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 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.

Objective To investigate the effect of preoperative carbohydrate fluid intake on postoperative insulin resistance and immune function.Methods Sixty elective gastroenteric tumor resection patients were randomly divided into test (n =30) and control (n =30) groups.Control group were fasted before surgery,while test group were given oral carbohydrate before surgery.The blood samples were collected to measure the levels of fasting blood glucose (FBG),fasting insulin (FINS),and cellular immunity (CD3 +,CD4 +,CD8 +,and CD4 +/CD8 +) before operation and 1,3,7 day postoperation,respectively.Homeostasis model assessment (HOMA) was applied to assess the status of insulin resistance.Results Compared to preoperation,the levels of CD4 +,CD4 + / CD8 +,and HOMA-IR at 1 day postoperation in both control and test groups were significantly higher (P ＜ 0.05).Compared to test group,the levels of CD4 +,CD4 +/CD8 +,and HOMA-IR at 1,3 day postoperation in control group were significantly higher (P ＜ 0.05).At the seventh day after surgery,HOMA-IR levels in the test group were returned to the preoperative level (P ＞ 0.05),while the control group was still higher than before surgery (P ＜ 0.05).There were no differences in CD4 + and CD4+/CD8 + at seventh days after surgery between two groups (P ＞ 0.05).Conclusions Preoperative carbohydrate administration may shorten the insulin resistance duration after gastrointestinal cancer surgery,reduce the intensity of insulin resistance,and improve immune function.Thus contributes to the rehabilitation of patients.

Objective: To investigate the combined preventive effect of folic acid (FA) and vitamin B12 (VB12) on the developmental toxicity of alcohol. Methods: To build animal model with the developmental toxicity by giving 5g/kg bw alcohol (25% ethanol) intragastrically (IG) to ICR mice during gestational day (GD) 6-15. In addition to alcohol, three groups were given FA 60 mg/kg bw, VB12 1 mg/kg bw, FA 60 mg/kg bw + VB12 1 mg/kg bw respectively by IG during GD1-GD16. An alcohol model group and a negative control group were set. All dams were killed at GD18. Results: Compared to the alcohol model group, the pregnant mice of FA + VB12 combined intervention group put on more weight during pregnancy; the live fetal rate; the fetal weight, body length and tail length were all increased; the abnormal ossification rate of sternum, occipital bone, and four limbs dropped (P

Objective This study was designed to explore the effects of phytoestrogen genistein on proliferation of post-menopausal osteoblast and the likely molecular mechanisms. Methods Osteoblast cultures were prepared from the upper femur of postmenopausal patients. Osteoblast proliferation was determined by Methyl Thiazolyl Tetrazolium(MTT)assay and cell cycle distribution by cytometry. The protein expressions of cyclin D and E were examined using Western-blot. Results Genistein (10~(-8) to 10~(-6) mol/L) stimulated cell viability of postmenopausal osteoblasts and increased the distribution ratio of the cells at the G2/M and S phases (P

OBJECTIVEThis study was designed to explore the effects of zinc on bone development.

METHODSForelimbs of mice with 16 d gestation were cultured by a rotating device.

RESULTSContents of OC and (45)Ca and activities of AKP in the bone tissues cultured at zinc-deficiency media and at media with 120 micro mol/L of Zn(2+) decreased significantly. Synthesis of OC, absorption of calcium and activities of AKP in the bone tissues cultures at media with 45 and 70 micro mol/L of Zn(2+) increased significantly. Radiograph of bone tissues showed that length of long bone cultured at zinc-deficiency media and at media with 120 micro mol/L of Zn(2+) shortened and their density reduced, and those cultured at media with 45 and 70 micro mol/L of zinc increased, as compared with self-control bone. Histological analysis showed the death of bone cells and loss of stroma in the bone tissues cultured at media with 120 micro mol/L of Zn(2+), and active proliferation and differentiation of bone cells, and secretion and synthesis of osteoid increased in the bone tissues cultured at media with 45 and 70 micro mol/L of zinc.

CONCLUSIONSAdequate supplementation of zinc could promote formation and development of bone tissues and deficiency or excess of zinc could alter their growth and development and normal metabolism.

Animals ; Bone Density ; drug effects ; Bone Development ; drug effects ; Calcium ; metabolism ; Embryo, Mammalian ; drug effects ; physiology ; Extremities ; embryology ; Female ; Insulin-Like Growth Factor II ; Male ; Mice ; Proteins ; metabolism ; Zinc ; pharmacology

AIM: To observe the changes of interleukin-6(IL-6), IL-8 and tumor necrosis factor-?(TNF-?) in serum and lung at different time, and the effects of anisodamine (654-2) treatment in rats with oleic acid-induced ARDS. METHODS: The ARDS model induced by intravenous injection of oleic acid in the rat was used and levels of IL-6, IL-8, TNF-? in serum and lung tissue supernatant were measured using enzyme linked immunosorbent assay (ELISA). RESULTS: Levels of serum and lung tissue IL-6, IL-8, TNF-? in oleic acid type ARDS 4 h group were increased significantly. These cytokines in oleic acid type ARDS 8 h group were lower than that of ARDS 4 h group, but serum IL-6, TNF-? and lung tissue IL-6 were still higher than that of control group . In oleic acid type ARDS 16 h group, serum IL-6, TNF-? were lower than that of the ARDS 8 h group and serum TNF-? and lung tissue IL-6 were higher than that of control group. After 654-2 treatment, the levels of serum and lung tissue IL-6, TNF-? were decreased significantly. CONCLUSION: IL-6, IL-8 and TNF-? might play important roles in the oleic acid-induced ARDS in the rat. 654-2 might alleviate ARDS by inhibiting excess production of IL-6 and TNF-?.

AIM: To observe the dynamic changes of interleukin-4(IL-4),IL-10,IL-12 in rat serum and lung tissues during acute respiratory distress syndrome (ARDS). METHODS: The ARDS model of rats was induced by intravenous injection of oleic acid. The levels of IL-4 ,IL-10,IL-12 in serum and the supernatant of lung tissues were measured by enzyme linked immunosorbent assay (ELISA). RESULTS: The Levels of serum and lung IL-10,IL-12 in ARDS rats were increased in 4 h ,8 h,16 h group compared with control group . The levels in IL-10 in serum in 16 h group and IL-10 in lung tissues of 8 h group were lower than that in 4 h group. The Levels of IL-4 in serum in 4 h, 8 h group were higher than that in control group , while IL-4 in 16 h group was lower than that in 8 h group. IL-4 of lung tissues in 4 h,8 h,16 h group were increased significantly,but in 16 h group were lower than that in 8 h group. The biggest changes of pulmonary coefficient and histopathology were observed at 4 h after injection of oleic acid. CONCLUSIONS: IL-4,IL-10 and IL-12 might play important roles in inflammatory reaction induced by oleic acid. The pro- and anti-inflammatory cytokines produced successively during ARDS. The relationship between unbalanced cytokines and lung injury in ARDS needs to be further studied.

Objective To study the effects of dietary estrogens genistein(GS)and zearalenone(ZEA)on apoptosis in-duced by estrogen depletion in PEO4cells.Methods The monolayer ovarian cancer cell line PEO4cells were cultured in DMEM medium containing10%bovine serum.Before the addition of the testing compounds the cells were washed in phosphate-buffered saline and the medium was displaced with a phenol red-free DMEM medium containing5%dextral charcoal-stripped FBS and the cells were cultured for5days in order to exhaust the estrogen stored in the cells,and then cells were divided into5groups,including solvent control group,estrogen control group,anti-estrogen control group and2experimental groups.After treatment the apoptotic features of the cells were observed by cellular morphology,DNA fragmentation and location and height of cell hypodiploid were indicated by flow cytometry.Results The typical characteristics of apoptosis in PEO4cells were observed after estrogen deletion and then disappeared following exposure of the PEO4cells to32?10 -9 mol/L and96?10 -9 mol/L ZEA for72hrs.32?10 -6 mol/L and96?10 -6 mol/L GS could significantly aggravate apoptosis in PEO4cells.Conclusion Zearalenone is a kind of mycoestrogen that has estrogenic activity to inhibit apoptosis in PEO4cells.Genistein is a kind of phytoestrogen that has anti-estrogen activity(tamoxifen-like)to promote apoptosis in PEO4cells with the high doses range.