severe infection group (n=22) according to the severity of postoperative intracranial infection. The patients in the infection group were divided into good prognosis group (n=53) and poor prognosis group (n=52) according to different outcomes. The subsets of ILCs (ILC1, ILC2, ILC3), interleukin (IL)-12, IL-33, IL-1β levels in peripheral blood were compared among the groups. The correlation between each index and the severity of intracranial infection and the predictive value of poor prognosis in infected patients were analyzed. Results Compared with the non-infection group, the levels of ILC2 and ILC3 in the infection group were reduced, while the levels of IL-12, IL-33 and IL-1β were increased (P < 0.05). The levels of ILC2 and ILC3 in infected patients decreased with the increase of infection severity, while the levels of IL-12, IL-33 and IL-1β increased with the increase of infection severity (P < 0.05). The levels of ILC2 and ILC3 in patients with intracranial infection werenegatively correlated with the severity of infection (r=-0.721, -0.596, P < 0.001), while the levels of IL-12, IL-33 and IL-1β were positively correlated with the severity of infection (r=0.576, 0.483, 0.553, P < 0.001). The combined prediction of peripheral blood ILC2, ILC3, IL-12, IL-33 and IL-1β for poor prognosis in infected patients had a higher AUC (0.930) and specificity (84.91%) than each index alone. Conclusion The levels of peripheral blood ILC2, ILC3, IL-12, IL-33 and IL-1β in patients with postoperative intracranial infection after hypertensive basal ganglia hemorrhage are associated with the severity of infection. The combined detection of five indicators has a good predictive value for poor prognosis in infected patients.

Detailed characterizations of genomic alterations have not identified subtype-specific vulnerabilities in adult gliomas. Mapping gliomas into developmental programs may uncover new vulnerabilities that are not strictly related to genomic alterations. After identifying conserved gene modules co-expressed with EGFR or PDGFRA (EM or PM), we recently proposed an EM/PM classification scheme for adult gliomas in a histological subtype- and grade-independent manner. By using cohorts of bulk samples, paired primary and recurrent samples, multi-region samples from the same glioma, single-cell RNA-seq samples, and clinical samples, we here demonstrate the temporal and spatial stability of the EM and PM subtypes. The EM and PM subtypes, which progress in a subtype-specific mode, are robustly maintained in paired longitudinal samples. Elevated activities of cell proliferation, genomic instability and microenvironment, rather than subtype switching, mark recurrent gliomas. Within individual gliomas, the EM/PM subtype was preserved across regions and single cells. Malignant cells in the EM and PM gliomas were correlated to neural stem cell and oligodendrocyte progenitor cell compartment, respectively. Thus, while genetic makeup may change during progression and/or within different tumor areas, adult gliomas evolve within a neurodevelopmental framework of the EM and PM molecular subtypes. The dysregulated developmental pathways embedded in these molecular subtypes may contain subtype-specific vulnerabilities.
Humans ; Brain Neoplasms/pathology* ; Neoplasm Recurrence, Local/metabolism* ; Glioma/pathology* ; Neural Stem Cells/pathology* ; Oligodendrocyte Precursor Cells/pathology* ; Tumor Microenvironment

Objective:To investigate the diagnostic accuracy of serological indicators and evaluate the diagnostic value of a new established combined serological model on identifying the minimal hepatic encephalopathy (MHE) in patients with compensated cirrhosis.Methods:This prospective multicenter study enrolled 263 compensated cirrhotic patients from 23 hospitals in 15 provinces, autonomous regions and municipalities of China between October 2021 and August 2022. Clinical data and laboratory test results were collected, and the model for end-stage liver disease (MELD) score was calculated. Ammonia level was corrected to the upper limit of normal (AMM-ULN) by the baseline blood ammonia measurements/upper limit of the normal reference value. MHE was diagnosed by combined abnormal number connection test-A and abnormal digit symbol test as suggested by Guidelines on the management of hepatic encephalopathy in cirrhosis. The patients were randomly divided (7∶3) into training set ( n=185) and validation set ( n=78) based on caret package of R language. Logistic regression was used to establish a combined model of MHE diagnosis. The diagnostic performance was evaluated by the area under the curve (AUC) of receiver operating characteristic curve, Hosmer-Lemeshow test and calibration curve. The internal verification was carried out by the Bootstrap method ( n=200). AUC comparisons were achieved using the Delong test. Results:In the training set, prevalence of MHE was 37.8% (70/185). There were statistically significant differences in AMM-ULN, albumin, platelet, alkaline phosphatase, international normalized ratio, MELD score and education between non-MHE group and MHE group (all P<0.05). Multivariate Logistic regression analysis showed that AMM-ULN [odds ratio ( OR)=1.78, 95% confidence interval ( CI) 1.05-3.14, P=0.038] and MELD score ( OR=1.11, 95% CI 1.04-1.20, P=0.002) were independent risk factors for MHE, and the AUC for predicting MHE were 0.663, 0.625, respectively. Compared with the use of blood AMM-ULN and MELD score alone, the AUC of the combined model of AMM-ULN, MELD score and education exhibited better predictive performance in determining the presence of MHE was 0.755, the specificity and sensitivity was 85.2% and 55.7%, respectively. Hosmer-Lemeshow test and calibration curve showed that the model had good calibration ( P=0.733). The AUC for internal validation of the combined model for diagnosing MHE was 0.752. In the validation set, the AUC of the combined model for diagnosing MHE was 0.794, and Hosmer-Lemeshow test showed good calibration ( P=0.841). Conclusion:Use of the combined model including AMM-ULN, MELD score and education could improve the predictive efficiency of MHE among patients with compensated cirrhosis.

ObjectiveTo establish a modified BISAP scoring system， and to investigate the value of the BISAP scoring system versus the modified BISAP scoring system in assessing the severity and condition of acute pancreatitis （AP）. MethodsFor the establishment of the new scoring system， a retrospective analysis was performed for the clinical data of 1 033 patients with AP who were admitted to Third Xiangya hospital of central South University from January 2019 to December 2021， and according to the revised Atlanta classification， they were divided into mild acute pancreatitis （MAP） group with 827 patients and severe acute pancreatitis （SAP） group with 206 patients. The two groups were compared in terms of clinical features， laboratory markers， and imaging data. A binary logistic regression analysis was performed for the statistically significant indicators to screen for the independent risk factors for SAP. The receiver operating characteristic （ROC） curve was used to obtain the optimal cut－off value corresponding to the maximum Youden index for each independent risk factor， and a score of 0 or 1 was assigned depending on different situations， which was integrated into the BISAP scoring system to establish a modified BISAP scoring system. For the validation of the new scoring system， a retrospective analysis was performed for the clinical data of 473 patients with AP who were admitted to Third Xiangya hospital of central South University from January 2017 to December 2018. BISAP score and modified BISAP score were determined for each patient， and the area under the ROC curve （AUC） was used to compare the value of the two scoring systems in predicting the severity and prognosis of AP. The chi－square test or the Fisher’s exact test was used for comparison of categorical data between two groups， and the independent－samples t test and the Mann－Whitney U test were used for comparison of continuous data between two groups. ResultsFor the establishment of the new scoring system， there were significant differences between the MAP group and the SAP group in mode of admission， length of hospital stay， ICU admission rate， number of deaths， underlying diseases， and incidence rate of complications （all P<0.05）. The binary logistic regression analysis showed that body temperature， neutrophil－to－lymphocyte ratio （NLR）， C－reactive protein （CRP）， albumin， triglycerides， D－dimer， fibrinogen， and MCTSI score were independent risk factors for SAP （all P<0.05）. The ROC curve analysis showed that CRP （AUC=0.921）， NLR （AUC=0.798）， D－dimer （AUC=0.768）， and MCTSI score （AUC=0.931） had a good predictive value for SAP， and the combination of these four indicators had an AUC of 0.976 and showed a significantly higher diagnostic efficiency than each indicator alone or the combination of two or three indicators （all P<0.05）. For the validation of the new scoring system， a total of 473 patients were enrolled， with 408 in the MAP group and 65 in the SAP group， and there were significant differences between the two groups in mode of admission， length of hospital stay， ICU admission rate， number of deaths， and incidence rate of complications （all P<0.05）. The modified BISAP score was better than the BISAP score in predicting SAP （AUC： 0.972 vs 0.887， P<0.05）， with an optimal cut－off value of >3 points. The modified BISAP score also had a relatively high value in predicting the mortality of AP patients （AUC=0.910）， but there was no significant difference between the modified BISAP score and the BISAP scoring system （AUC： 0.910 vs 0.896， P=0.707）. ConclusionThe modified BISAP score is better than the BISAP score in predicting the severity of AP and has a relatively high value in predicting the mortality of AP patients， giving a more accurate， objective， and early assessment of the condition of AP patients.