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Objective To investigate how the m6A methylation enzyme Methyltransferase like protein 16(METTL16) exerts its effects on the proliferation, migration and invasion of hepatocellular carcinoma(HCC) cells HepG2 and HCC-LM3, and to further explore the underlying molecular mechanism. Methods The overexpression and RNA interference vectors targeting METTL16 were transfected into HepG2 and HCC-LM3 cells and screened the stable cell lines by purimycin. The expressions of METTL16 were detected by means of qRT-PCR and Western blot assay; in HCC cell lines, Cell counting kit-8(CCK-8), Transwell assays, and flow cytometry were used to observe the effects in the proliferation, migration, invasion and cell cycle after transfection; Western blot assay was used todetect the effect of expression of VEGFA-VEGFR2 pathway-related proteins in hepatocellular carcinoma cells; Gene Expression Omnibus database was used to analyzethe expression levels of METTL16 in human liver cancer tissues and paraneoplastic tissues.Log-rank test was used to compare the clinic pathological characteristics between patients with high and low expression of METTL16 in hepatocellular carcinoma. Results Western blot and real-time quantitative PCR experiments showed that METTL16 overexpressing cell lines and interfering cell lines were successfully constructed in HepG2 and HCC-LM3 cells. CCK-8, Transwell and flow cytometry results showed that overexpression of METTL16 resulted in a decrease in the number of proliferating, migrating and invasive cells, and the number of cells in G2/M phase proportion increased. Western blot showed that overexpression of METTL16 inhibited the expression of VEGFA-VEGFR2 pathway-related proteins VEGFR2, p-AKT, Cyclin B, and CDK1 in HepG2 cells, but knockdown of METTL16 reversed the inhibition effect on these proteins.Compared to the matched non-tumor liver tissues, METTL16 was downregulated in HCC tissues; however, the levels of METTL16 were not significantly associated with the clinic pathological characteristics of HCC patients. Conclusion METTL16 may inhibit the proliferation, migration and invasion of HCC cells by inhibiting the VEGFR2 pathway.

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Objective To explore the mechanism of motor symptoms in Parkinson′s disease(PD) aggravated by decreased glial cell line-derived neurotrophic factor(GDNF) in striatum. Methods Male C57/BL mouse(6-8 weeks), were administered of PBS, AAV-GDNF or AAV-shGDNF in striatum by brain stereoscopic injection, combinated with sub-acute PD model, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) 30 mg/(kg·d)was administered through intraperitoneal injection for five consecutive days; subsequently, mice were randomly divided into PBS group, negative control(NC) group, AAV-shGDNF group, MPTP group, MPTP+AAV-GDNF group, MPTP+AAV-shGDNF group. Behavior tests(rotarod, pole and field) were applied for assessing the motor ability of mice; ELISA kit was used to detect striatal glutamic acid(Glu) content; Western blot and other techniques were carried out to detect the expression and distribution of GLAST, GLT-1 and GluN2 B in striatum; TUNEL staining was applied for observing the apoptosis of neurons in striatum. Results Compared with the NC group,the mice of the AAV-sh GDNF group,with down-regulation of GDNF in striatum,had poor motor ability,decreased Glu transporter( GLAST and GLT-1),and increased Glu content. Compared with the PBS group,the mice of the MPTP group had increased Glu content and decreased Glu N2B in striatum. Compared with the MPTP group,the mice of the MPTP + AAV-GDNF group showed enhanced motor ability,along with decreased Glu content,increased Glu N2B and less neurons apoptosis in striatum; while,the mice of the MPTP + AAV-GDNF group showed worse motor ability,along with augmented Glu content,reduced Glu N2B and more neurons apoptosis in striatum. Conclusion In PD pathological process,decreased striatal GDNF may promote the neurons apoptosis by enhancing Glu excitotoxicity,thereby leading to the aggravation of motor symptoms.

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Objective To investing ate the milk-derived hexapeptide PGPIPN and its truncated pentapeptide PGPIP to alleviate chronic alcoholic liver injury in mice and its associated molecular mechanisms. Methods Sixty Kunming mice were randomly divided into control group, model group, GSH group, PGPIPN group, and truncated pentapeptide PGPIP group. The model of chronic alcoholic liver injury in mice was established by gavage with gradient alcohol. Drug intervention was given at the same time for 12 weeks. Liver HE staining was used to analyze the pathological effects of each treatment group on alcoholic liver injury in mice. Primary mouse hepatocytes and human normal hepatocyte line L-02 were isolated and culturedin vitro. The appropriate PGPIPN induction concentrations of both cells were determined by WST-1 method. L-02 cells were induced at different times. The expression of FoxO3 a and phosphorylated FoxO3 a protein were detected by Western blot to determine the appropriate induction time. The subcellular localization of FoxO3 a in L-02 cells was detected by cellular immunofluorescence. The mRNA changes of FoxO3 a and MnSOD genes in primary hepatocytes and L-02 cells of mice in different treatment groups were detected by qRT-PCR. Results The pathological examination of PGPIPN group and PGPIP group was similar to that of GSH group, and the liver injury of mice was significantly reduced. Medium and high concentrations of PGPIPN were respectively selected to induce mouse primary hepatocytes and L-02 cells. At 16 hours, the expression of FoxO3 a protein in L-02 cells increased significantly. FoxO3 a protein was mainly expressed in the nucleus. In addition, mRNA levels in both types of cells increased significantly after induction with the corresponding dose of PGPIPN. Conclusion PGPIPN and truncated pentapeptide PGPIP can reduce chronic alcoholic liver injury in mice. The mechanism may be to reduce alcohol-induced oxidative stress through FoxO3 a-MnSOD signaling pathway.

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Objective To investigate the role of N-methyladenosine(m6A) demethylase ALKBH5 in the proliferation and activation of cardiac fibroblasts(CFs) in rats. Methods The CFs taken from SD rats in 1 to 3 days were isolated by differential adhesion and observed under microscope. After cells were adherently grown to appropriate density, the cells were induced by TGF-β1 for modeling. The model cells were divided into the overexpression of ALKBH5 group infected by lentivirus and the negative control group for 24-48 hours. RT-qPCR was used to detect mRNA expression of ALKBH5, α-smooth muscle actin(α-SMA), type I collagen(Collagen Ⅰ) and proliferating cell nuclear antigen(PCNA). The expression of ALKBH5、α-SMA、Collagen Ⅰ and PCNA were assayed by Western blot. The cell proliferation activity was tested by CCK-8 assay and EdU. Results Compared with the control group, the protein and mRNA of ALKBH5 were reduced in the model group active by TGF-β1. Meanwhile, the biomarkers of activation, such as PCNA,α-SMA and Collagen Ⅰ, increased significantly. Besides, the protein and mRNA of PCNA、α-SMA and Collagen Ⅰ were lower in overexpression of ALKBH5 group than those of the negative control group. CCK-8 assay and EdU suggested that the proliferation viability of CFs was reduced evidently in overexpression of ALKBH5 group, compared with the negative control group. Conclusion Overexpression of ALKBH5 can inhibit the proliferation of CFs, suggesting that ALKBH5 may be a key regulatory point in the development of myocardial fibrosis.

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Objective To explore the effect of liver-specific knockout of transcription factor EB(Tfeb) on high-fat diet(HFD)-induced hepatic steatosis in mice. Methods Wild-type C57 BL/6 J mice and liver-specific Tfeb knockout C57 BL/6 J mice were fed with HFD or normal chow for 12 weeks, respectively, and then the serum triglyceride(TG), total cholesterol(TC), monocyte chemoattractant protein-1(MCP-1), tumor necrosis factor-α(TNF-α), aspartate aminotransferase(AST) and alanine transaminase(ALT) were measured in each group of mice; Western blot was used to detect Tfeb protein expression levels in liver tissues of mice in each group, HE staining was used to monitor histopathological changes in liver tissues of mice in each group, oil red O staining was used to monitor lipid deposition in liver tissues of mice in each group, and F4/80 fluorescence staining was used to monitor macrophage infiltration in liver tissues of mice in each group. Results There was no expression of Tfeb gene in liver of liver-specific knockout Tfeb mice, suggesting that the effect of Tfeb gene knockout in liver tissue was better. Compared with the normal control group, the expression of Tfeb protein in the liver tissue of the model control group was down-regulated.Compared with the normal control group, both the HE staining results and the oil red O staining results showed that the liver specific Tfeb caused lipid deposition and liver lobule disorder in mice, which was similar to the liver changes in the model control mice, however, liver-specific knockout of Tfeb mice at 12 weeks of HFD had more severe liver lipid deposition and hepatic lobular structural disorder. The results of F4/80 fluorescence staining indicated that the specific knockout of liver Tfeb could aggravate the infiltration of macrophages in the liver of mice induced by high-fat feeding. At the same time, the serological test results indicated that compared with the normal control group, the serum levels of TC, TG, TNF-α, MCP-1, AST and ALT in the liver-specific knockout Tfeb group and the model control group increased, and these changes were further elevated in Tfeb knockout mice after HFD feeding. Conclusion Liver-specific knockout of Tfeb aggravates HFD-induced hepatic steatosis in mice.

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Objective To investigate the role of endoplasmic reticulum stress in hepatic insulin resistance induced by intermittent hypoxia in rats. Methods Twenty-four SD rats were randomly divided into control group(NC group) and intermittent hypoxia group(CIH group). The NC group was placed in a normoxia environment for 12 weeks, and the CIH group was given intermittent hypoxia for 8 weeks, and then returned to normoxia until the 12 th week. In both groups, fasting blood glucose(FBG), fasting insulin(FINS), and liver inositol-requiring enzyme-1α(IRE1α), X-box binding protein 1 s(XBP1 s), forkhead box transcription factor O1(FoxO1), activating transcription factor-6( ATF6),c AMP-response element binding protein( CREB),CREB-regulated transcription coactivator-2( CRTC2),double-stranded RNA-dependent protein kinase-like ER kinase( PERK),eukaryotic initiation factor 2α( e IF2α),protein kinase B( AKT),phosphoenolpyruvate carboxykinase( PEPCK),glucose-6-phosphatase( G6Pase) mRNA were measured at baseline,week 8,and week 12. Results There was no significant difference in each observation index between the two groups at baseline; at 8 weeks,the levels of FBG,FINS and the mRNA levels of IRE1α,XBP1s,ATF6,PERK,e IF2α,PEPCK and G6Pase in the CIH group were higher than those in the NC group( P<0. 05),while the mRNA levels of CREB,CRTC2 and AKT were lower than those in the NC group( P<0. 05); at 12 weeks,there was no significant difference in each observation index between the two groups. Pearson correlation analysis showed( 8th week of intermittent hypoxia group) : homeostasis model assessment-insulin resistance( HOMA-IR) was positively correlated with Fox O1,CREB,CRTC2 and PERK,e IF2αmRNA levels( r = 0. 172,0. 595,0. 183,0. 702,0. 608; P<0. 05) while it was negatively correlated with IRE1α,XBP1s,ATF6,AKT mRNA levels( r =-0. 422,-0. 327,-0. 309,-0. 399; P<0. 05). Conclusion Intermittent hypoxia can lead to insulin resistance,and endoplasmic reticulum stress may mediate this effect.

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Objective To analyze interacting proteins of tropomodulin1(TMOD1) in Raw264.7 mouse monocyte macrophage line by mass spectrometry and GeneCards database. Methods Immunoprecipitation combined with mass spectrometry was used to find interacting proteins of TMOD1 after overexpress TMOD1 in Raw264.7 cells. GeneCards database was used to search for known genes for macrophage migration. Bioinformatics&Systems Biology was used to analyze correlation between known targets and mass spectrometry proteins to find common differentially expressed proteins(CO-DEPs). WoLF PSORT was used to predict subcellular localization of CO-DEPs. EggNOG databasewas used to analyze eukaryotic orthologous group(KOG) of CO-DEPs. DAVID database was used to analyze gene ontology(GO) enrichment kyoto encyclopedia of genes and genomes(KEGG) pathway of CO-DEPs. String database was used to analyze protein interaction network and CytoScape software drawing. Results There were 41 CO-DEPs in mass spectrometry and GeneCards database. Subcellular localization of CO-DEPs was mainly distributed in cytoplasm, nucleus and mitochondria. KOG notes were mainly O: post-translational modification, Z: cytoskeleton and J: translation. GO enrichment found that CO-DEPs was mainly involved in poly(A) RNA binding, protein folding and focal adhesion. KEGG was mainly enriched in arrhythmogenic right ventricular cardiomyopathy(ARVC) and tight junction. ACTB was a protein with large protein interaction. Conclusion The proteins interacting with TMOD1 in macrophages mainly include myosin heavy chain-9(MYH9), α-actinin 1(ACTN1) and β-actin(ACTB), etc, suggesting that TMOD1 is related to macrophages migrate.

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Objective To investigate the expression of programmed death 1(PD1) on CXCR5-CD4+T cells from the patients with rheumatoid arthritis(RA) and to analyze the clinical relevance to disease severity. Methods The expression of PD1 on CXCR5-CD4+T cells were examined from 82 RA patients and 46 healthy controls(HC) by the technique of flow cytometry. The expression of PD1 including mean fluorescence intensity(MFI) on CXCR5-CD4+T cells and percentage of PD1+CXCR5-CD4+T cells were compared between RA patients and HC. Moreover, its correlation with laboratory inspection was analyzed. Results (1) The percentage of PD1+CXCR5-CD4+T cells and the MFI of PD1 on CXCR5-CD4+T cells from RA patients were significantly elevated compared with HC(P<0.000 1,U=1 082;P<0.000 1,U=917.5).(2) The percentage of PD1+CXCR5-CD4+T cells in RA patients were positively associated with rheumatoid factors(RF)(rs=0.267 9,P=0.022 9), increased RF(rs=0.419,P=0.001 1), increased anti-cyclic citrullinated peptide(anti-CCP)(rs=0.260 7,P=0.040 7), the percentage of plasmablasts(rs=0.302 4,P=0.029 3), DAS28-ESR(rs=0.244 7,P=0.042 7).(3) The MFI of PD1 on CXCR5-CD4+T cells in RA patients were negatively with lymphocytes number(rs=-0.338 7,P=0.002 0), lymphocytes percentage(rs=-0.347 9,P=0.001 4), lymphocyte-to-monocyte ratio(rs=-0.263 7,P=0.017 4). The MFI of PD1 on CXCR5-CD4+T cells in RA patients were positively associated with neutrophil percentage(rs=0.304 7,P=0.005 4), neutrophilto-lymphocyte ratio(rs=0.341 1,P=0.001 8), platelet-to-lymphocyte ratio(rs=0.232 1,P=0.037 1), systemic immune inflammation index(rs=0.288 0,P=0.009 1), derived Neutrophil to lymphocyte ratio(rs=0.320 3,P=0.003 6), erythrocyte sedimentation rate(rs=0.259 5,P=0.019 3), patient visual analogue scale(rs=0.241 6,P=0.043 9). The MFI of PD1 on CXCR5-CD4+T cells from high active RA patients was significantly elevated compared with non-high active group(P=0.040 7,U=406.0). Conclusion The abnormal expression of PD1 on CXCR5-CD4+T cells are observed in patients with RA. Increased expression of PD1 on CXCR5-CD4+T cells are associated with the production of antibody, the percentage of plasmablasts, inflammation, and also disease activity.

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Objective To screen the target of helicid in the intervention of depression based on network pharmacology and molecular docking, and to study the effect of helicid on the expression level of related targets in hippocampus, prefrontal cortex, striatum and habenular nucleus of chronic unpredictable mild stress(CUMS) rats. Methods The target of helicid was predicted by SwissTargetPrediction database, and the depression related targets were screened by GeneCards、DisGeNet、TTD and DRUGBANK databases; the metascape platform was used for gene enrichment analysis, and the "helicid-depression-pathway" network was constructed; Autodock Vina was used for molecular docking research; qRT-PCR was used to detect the effect of helicid on the mRNA expression of HTR1 A, ADORA1 and ADORA2 A in rat tissues. Results The 81 helicid targets and 1 640 depression targets were obtained, including 40 intersecting targets; the key targets were mainly enriched in cAMP signal pathway, PI3 K-Akt signal pathway, MAPK signal pathway and so on; the results of molecular docking showed that the binding activity of helicid with most targets was good; helicid up-regulated the expression levels of HTR1 A, ADORA1 and ADORA2 A mRNA in hippocampus, prefrontal cortex, striatum and habenular nucleus of CUMS rats. Conclusion Helicid may act on cAMP, PI3 K-Akt, MAPK and other signal pathways to intervene depression through HTR1 A, ADORA1 and ADORA2 A.

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Objective To investigate the diagnostic endoplasmic reticulum aminopeptidase-1(ERAP1) in patients with hepatocellular carcinomae(HCC). Methods Enzyme-linked immunosorbent assay(ELISA) was used to detect the serum levels of ERAP1 in HCCpatients, cirrhosis patients and healthy controls(HC).Multivariate logistic regression was used to analyze the independent risk factors of the severity and prognosis, and receiver operating characteristic curve(ROC) was used to evaluatesensitivity and specificity of ERAP1 in the diagnosis of different degree of disease and prognosis. Results The serum ERAP1 level of HCC was related to tumor stage, tumor size and number of cancer focal(P<0.05). ERAP1 level of HCC patients was positivecorrelated with ALT, AST, TBIL and AFP, while negative correlated with ALB(P<0.05). ERAP1 was found to be an independent predictor of different severity and prognosis. When joint diagnosing HCC with AFP, the area under the curve(AUC) was 0.932. For the diagnosis of poor prognosis, the AUC was 0.742. Conclusion Serum ERAP1 level has important clinical significance and potential application value in evaluating the severity and prognosis of HCC patients.

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Objective To explore the relationship betweenPorphyromonas gingivalis(Pg)and mitophagy in esophageal cancer cells, and to explore new therapeutic targets for esophageal cancer. Methods (1) Western blot was used to detect the phosphorylation of unc-51-like authophagy activating kinase1(ULK1) in mitochondria of the Pg infected cells and immunohistochemical method was used to detect the correlation between the expression of Pg and the phosphorylation status of ULK1 in esophageal cancer tissues.(2) Western blot, ICC and ELISA were used to detect the transfer of nucleotide blinding domain and leucine rich repeat containing family member X1(NLRX1) from cytoplasm to mitochondria, mitophagy, and the secretion levels of interleukin(IL)-6 and reactive oxygen species(ROS) under Pg infection.(3) Pg colonization in esophageal tissues of mice in each group was detected by qPCR and Pg colonization in esophageal squamous epithelial cells of mice by RNAscope. Results Compared with the untreated group, the phosphorylation level of mitochondrial ULK1(P<0.01), NLRX1 expression(P<0.001) and mitophagy(P<0.001) of esophageal cancer cells increased after Pg infection. Compared with the control group, the combined intervention group could inhibit Pg colonization in esophageal tissue and esophageal squamous epithelial cells of mice(P<0.001). Conclusion Pg promotes the translocation of NLRX1 from cytoplasm to mitochondria by up-regulating the phosphorylation level of ULK1 in the mitochondria of esophageal cancer cells, and then induces mitophagy, leading to the reduction secretion of IL-6 and ROS, and ultimately maintaining Pg colonization.

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Objective To explore the effect of sericin on podocyte epithelial-mesenchymal transition induced by high glucose. Methods Conditional immortalized mouse podocytes were used as the research object to construct podocyte injury model by high glucose stimulation. The podocytes were divided into normal( NG) group,hypertonic control( MG) group,high glucose injury( HG) group,low,medium and high dose sericin( LS,MS,HS)group. CCK-8 method was used to determine the viability of podocytes in each group; the morphological structure of podocytes in each group was observed by ordinary optical inverted microscope. The migration ability was detected by Transwell and cell scratch test. Western blot was used to detect the expression levels of EMT-related mesenchymal phenotype factors Snai1,α-SMA,FSP-1,MMP-9 and epithelial phenotype factors Nephrin,E-cadherin,and WT-1 in podocytes of each group. Results Compared with NG group,HG group showed lower podocyte activity( P<0. 01),worse podocyte status and enhanced podocyte migration ability,up-regulated Snai1,α-SMA,FSP-1and MMP-9 protein expressions( P<0. 01),and down-regulated Nephrin,E-cadherin and WT-1 protein expressions( P<0. 01). Compared with HG group,the viability of podocytes in sericin group increased( P<0. 05),the state of podocytes was improved,the migration ability of podocytes was weakened,the expression of Snai1,α-SMA,FSP-1 and MMP-9 was down-regulated( P<0. 05),and the expression of Nephrin,E-cadherin and WT-1was up-regulated( P<0. 05). Conclusion Sericin can alleviate podocyte epithelial-mesenchymal transition induced by high glucose.

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Objective To investigate the expression of milk fat globule epidermal growth factor 8 protein(MFGE8) in osteosarcoma cell lines and its effects on the proliferation, apoptosis, invasion and migration ability of osteosarcoma cells(U2 OS). Methods The expression of MFGE8 protein in normal osteoblasts and osteosarcoma cell lines was examined by Western blot method. The relationship between MFGE8 mRNA expression and prognosis of osteosarcoma patients was analyzed by GEO database. The effects of down-regulation of MFGE8 on proliferation, apoptosis, migration and invasion of U2 OS cells were examined. Results Western blot method showed that the expression of MFGE8 protein was higher in osteosarcoma cells than that in normal osteoblasts and its was negatively correlated with the prognosis of osteosarcoma patients(allP<0.05); compared with sh-Ctrl(U2 OS cells transfected with the null group) group, the U2 OS cells in sh-MFGE8(U2 OS cells transfected with sh-MFGE8) group had decreased healing rate and invasion number while increased proliferation inhibition rate and apoptosis rate, and all differences were statistically significant(P<0.05). Conclusion MFGE8 is highly expressed in U2 OS of osteosarcoma cells, and down-regulation of MFGE8 can inhibit the proliferation, invasion and migration of U2 OS and induce their apoptosis.

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Objective To investigate the association between single nucleotide polymorphism(SNP) rs558614, rs9552315, rs7317471 and rs9509492 in large tumor suppressor kinase 2(LATS2) gene and the risk of colorectal cancer. Methods A total of 390 colorectal cancer patients and 413 healthy subjects were genotyped by Taqman method. The odds ratio(OR) and its 95%CIwere calculated by unconditional logistic regression, to estimate the associations between SNP rs558614, rs9552315, rs7317471, rs9509492 in LATS2 gene and the risk of colorectal cancer, rectal cancer, as well as colon cancer under codominant, dominant, recessive, overdominant, and log-additive genetic models. Haplotypes were constructed by haploview software 4.2. Results SNP rs558614, rs7317471, rs9552315 and rs9509492 in LATS2 gene were not associated with the risk of colorectal cancer, rectal cancer and colon cancer under codominant, dominant, recessive, overdominant, and log-additive genetic models. No haploid blocks were formed between the 4 SNPs. Conclusion SNP rs558614, rs7317471, rs9552315, rs9509492 in LATS2 gene may not play a major role in the development of colorectal cancer, rectal cancer and colon cancer.

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Objective To investigate the expression and clinical significance of erbb2 interacting protein(ERBIN) and PDZ domain containing 2 protein(PDZD2) in colorectal cancer tissues, as well as the effects of ERBIN and PDZD2 on the progression of colorectal cancer and the corresponding mechanisms. Methods Western blot was used to detect the expression of ERBIN and PDZD2 proteins in tumor tissues and corresponding adjacent tissues from 86 colorectal cancer patients, as well as normal human colon fibroblasts(CCD-18 Co) and human colorectal cancer cell lines(SW480, HCT116, SW620 and HT29); SW620 and HCT116 cells were transfected with ERBIN or PDZD2 overexpression vector, and then cell proliferation, cell invasion and apoptosis were detected. The expression of ERBIN or PDZD2 protein in SW620 cell was observed by immunofluorescence chemistry. Co-immunoprecipitation assay was used to detect the interaction between ERBIN and PDZD2 proteins. Results The expression levels of ERBIN and PDZD2 proteins in colorectal cancer tissues were lower than those in adjacent tissues(P<0.01), and the expression levels of ERBIN and PDZD2 in colorectal cancer cells were lower than those in CCD-18 Co cells(P<0.01). The expression levels of ERBIN and PDZD2 proteins were correlated with the depth of invasion, differentiation, TNM staging and lymph node metastasis of colorectal cancer(P<0.001); ERBIN or PDZD2 overexpression reduced the proliferation and invasion of SW620 and HCT116 cells(P<0.01), increased cell apoptosis(P<0.01); ERBIN directly bound to PDZD2, and overexpression of ERBIN increased the expression level of PDZD2 protein. Conclusion ERBIN can inhibit the proliferation and invasion of colorectal cancer cells and promote apoptosis by promoting the expression of PDZD2 protein.

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Objective To investigation of the interaction between interstitial cells of Cajal(ICCs) and connexin 43(Cx43) in bladder contraction and its significance. Methods Eighty male guinea pigs were randomly divided into blank control group, Glivec group, Gap27 group and Glivec+ Gap27 group. Four groups of guinea pigs were perfused with saline, Glivec, Gap 27, and Glivec+Gap 27 every morning for 2 months. Success of urodynamic testing model after 2 months. Bladder tissue was collected for an in vitro muscle strip test to detect muscle contraction in each group. Correlation between c-Kit and Cx43 was detected by immunofluorescence. The interaction between c-Kit and Cx43 in the bladder was further validated by qRT-PCR and Western blot. Ultrastructural changes in the muscle layer of the bladder were observed by electron microscopy. Results Urodynamics revealed increased blad-der compliance in the experimental group compared to the blank control group(P<0.05); bladder compliance increased in the Glivec + Gap27 group compared to the Glivec and Gap27 groups( P<0. 01). In vitro muscle strip experiments revealed that the frequency and tone of bladder muscle strip contractions were lower in the experimental group compared to the blank control group( P<0. 05),and that muscle strip contractions were weaker in the experimental group after administration of acetylcholine( ACH) compared to the control group( P<0. 05). Immunofluorescence showed that c-Kit was co-expressed with Cx43 on ICCs cells. qRT-PCR and Western blot suggested that the protein expression level and gene expression level of Cx43 in bladder tissues were lower after inhibition of c-Kit than in the blank control( P<0. 05); after inhibition of Cx43,the protein expression level and gene expression level of c-Kit in bladder tissues the levels of c-Kit protein expression and gene expression in bladder tissues were lower than those in the blank control group( P<0. 05). Electron microscopy revealed that the mitochondrial structure of bladder smooth muscle was disrupted after simultaneous inhibition of c-Kit and Cx43. Conclusion Cx43 is expressed on bladder ICCs and the two may be jointly involved in regulating bladder contractile function;the joint reduction of Cx43 and c-Kit may have disrupted the mitochondria of bladder smooth muscle,affecting its function and consequently bladder contractile function.

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Objective To investigate the sex difference of the effects of chronic pregnancy stress on depression-like behavior in offspring adolescent mice and whether the amygdala is involved in mediating depression-like behavior and its possible mechanism. Methods Male and female of C57 BL/6 J mice were put in cage together. Pregnant mice were randomly divided into normal control group(CON group) and chronic pregnancy stress group(CPS group). The day of delivery was recorded as post-natal day(PND0). The offspring of different groups were divided into Female group and Male groupaccording to sex, respectively. From PND35, the depressive-like behavior of offspring was monitored in different groups. Morphological structure of basolateral amygdala(BLA)cone neurone was observed by Golgi-Cox staining, and apoptosis of BLA neurone was detected by TUNEL.Serum corticotrophin-releasing hormone(CRH)was detected by ELISA. The level of protein associated with amygdala mammalian target of rapamycin(mTOR) and phosphorylated mammalian target of rapamycin [p-mTOR(Ser2448)] was detected by Western blot. Results Depression-like behavior was appeared in different sexual offspring by chronic pregnancy stress, and there was an interaction between chronic pregnancy stress and gender. In the forced swimming test, the immobility time of offspring in the CPS group prominently increased(Female:P<0.05,Male:P<0.001). Interestingly, compared with female offspring, despairing behavior of male offspring was much more clearly observed in CPS group(P<0.05). Compared with offspring of CON group, the rate of sucrose preference was significantly reduced in the female offspring of CPS group(P<0.05), while no obvious difference was observed in the male offspring. Compared with the CON group, the density of neuronal dendrite branches in the BLA of offspring mice in the CPS group decreased(Female:P<0.01,Male:P<0.01) and the degree of neuronal apoptosis increased(Female:P<0.001,Male:P<0.001), the expression level of p-mTOR in amygdala of offspring mice in CPS group significantly decreased(Female:P<0.001,Male:P<0.001). Chronic pregnancy stress increased the serum CRH level of offspring mice(P<0.001), and the gender had significant influence on serum CRH level, the serum CRH level of female in CPS group was higher than that of male(P<0.05). Conclusion Chronic pregnancy stress leads to depression-like behavior in offspring adolescent mice, and the depression-like behavior has gender differences. In addition, chronic pregnancy stress leads to dendrite atrophy and apoptosis of BLA neurons in offspring mice, and the mechanism may be that the activation of mTOR in the amygdala of offspring mice is inhibited. CRH may be involved in mediating sex differences in depression-like behavior and BLA neuron damage in offspring induced by chronic pregnancy stress.

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Objective To investigate the influence and molecular mechanism of hypoxia-inducing factor-1α(HIF-1α) gene silencing combined with methyl selenenic acid(MSA) on cervical cancer cell proliferation, apoptosis and cell migration. Methods HeLa cells were transfected with HIF-1 interference RNA and negative control RNA. After transfection for 48 h, cells were stimulated with MSA for 24 h, and cell proliferation was determined by CCK-8 assay and colony formation. Apoptosis was determined by flow cytometry combined with Annexin V-FITC/PI. The expression levels of HIF-1α, Bcl-2, and E-cadherin were detected by Western blot assay. Cell migration ability was determined by Transwell assay. RNA-seq analysis was used to investigate the differentially expressed genes and differential signaling pathways. Results Compared with the control group, interfering with HIF-1α combined with MSA significantly inhibited cell proliferation(P<0.01). Flow cytometry results showed that the combined drug group significantly induced apoptosis. Transwell results showed that interfering with HIF-1α combined with MSA inhibited He La cell migration. Compared with the control group,interfering with HIF-1α combined with MSA downregulated the expression of Bcl-2 and up-regulated the expression of E-cadherin. RNA-sequencing combined with signal pathway enrichment results showed that the expression of apoptotic signal pathway and downstream genes was inhibited. Conclusion HIF-1α gene silencing combined with MSA can synergically inhibit the proliferation and induce apoptosis of cervical cancer cells,and its regulatory mechanism may be related to the expression of Bcl-2family proteins and the inhibition of p53 signaling pathway.

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Objective To investigate whether NaHS, a hydrogen sulfide donor, can improve myocardial injury in sepsis by inhibiting oxidative stress and activating the Xc-/GPX4 signaling pathway in ferroptosis. Methods Lipopolysacc-haride( LPS) induced H9c2 in rat cardiomyocytes to form an in vitro model of myocardial injury in sepsis,which was divided into Control group,LPS group and LPS + Na HS group. The kits were applied to detect the changes of cardiomyocyte viability,Fe2 +,LDH and CK-MB,determine the levels of oxidative stress indexes GSH and MDA,detect the changes of cellular ROS and mitochondrial membrane potential levels by fluorescent probes,and detect the expression levels of ferroptosis regulatory proteins SLC7A11 and GPX4 by Western blot. Results Compared with the Control group,H9c2 cell viability decreased,Fe(2 +) concentration increased,GSH,MDA and ROS levels increased,mitochondrial JC-1 monomer increased,expression levels of ferroptosis regulatory proteins SLC7A11 and GPX4 decreased,and cell damage increased after LPS stimulation( P<0. 05). Compared with the LPS group,Na HS attenuated LPS-induced H9c2 cell injury and elevated Fe2+concentration,decreased the level of LPS-induced oxidative stress in H9c2 cells,and increased the expression levels of ferroptosis regulatory proteins SLC7A11 and GPX4( P<0. 05). Conclusion The mechanism by which Na HS attenuates myocardial injury in sepsis may be related to the inhibition of oxidative stress and activation of the Xc-/GPX4 signaling pathway in ferroptosis.

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Objective To screen differentially expressed miRNAs and explore its effect and mechanism on cell migration in lung adenocarcinoma(LUAD). Methods Differentially expressed miRNAs in LUAD tissues and normal lung tissues were screened by miRNA microarrays, and then bioinformatics analysis was used to predict their potential biological functions and signaling pathways. The cancer genome atlas(TCGA) analysis and quantitative real-time PCR(qRT-PCR) verified the expression level of hsa-let-7 e-5 p in LUAD tissues and cell lines. The effect of hsa-let-7 e-5 p cell migration in LUAD was examined by would healing experiment. After screening the underlying target genes by bioinformatics analysis, the targeting relationship between hsa-let-7 e-5 p and DTX2,NME6, C8 orf58, GATM and DHX57 were verified by qRT-PCR. Results The miRNA microarray results showed that 347 miRNAs were down-regulated while 229 miRNAs were up-regulated in lung adenocarcinoma tissues. Compared with normal lung tissue and cells, the expression level of hsa-let-7 e-5 p was significantly down-regulated. Besides, overexpression of hsa-let-7 e-5 p inhibitedLUAD cell migration. Conclusion Non-coding RNA hsa-let-7 e-5 p is down-regulated in LUAD and inhibits the migration of lung adenocarcinoma cells. DTX2, NME6, C8 orf58, GATM and DHX57 are the potential target genes of hsa-let-7 e-5 p.

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Objective To investigate whether exogenous calcium-binding protein A8/A9(S100 A8/A9) promotes the migration and invasion of oral squamous cell carcinoma(OSCC) cells CAL-27 and SCC-25 through Wnt/β-catenin. Methods CAL-27 and SCC-25 were cultured with S100 A8 and S100 A9 proteins respectively or together, and the expression of β-catenin was detected by Western blot. 20 μg/ml Wnt/β-catenin pathway inhibitor DKK-1 was added, the expression of β-catenin was detected by Western blot and qPCR; Transwell assay was used to compare the cell invasion and migration ability of normal control group, S100 A8/A9 protein added group, DKK-1 added group, and DKK-1 added and S100 A8/A9 protein recovery group; Western blot was used to detect the expression of c-Myc and cyclinD1. Results S100 A8 and S100 A9 could activate the Wnt/β-catenin pathway. Compared with S100 A8 and S100 A9 cultured separately, the expression of β-catenin in CAL-27 and SCC-25 cells was up-regulated with time when co-cultured, and the qPCR results were consistent with them; in the immunofluorescence experiment, changes in the expression of β-catenin were observed in the cytoplasm and nucleus of SCC-25 and CAL-27 cells, and the expression of β-catenin protein in SCC-25 and CAL-27 cells increased at 48 h(P<0.05). Adding DKK-1, Western blot and qPCR showed that the expression of β-catenin decreased(P<0.001); the number of cell migration and invasion in the S100 A8/A9 group was more than that in the blank group(P<0.001), the number of cells invading and migrating decreased in the DKK-1 inhibitor group, and the number of cells in the DKK-1 and S100 A8/A9 protein recovery group decreased, suggesting that DKK-1 could inhibit the promoting effect of exogenous S100 A8/A9 protein on the invasion and migration ability of SCC-25 and CAL-27 cells, and the difference was statistically significant(P<0.001); adding Wnt/β-catenin pathway inhibitor DKK-1, Western blot experiments showed that the expressions of key molecules c-Myc and cyclinD1 in the Wnt/β-catenin pathway decreased(P<0.001). Conclusion Exogenous S100 A8/A9 can promote the invasion and migration of SCC-25 and CAL-27 cells by activating the Wnt/β-catenin signaling pathway.

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Objective To study the differential expression and diagnostic value of miR-26 a-5 p and miR-151 a-3 p in plasma exosomes of tuberculosis. Methods Differentially expressed miRNAs(miR-26 a-5 p and miR-151 a-3 p) in tuberculosis exosomes were identified through bioinformatics website. Furthermore, 70 tuberculosis patients(tuberculosis group) and 58 healthy subjects(healthy control group) were selected for clinical validation: Plasma exosomes, extracted by differential centrifugation, were identified by Western blot, transmission electron microscopy, nanoparticle tracking analysis.The expression levels of miR-26 a-5 p and miR-151 a-3 p in plasma exosome were detected by RT-qPCR in the tuberculosis group and the control group. The diagnostic value of miR-26 a-5 p and miR-151 a-3 p in tuberculosis was evaluated by ROC curve. Results The characteristics of exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis and Western blot, which showed that plasma exosomes were extracted successfully. In clinical validation, the internal and external parameters were calibrated simultaneously, the expression level of miR-26 a-5 p in plasma exosome of patients with tuberculosis was lower than that in control group(P<0.000 1), and the expression level of miR-151 a-3 p in plasma exosome of patients with tuberculosis was higher than that in control group(P<0.000 1), which were consistent with the bioinformatics results. As a diagnostic marker, taking internal reference as the benchmark, the AUC of plasma exosome miR-26 a-5 p and miR-151 a-3 p for tuberculosis differentiation was 0.872 and 0.709, the AUC of the combined miR-26 a-5 p and miR-151 a-3 p was 0.915(P<0.000 1). Taking external reference as the benchmark, the AUC of plasma exosomes miR-26 a-5 p and miR-151 a-3 p were 0.829 and 0.854 respectively, the AUC of the combined miR-26 a-5 p and miR-151 a-3 p was 0.911(P<0.000 1). Conclusion The expression level of plasma exosome-derived miR-26 a-5 p decreases in tuberculosis patients and the expression levelof miR-151 a-3 p increases, and the clinical diagnostic efficiency of bothis high, which may be a potential biomarker for the diagnosis of tuberculosis.

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Objective To explore the spatial and temporal heterogeneity of pathological phenotype of epidermal growth factor receptor(EGFR)-mutant transformed locally advanced or metastatic small-cell lung cancer(SCLC) or neuroendocrine carcinoma(NEC). Methods This study retrospectively analyzed EGFR-mutant LUAD patients who were treated with EGFR-TKIs and underwent a transformation into SCLC/NEC. Baseline clinicopathological characteristics after initial transformation were summarized. According to the different pathological components of initial SCLC/NEC transformation, it was defined as incomplete transformation and complete transformation. Incomplete transformation was defined as three categories: intratumoral transformation, intertumoral transformation and effusional transformation. Results This study collected 49 patients who met the criteria. Among forty-nine patients, incomplete transformation was 34.7%(17/49) and complete transformation was 65.3%(32/49). In addition, intratumoral transformation, intertumoral transformation and effusional transformation were respectively 82.3%(14/17), 5.9%(1/17) and 11.8%(2/17).It was mean that the spatial heterogeneity of pathological phenotype of initially transformed SCLC/NEC was 34.7%. After initial transformation, among the fifteen patients in dynamic histological and/or cytological biopsies, the temporal heterogeneity of pathological phenotype of initially transformed SCLC/NEC was 46.7%(7/15). In addition, there was no significant difference in first-line treatment post-transformation progression-free survival and post-transformation overall survival between incomplete transformation group and complete transformation group. Conclusion EGFR-mutant transformed locally advanced or metaststic SCLC/NEC has spatial and temporal heterogeneity in pathological phenotype.

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Objective To construct a cell-free DNA(cfDNA) methylation model for early screening in male patients with gastric cancer by using novel cfDNA methylation detection technology. Methods Methylation information of the whole genome of gastric cancer patients were detected by cell-free methylated DNA immunoprecipitation and highthroughput sequencing(cfMeDIP-seq) technology and locate gastrogenic cfDNA. Then bioinformation methods were used to extract specific methylation labels which could distinguish GC patients and establish diagnosis model by random forest algorithm. Related validation clinical researches were also conducted. Results 63 most significant DMR were selected to construct the cfDNA methylation model based on GC samples and normal control samples, the goal sensitivity was above 85 percent while the goal specificity was above 95%. The sensitivity and specificity of the validation set were 98.7% and 99.0% while the area under curve(AUC) was 0.999. Conclusion The cfDNA methylation model constructed in this study has good performance in predicting GC.

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Objective To explore the clinical value of monocyte count and high-density lipoprotein cholesterol ratio(MHR) in the diagnosis and treatment of chronic heart failure in patients with dilated cardiomyopathy(DCM). Methods A total of hosphospitalized 300 patients with chronic heart failure in DCM was selected and divided into NYHA Ⅱ, NYHA Ⅲ and NYHA Ⅳ group(100 patients), according to the cardiac function classification of New York Heart Association(NYHA). In the same period, 100 patients with organic heart disease and chronic heart failure were selected as the control group.The level of MHR, the relationship between MHR and N-terminal precursor B-type natriuretic peptide(NT-pro BNP), and the relationship between MHR and echocardiographic indexesrelated to cardiac remodeling and cardiac function were observed and analyzed in DCM patients with chronic heart failure. Results The MHR of DCM patients with chronic heart failure was significantly higher than that of the control group(P<0.001), and there were differences among different grades of heart failure in the case group(P<0.05). After controlling the influence of gender, it was found that MHR was positively correlated with left atrial diameter(LAD), left ventricular end-diastolic diameter(LVEDD), left ventricular end-systolic diameter(LVESD) and NT-pro BNP. It was negatively correlated with LVEF.Correlation analysis showed that MHR was positively correlated with left atrial diameter(LAD), left ventricular end-diastolic diameter(LVEDD), left ventricular end-systolic diameter(LVESD) and NT-pro BNP(P<0.05), and negatively correlated with LVEF(P<0.05). The diagnostic value of MHR combined with NT-pro BNP(AUC=0.983) was higher than that of NT-pro BNP alone(AUC=0.974)(P<0.05). These results suggested that MHR might be associated with cardiac remodeling and cardiac function in DCM.The higher the MHR, the more pronounced the cardiac remodeling of DCM, and the more severe the heart failure. The combination of MHR and NT-pro BNP might further improve the diagnostic efficacy of NT-pro BNP in chronic heart failure in DCM. Conclusion Clinically, MHR can be used as an evaluation index of the presence of chronic heart failure and the severity of HF in DCM patients. The combined measurement of MHR and NT-pro BNP may be more conducive to improve the sensitivity and specificity of the clinical diagnosis of DCM chronic HF, so as to identify and accurately implement the standardized treatment of DCM chronic HF earlier.

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Objective To explore the independent risk factors affecting the prognosis, and to construct a nomogram model predicting overall of patients with rectal cancer at T1 and T2 stage. Methods Retrospective analysis was made on the data of 353 patients diagnosed as rectal cancer, who received the radical rectal resection. The collected data were as follows: age, body mass index(BMI),carcinoembryonic antigen(CEA), tumor size, histological type, T stage, N stage, tumor location and number of lymph nodes detected, which were used to perform Kaplan-Meier curve and Log-rank test for univariate analysis and Cox regression for multivariate analysis. The nomogram model was established to predict the overall survival of patients. Results Age≥60 years, Mucinous adenocarcinoma, poorly differentiation, T2 stage, lymph node metastasis, BMI≥25 kg/m2, CEA≥5 μg/L and number of lymph nodes detected<12 were associated with overall survival of patients with rectal cancer at T1 and T2 stage(allP<0.05).Cox regression showed that age≥60 years, T2 stage, mucinous adenocarcinoma, lymph node metastasis, CEA≥5 μg/L, BMI≥25 kg/m2and lymph node detection number<12 were independent risk factors. Based on the above independent risk factors, the nomogram model was constructed, and the predicted curve was in good agreement with the actual survival curve(C-index=0.779). Conclusion Age≥60 years, T2 stage, mucinous adenocarcinoma, lymph node metastasis, CEA≥5 μg/L, BMI≥25 kg/m2and the number of lymph nodes detected<12 are independent risk factors, and the nomogram established in this study can effectively predict the prognosis of patients with rectal cancer at T1 and T2 stage.

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Objective To explore the characteristics of impaired attention network function in chronic insomnia patients. Methods Polysomnography(PSG), sleep scale, attention network test(ANT) and neuropsychological cognitive scale were used to evaluate the sleep quality, attention network function and cognitive function of 73 patients with chronic insomnia and 65 healthy subjects. Results Compared with normal control group, the scores of pittsburgh sleep quality index(PSQI) and insomnia severity index(ISI) in the chronic insomnia group increased, total sleep time reduced, sleep latency prolonged, sleep efficiency decreased, wake after sleep onset and arousal index increased, the proportion of non-rapid eye movement sleep stage 1 increased, the proportion of non-rapid eye movement sleep 3 and the proportion of rapid eye movement sleep decreased, and differences were statistically significant(allP<0.05). In the attention network test, the efficiency of executive control network decreased in the chronic insomnia group, and the difference was statistically significant(allP<0.05). In neuropsychological tests, patients in the chronic insomnia group had spent more time on the Stroop color word test-word and trail making test-B. Correlation analysis showed that executive control function was associated with decreased non-rapid eye movement sleep stage 3 andincreased PSQI scores in chronic insomnia group. Conclusion Chronic insomnia patients have a certain degree of cognitive impairment, mainly executive control network impairment, which is associated with slow-wave sleep reduction.

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Objective To investigate the expression levels and potential clinical significance of pre-B-cell leukemia homologous box 3( PBX3) in bone marrow samples from acute myeloid leukemia( AML). Methods The mRNA expression levels of PBX3 were determined by bioinformatics which analyzed the RNAseq data of 33 malignancies from the cancer genome atlasdatabase.( TCGA) The correlations among the expression levels of PBX3,the clinical parameters and prognosis of AML patients were further analyzed. The differentially expressed genes in the whole transcriptome were analyzed to identify the molecular network in AML caused by PBX3 expression abnormalities.Results The mRNA expression levels of PBX3 were up-regulated in 12 malignancies,and the altitudes increased most significantly in AML than any other cancer types. Patients with high PBX3 expression showed shorter overall survival and disease-free survival than patients with low PBX3 expression. High PBX3 expression was significantly associated with FLT3,NPM1,and DNMT3A mutation. PBX3 expression was positively correlated with multiple homeobox genes( including most HOXA and HOXB genes,MEIS1),and the expression levels of these homeobox genes were all negatively correlated with AML patients overall survival. Conclusion PBX3 high expression in the bone marrow of AML patients is a potential biomarker for poor prognosis,and it may have extensive interactions with other homeobox genes.