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 design and synthesize 89Zr-deferoxamine(DFO)-G4C2, a novel molecular probe targeting programmed cell death receptor 1(PD-1), and evaluate its in vivo biodistribution and microPET/CT imaging characteristics in tumor-bearing mice. Methods:DFO-G4C2 was prepared by coupling DFO with G4C2, a monoclonal antibody targeting PD-1. The affinity and binding specificity of this amalgamation were subsequently assessed through the implementation of flow cytometry and surface plasmon resonance techniques. The molecular probe 89Zr-DFO-G4C2 was achieved by labeling DFO-G4C2 with the radioisotope 89Zr, and the labeling efficiency and in vitro stability of 89Zr-DFO-G4C2 were determined. Mouse models laden with CT26 colorectal cancer cells expressing PD-1 were established, followed by in vivo biodistribution and microPET/CT imaging studies, to explore the potential clinical value of 89Zr-DFO-G4C2. Additionally, the validity of this molecular probe was verified in 4T1 breast cancer models, affirming its efficacy as an imaging tool across different tumor models. Independent-sample t test was used to analyze the data. Results:DFO-G4C2 exhibited an affinity constant KD of (0.55±0.02) μmol/L, indicating a strong binding affinity. The binding rate to mouse PD-1 protein was determined to be (61.82±8.49)%. The labeling rate of 89Zr-DFO-G4C2 reached a high level of (98.76±0.51)%. Furthermore, the labeling rates in lysate and human serum after 144 h were measured to be (93.07±2.16)% and (83.42±3.21)%, respectively. MicroPET/CT imaging of CT26 tumor-bearing mice injected with 89Zr-DFO-G4C2 showcased pronounced radioactivity uptake in the tumor tissue. At 72 h post-injection, the tumor uptake value reached (10.47±0.34) percentage activity of injection dose per gram of tissue (%ID/g). The tumor uptake observed in the blocked experimental group, wherein an excess of unlabeled antibody was administered, was significantly lower at (6.26±1.03) %ID/g in comparison to the non-blocked group ( t=6.67, P=0.003). The in vivo biodistribution results were consistent with the observed microPET/CT imaging outcomes. MicroPET/CT imaging observations in the 4T1 breast cancer bearing mouse model were analogous to those obtained from the CT26 model. Conclusion:ImmunoPET based on the 89Zr-DFO-G4C2 molecular probe can non-invasively and visually assess the PD-1 expression level of tumors in vivo, and it is expected to be a new molecular imaging technique for immunotherapy monitoring of PD-1 inhibitors.

Objective:To investigate the perioperative bleeding of patients undergoing transurethral plasma kinetic prostatectomy (TUPKP) and analyze the related influencing factors.Methods:Retrospectively analyzed the clinical data of 206 patients with TUPKP admitted to Xi′an Lintong District People′s Hospital from March 2021 to March 2024, all patients were aged from 51 to 83 years old, with an average of (64.33±9.74) years; patients′ course of disease was 1 to 7 years, averaged (4.57±1.59) years. and the perioperative bleeding of patients was observed and divided into bleeding group ( n=108) and non-bleeding group( n=98). By comparing the general data and laboratory data between groups, the influencing factors of perioperative bleeding in TUPKP patients were analyzed by multivariate Logistic regression. Results The proportion of diabetes and postoperative constipation in bleeding group was higher than that in non-bleeding group, and the prostate volume and operation time were higher than that in non-bleeding group ( P<0.05). Serum calcium, platelet count (PLT) and activated partial thromboplastin time (APTT) in bleeding group were lower than those in non-bleeding group ( P<0.05). Multivariate Logistic regression analysis showed that diabetes ( OR=4.283), prostate volume ( OR=1.111), operation time ( OR=1.091) and postoperative constipation ( OR=4.144) were the risk factors for perioperative bleeding in TUPKP patients ( P<0.05). Serum calcium ( OR=0.001) and PLT ( OR=0.980) are the protective factors for perioperative bleeding in TUPKP patients ( P<0.05). Conclusion:The rate of perioperative bleeding in TUPKP patients is of great risk. Diabetes, prostate volume, operation time and postoperative constipation are its risk factors, and serum calcium and PLT are its protective factors.

Oxidative stress, due to the disruption of the balance between reactive oxygen species (ROS) generation and the antioxidant defense system, plays an important role in the pathogenesis of rheumatoid arthritis (RA). Excessive ROS leads to the loss of biological molecules and cellular functions, release of many inflammatory mediators, stimulate the polarization of macrophages, and aggravate the inflammatory response, thus promoting osteoclasts and bone damage. Therefore, foreign antioxidants would effectively treat RA. Herein, ultrasmall iron-quercetin natural coordination nanoparticles (Fe-Qur NCNs) with excellent anti-inflammatory and antioxidant properties were constructed to effectively treat RA. Fe-Qur NCNs obtained by simple mixing retain the inherent ability to remove ROS of quercetin and have a better water-solubility and biocompatibility. In vitro experiments showed that Fe-Qur NCNs could effectively remove excess ROS, avoid cell apoptosis, and inhibit the polarization of inflammatory macrophages by reducing the activation of the nuclear factor-κ-gene binding (NF-κB) pathways. In vivo experiments showed that the swollen joints of mice with rheumatoid arthritis treated with Fe-Qur NCNs significantly improved, with Fe-Qur NCNs largely reducing inflammatory cell infiltration, increasing anti-inflammatory macrophage phenotypes, and thus inhibiting osteoclasts, which led to bone erosion. This study demonstrated that the new metal-natural coordination nanoparticles could be an effective therapeutic agent for the prevention of RA and other diseases associated with oxidative stress.

Objective:To explore the potential clinical value of 99Tc m-methoxyisobutylisonitrile(MIBI) SPECT/CT muscle imaging in the diagnosis of cervical dystonia (CD). Methods:From January 2021 to April 2022, 50 patients with CD (14 males, 36 females; age (45.8±12.5) years) who were treated in Second Affiliated Hospital of Soochow University were prospectively included. The 99Tc m-MIBI SPECT/CT muscle imaging results of 400 pieces of muscle (bilateral sternocleidomastoid, musculus scapulae, splenius capitis and musculus trapezius; each of 100 pieces) in 50 patients were analyzed and divided into the dystonic muscle group and normal muscle group according to the electromyography (EMG). Toronto western spasmodic torticollis rating scale (TWSTRS) score, SUV max and target-to-background ratio (TBR) of single superficial cervical muscle and total cervical muscle, and EMG diagnosis results were obtained before botulinum toxin injection. ROC curves of SUV max and TBR of dystonic muscles were constructed to determine AUCs and the difference was compared by Delong test. Differences of SUV max and TBR between 2 groups were analyzed by Mann-Whitney U test. Spearman rank correlation analysis was used to analyze the correlation of SUV max, TBR and TWSTRS scores of total cervical muscle. Results:There were 205 pieces of muscle in dystonic muscle group and 195 pieces of muscle in normal muscle group. The uptake of 99Tc m-MIBI in dystonic muscle was significantly increased in CD patients, and the non-whole uptake of 99Tc m-MIBI was increased in some dystonic muscles, which was manifested as uneven uptake of the whole muscle. The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of visual analysis were 95.12%(195/205), 75.90%(148/195), 85.75%(343/400), 85.58%(195/242) and 93.67%(148/158), respectively. There were significant differences of SUV max (1.74(1.42, 2.12) vs 0.92(0.81, 0.99)) and TBR (2.55(1.92, 3.27) vs 1.44(1.22, 1.73)) between the dystonic muscle group and the normal muscle group ( z value: -15.29, -12.69, both P<0.001). The diagnostic efficiency of SUV max in dystonic muscle was better than TBR (AUC: 0.942 vs 0.867; z=5.03, P<0.001). SUV max, TBR and TWSTRS score in the neck muscles of patients with CD showed positive correlation ( rs values: 0.44, 0.45, both P<0.001). Conclusion:99Tc m -MIBI SPECT/CT muscle imaging is a good diagnostic method for dystonic muscle in patients with CD.

Objective To investigate the correlation between serum lectin like oxidized low density lipoprotein receptor-1 ( sLOX-1 ) and left ventricular diastolic function in patients with essential hypertension. Methods From January 2016 to July 2017, one hundred and forty-six patients with essential hypertension were selected and divided into two groups according to the ratio of E/ A,the left ventricular diastolic function group (76 cases) and the left ventricular diastolic function group (70 cases),the sLOX-1 level of the patients was detected by ELISA method, and the left ventricular diastolic function was evaluated by echocardiography. The correlation between sLOX-1 and left ventricular diastolic function was analyzed. Results The serum sLOX-1 levels in left ventricular diastolic function group (( 208. 12 ± 13. 48 ) μg/ L ) were significantly higher than those inthe left ventricular diastolic function group ((152. 12 ± 12. 96) μg/ L) . The difference between the two groups was statistically significant (t= 6. 586,P= 0. 000). Logistic regression analysis showed that serum sLOX-1 was a influence factor of left ventricular diastolic function (OR= 2. 42,95%CI 1. 42-2. 82,P = 0. 036) . Conclusion The levels of serum sLOX-1 can be used as a indicator of left ventricular diastolic function.

Objective To analyze Clopidogrel Resistance (CR) and influencing factors of coronary heart disease (CHD) with diabetes (DM) patients and evaluatc the relationship of CR and major adverse cardiovascular events (MACE) and readmission of CHD with DM patients.Methods 270 CHD patients were enrolled.Clinical conditions of CR were measured by adenosine diphosphate (ADP) induced maximum platelet aggregation rate (MPAR).After 1-year follow-up,MACE events and rehospitalization were recorded.Results CR of NDM and DM patients were 45 (33.1％) and 78 cases (58.2％) respectively,and the difference was significant (P ＜ 0.001).Factors of CR of CHD DM patients included heart rate,TG level,the number of severe coronary artery disease.MACE events of CS and CR patients were 35 (23.8％) and 47 patients (38.2％) respectively,and the difference was significant (P =0.010).The readmitted patients of CS and CR groups were 15 cases (10.2％) and 27 patients (22.0％) respectively,and the differcnce was significant (P =0.008).The MACE of CR and CS patients in DM group were 32 (41.0％) and 12 cases (21.4％) respectively,and thc difference was significant (P ＜ 0.05).The Readmitted cases of CR and CS patients in DM group were 19 (24.4％) and 5 (8.9％) respectively,and the diffcrcnce was significant (P ＜ 0.05).Conclusions CR of CHD DM patients increased significantly.The influencing factors of CR of CHDDM are including heart rate,TG level,the number of severe coronary artery disease.MACE events and rehospitalization rate were significantly increased in CHD patients with DM AR.Therefore,it should be further strengthened the anti-platelet therapy for CHD patients with DM.

Objective To analyze the dTP value in the patients with coronary heart disease (CHD) complicating diabetes mellitus (DM) and its relationship with major adverse cardiovascular events (MACE) and rehospitalization.Methods Two hundreds and seventy CHD patients were selected as the research subjects,including 136 cases of non-MD and 134 cases of DM.Their clinical condition was recorded.The indicators such as height,body mass,blood pressure and heart rate were measured.ECG,echocardiography,coronary angiography and other examiantions were carried out.The various indicators were detected.11-dh-TXB2 and 6-k-PGF1a levels were detected in the two groups and then dTP value was calculated.The 1-year follow-up was performed,MACE and rehospitalization were recorded.Epdate software was used for building a database and SPSS 17.0 software was applied for conducting the statistical analysis.Results The dTP level in the f non-DM and DM patients were 1.8 ± 0.6 and 2.0 ± 0.7 respectively,the difference was statistically significant (P＜ 0.05).For the non-DM CHD group,hs-CRP,systolic blood pressure,diastolic pressure,lesions number and severe lesions number were correlated with dTP level(P＜0.05).For the complicating DM CHD group,hs CRP,blood glucose,CHO level,lesions number and severe lesions number were correlated with dTP level(P＜0.05).After 1-year follow-up,MACE had 33 cases (24.3％) in the non-DM group and 44 cases (32.8％) in the DM group respectively,the difference was not statistically significant (P＞0.05).The rehospitalized cases had 12 cases (8.8％) in the non-DM group and 24 cases (17.9 ％).in the DM group respectively,the difference was statistically significant (P＜ 0.05).The dTP levels of MACE occurrence and non-MACE occurrence were 2.3 ± 0.8 and 1.8 ± 0.6 respectively,the difference was statistically significant (P＜0.05).The dTP levels of rehospitalized patients and non-rehospitalized patients were 2.4 ± 1.0 and 1.9 ±-0.6 respectively,the difference was statistically significant(P＜0.05).Conclusion The dTP level in the patients with CHD complicating DM is significantly increased,suggesting that platelet is obviously activated,moreover higher dTP level increases the risk of MACE and rehospitalization.So the anti-platelet therapy should be strengthened.

Objective: To investigate the mechanism of cleft palate in mice induced by excessive all-trans retinoic acid (atRA). Methods: The pregnant mice were randomly divided into atRA-treated group (n=27) and control group (n=27). atRA-treated group was given by gavage a single dose of atRA (100 mg/kg) at 8: 00 AM on gestation day 10 (GD10) and the control group was given by gavage the isopyknic corn oil. At GD13-GD15, the fetal mice palate development was observed by HE staining. The mouse embryonic palatal mesenchymal cell proliferation was detected by 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry. The expressions of Smad2, phospho-Smad2 (p-Smad2), Smad4 and Smad7 in mouse embryonic palatal mesenchyme were analyzed by Western blotting. Results: At GD13-GD15, compared with the control, the ratio of BrdU-positive cells in the palatal mesenchyme of atRA-treated fetuses decreased significantly (P<0.05), especially at GD14, atRA inhibition rate was (65.4±1.7)%. Moreover, atRA decreased the levels of p-Smad2 and Smad4 in embryonic palate mesenchymal cells, whereas the expression of Smad7 was significantly increased at GD14 and GD15. Conclusions atRA may lead to cleft palate by inhibiting the activation of Smad signaling pathway and affecting the proliferation of palatal mesenchymal cells.