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.

Purpose: Most lung adenocarcinoma (LUAD) patients are diagnosed at the advanced stage and have poor prognosis. DNA methylation plays an important role in the prognosis prediction of cancers. The objective of this study was to identify new DNA methylation sites as biomarkers for LUAD prognosis. Materials and Methods: We downloaded DNA methylation data from The Cancer Genome Atlas data portal. Cox proportional hazard regression model and random survival forest algorithm were applied to identify the DNA-methylation sites. Methylation of sites were validated in the Gene Expression Omnibus cohorts. Function annotation were done to explore the biological function of DNA methylated sites signature. Results: Six DNA methylation sites were identified as prognosis signature. The signature yielded acceptable discrimination between the high-risk group and low-risk group. The discrimination effect of this DNA methylation signature for the OS was obvious, with a median OS of 21.89 months vs. 17.74 months for high-risk vs. low-risk groups. This prognostic prediction model was validated by the test group and GEO dataset. The predictive survival value was higher for the prognostic prediction model than that for the tumor node metastasis stage. Adjuvant hemotherapy could not affect the prediction of the signature. Functional analysis indicated that these signature genes were involved in protein binding and cytoplasm. Conclusion We identified the prognostic signature for LUAD by combining six DNA methylation sites. This could service as potential robust and specificity signature in the prognosis prediction of LUAD.

OBJECTIVETo evaluate the toxic effects of mixture of volatile organic compounds (VOCs) on Mice Testis related enzymes and hormones.

METHODSAfter determining the median lethal dose (LD₅₀) of VOCs using the acute toxicity test, 40 male clean inbred Kunming mice were assigned to 1/8 LD₅₀ VOCs exposure group, 1/4 LD₅₀ VOCs exposure group, and 1/2 LD₅₀ VOCs exposure group, as well as positive control group with cyclophosphamide (60 mg/kg) and negative control group with tea oil, with 8 mice in each group. The mice were intraperitoneally injected with respective agents for 5 days. The levels of testis testosterone, estradiol, follicle stimulating hormone, and luteinizing hormone were determined by ELISA. Meanwhile, the activity of testicular marked enzymes such as lactate dehydrogenase, gamma-glutamyl transpeptidase, acid phosphatase, and glucose-6-phosphate dehydrogenase were examined.

RESULTSCompared with the negative control group, the 1/8 LD₅₀ exposure group had a significantly increased testis coefficient (P<0.05). Both the activity of testicular marked enzymes and the levels of testicular sex hormones in all exposure groups showed significant downward trends with increasing VOC doses compared with those in the negative control group (P<0.05).

CONCLUSIONVOCs have obvious toxicity to mouse testis by changing the levels of testicular sex hormones and the activity of testicular marked enzymes.

Animals ; Estradiol ; chemistry ; Follicle Stimulating Hormone ; chemistry ; Gonadal Steroid Hormones ; chemistry ; Luteinizing Hormone ; chemistry ; Male ; Mice ; Testis ; chemistry ; drug effects ; Testosterone ; chemistry ; Volatile Organic Compounds ; toxicity

BACKGROUNDMonoclonal antibodies (mAbs) such as DD3, raised against progastrin-releasing peptide(31-98) (ProGRP (31-98)) antigen, have been used to target small cell lung cancer (SCLC). However, as an intact mAb, DD3 is cleared slowly from the body, with an optimal radioimmunoimaging time of 72 hours. More recently, a single-chain antibody fragment has demonstrated reduced excretion time in blood and normal tissues and is increasingly used in diagnostic cancer research. Thereby, it potentially increases the radioimmunoimaging efficacy. However, there have been few studies with this antibody fragment. The aim of this study was to characterize the preliminary radioimmunoimaging and biodistribution of (131)I-anti-ProGRP(31-98) scFv in nude mice bearing SCLC xenografts.

METHODSAnti-ProGRP(31-98) scFv was used to detect ProGRP expression by flow cytometry analysis and immunohistochemistry. (131)I-anti-ProGRP(31-98) scFv was injected intravenously into healthy Kunming mice and the percentage injected dose per gram (%ID/g) in various organs was calculated. Similarly, the %ID/g and tumor/non-tumor ratio in xenograft-bearing mice was calculated. After injection of (131)I-anti-ProGRP(31-98) scFv, treated mice were imaged at 1, 24, and 30 hours. Then the tumor/base ratios were calculated.

RESULTSProGRP was highly expressed in NCI-H446 cells and xenograft tissue. The metabolism of (131)I-anti-ProGRP(31-98) scFv in healthy mice was consistent with a first-order and two-compartment model; T1/2α and T1/2β were 10.2 minutes and 5 hours 18 minutes, respectively. The %ID/g of (131)I-anti-ProGRP(31-98) scFv in xenografts was much higher than in healthy tissues at 12 hours after injection, reaching a maximum of (5.38±0.92) %ID/g at 24 hours. Successful imaging of xenograft tissue was achieved as early as 1 hour post-injection and persisted until 30 hours, with 24 hours proving optimal.

CONCLUSION(131)I-anti-ProGRP(31-98) scFv shows highly selective tumor uptake with low accumulation in normal tissues and rapid blood clearance, indicating that it could be a promising agent for SCLC radioimmunoimaging.

Animals ; Female ; Flow Cytometry ; Humans ; Immunoglobulin Fragments ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Peptide Fragments ; immunology ; Radioimmunodetection ; methods ; Recombinant Proteins ; immunology ; Small Cell Lung Carcinoma ; diagnostic imaging ; metabolism ; Xenograft Model Antitumor Assays

Objective To explore the clinical value of dual-phase 99Tcm-MIBI scintigraphy in the localization and diagnosis of secondary hyperparathyroidism (SHPT).Methods A total of 20 patients (8 males,12 females; average age 49.6 years) with uremic SHPT who underwent parathyroidectomy from 2010 to 2013 were retrospectively analyzed.All patients underwent 99Tcm-MIBI SPECT/CT and 19 underwent color Doppler ultrasonography (CDUS).Post-excisional histopathology was considered as the gold standard.The diagnostic efficacies of 99Tcm-MIBI and CDUS for SHPT were calculated.The correlation between T/NT ratio in delayed imaging and the volume of excised parathyroid and the intact PTH (iPTH) were analyzed.x2 test,Pearson or Spearson correlation analysis were used to analyze the data.Results The sensitivity,specificity,positive predictive value,negative predictive value and accuracy of 99Tcm-MIBI SPECT/CT and CDUS in the diagnosis of SHPT were 66.67％ (44/66),100％(14/14),100％ (44/44),38.89％(14/ 36),72.50％ (58/80) and 78.19％(43/55),52.38％(11/21),81.13％(43/53),47.83(11/23),71.05％ (54/76),respectively.There were significant differences in specificity and positive predictive value (x2 =9.33,9.26,both P＜0.05),but no significant differences in the sensitivity,negative predictive value and accuracy (x2 =1.97,0.04,0.46,all P＞0.05).T/NT ratio correlated with serum iPTH and parathyroid volume (r=0.638,rs =0.571,both P＜0.05).Conclusions The specificity of 99Tcm-MIBI SPECT/CT is superior to CDUS in the diagnosis of SHPT.Dual-phase 99Tcm-MIBI SPECT/CT could locate the hyperfunctional parathyroid gland and provide the basis for surgical treatment.

Objective To study the serum levels of progastrin-releasing peptide (pro-GRP) and neuron-specific enolase (NSE) for the clinical diagnosis,therapy monitoring and survival time analysis of small cell lung cancer (SCLC).Methods All 41 SCLC samples (30 males,11 females,age range from 46 to 78 years),95 NSCLC samples (55 males,40 females,age range from 42 to 88 years),and 127 normal individuals samples (80 males,47 females,age range from 35 to 78 years) which were diagnosed by People's Hospital of Zhengzhou from May 1,2008 to April 30,2011 were collected.Serum levels of pro-GRP,NSE and their changes in SCLC patients before and after therapy were evaluated.ANOVA analysis,randomized block design analysis of variance and the log-rank test were collected SPSS 16.0 to evaluate the survival time.Results The serum levels of pro-GRP (median 357.8 ng/L) and NSE (median 89.5 μg/L) in SCLC group were significantly higher than those in the NSCLC group (pro-GRP:39.9 ng/L;NSE:11.43 μg/L) and normal individuals group (pro-GRP:12.7 ng/L;NSE:10.03 μg/L) (P=0.000).The sensitivity of pro-GRP and NSE for the diagnosis of SCLC were 80.4％ and 78.0％,while the specificity were 92％ and 87％,respectively.There is a poor correlation between pro-GRP and NSE serum levels,but when combined the sensitivity can be 95％ and specificity can be 85％.Significantly statistical difference of pro-GRP levels was observed in the different stages of treatment (before and after therapy) in SCLC-LD patients (F =3.53,P =0.038),and significant statistical difference of NSE levels was also observed in SCLC-ED patients in different stages (F =16.049,P =0.000).In partied response SCLC patients,the group with NSE level lower than cut-off value had longer survival time than the other group with NSE level higher than cut-off value (P =0.001).Conclusions The sensitivity of the combined analysis of pro-GRP and NSE is better than single marker for the diagnosis of SCLC.The serum level of pro-GRP has better correlation with therapeutic effect of SCLC-LD patient than NSE.The serum level of NSE are well correlated with therapeutic effect in SCLC-ED patients.There are some certain value of NSE level for evaluation the survival time of SCLC patients who were in partial response.

ObjectiveTo study the biologic distribution of 131I-Herceptin in BALB/c-neu nude mice bearing HER-2 positive SK-BR-3 human breast cancer xenografts and the radioimmunoimaging characteristics of nude mouse bearing human SK-BR-3 breast cancer xenografts. MethodSK-BR-3 breast cancer cells were implanted subcutaneously to athymic mice to establish animal model.Tumor bearing mice were continuously imaged with SPECT. The radiocounting per minute (cpm) of different organ on a γ-arithmometer was measured at 4,12,24,48 h postinjection of 131I-Herceptin or 131I-mlgG,and the T/NT ratios and the uptake percentages per gram of the injection dose (％ ID/g) was gained. ResultsModel was established in 96％ nude mouse.Compared with the control group,there was a significantly stronger contrast enhancement of tumor imaging,bigger T/NT and ％ ID/g in experimental group ( P ＜ 0.0l ).Conclusions 131I-Herceptin concentrates obviously in implanting tumor tissues of nude mouse,hence it is a good radiopharmaceutical agent targeting SK-BR-3 xenografts.

23 cases of the patients with supraorbital neuralgia were treated by puncturing Yangbai (GB 14) toward Yuyao (Extra), Zanzhu (BL 2), Taiyang (Extra), Touwei (ST 8),Zhongzhu (TE 3) and Neiting (ST 44) on the sick side, plus laser radiation on a site about 1 cm apart from the midpoint of the eyebrow of the sick side. After 10 treatments, the results showed cure in 19 cases and remarkable effect in 4 cases.

Objective:To label monoclonal anti-GPⅢa antibody (SZ21) with 99 Tcm and evaluate the value of 99 Tcm-SZ-21 for detecting thrombi in vivo.Methods:SZ-21 was modified with 2-iminotholane and labeled with 99 Tcm-GH and radiochemical purity was determined by ITLC-SG.99Tcm-SZ-21 was injected via ear edge vein in 5 rabbits in which thrombi were induced in the right femoral arteries.As control,99 Tcm-GH was administered in 1 rabbit.Static images were acquired and irregularly shaped ROIs were drawn on the images to calculate the ratios of T/M and T/B.Vein blood was drawn at 2 min,5 min,10 min,30 min and 1 h-3 h after injection in 2 rabbits so as to observe the blood clearance of 99 Tcm-SZ-21.Rabbits were sacrificed after 3 h of imaging.The vessels including clots were harvested and imaged.Cardiac muscle,liver,spleen,lung,kidney,etc.were excised and weighed.Radioactivity counts were measured to calculate % ID/g.Results:The radiochemical purity of 99 Tcm-SZ21 was beyond 90% and stable in vitro.Thrombi could be visualized at 30 min after injection,and at 2 h image of thrombi was clearly visualized,T/M (2.55?0.72) and T/B (1.94?0.51) ratios were high.In vitro imaging showed that T/B was 4.43?1.5.Conclusion:99Tcm-SZ-21 could be a potential agent for imaging diagnosis of thrombotic disease.