Haplotype analysis of long-chain non-coding RNA NONHSAT102891 promoter polymorphisms and depression in Chinese individuals: A case-control association study

BACKGROUND Our previous study reported that the single-nucleotide polymorphism(SNP)rs155979 GC in the promoter region of long-chain non-coding RNA(lncRNA)NONHSAT102891 affects depression susceptibility in a Chinese population.AIM To explored associations of two SNPs and haplotypes in the lncRNA NONHSAT102891 promoter region with depression susceptibility in Chinese population.METHODS This this case-control association study was approved by the Ethics Committee of Chengdu Medical College(approval number:201815).Patient diagnosis was based on DSM-IV criteria.We selected a total of 480 patients with depression and 329 healthy controls with no history of psychopathology,and performed genotyping of two SNPs by extracting peripheral venous blood samples from the subjects.The function of the two lncRNA NONHSAT102891 promoter G/C and A/T haplotypes was detected by dual-luciferase reporter assays of human embryonic kidney 293T transfected cells.RESULTS Stratified analysis of clinical and genotypic characteristics of our cohort showed that the degree of mild depressive episodes associated with the rs6230 TC/CC genotype increased by 1.59 times[TC/CC vs TT:odds ratio(OR)=1.59,95%confidence interval(CI):1.08-2.35,P=0.019].The haploid analysis revealed linkage disequilibrium between rs3792747 and rs6230,and the double SNP CG haplotype was more common in the control group compared to case group,indicating that this haplotype significantly reduced the risk of depression(C/G vs T/A:OR=0.42,95%CI:0.21-0.83,P=0.01).There was no significant difference in the dual-luciferase reporter activity of the G/C and A/T haplotypes compared with the control group(P>0.05),indicating that the double SNP haplotype has no transcrip-tional activity.CONCLUSION The rs3792747 and rs6230 CG haplotypes of the lncRNA NONHSA T102891 promoter may be related to a reduced risk of depression in the Han Chinese population.

Using multi-omics analysis to explore diagnostic tool and optimize drug therapy selection for patients with glioma based on cross-talk gene signature

Background:The heterogeneity of prognosis and treatment benefits among patients with gliomas is due to tumor microenvironment characteristics.However,biomarkers that reflect microenvironmental characteristics and predict the prognosis of gliomas are limited.Therefore,we aimed to develop a model that can effectively predict prognosis,differentiate microenvironment signatures,and optimize drug selection for patients with glioma.Materials and Methods:The CIBERSORT algorithm,bulk sequencing analysis,and single-cell RNA(scRNA)analysis were employed to identify significant cross-talk genes between M2 macrophages and cancer cells in glioma tissues.A predictive model was constructed based on cross-talk gene expression,and its effect on prognosis,recurrence prediction,and microenvironment characteristics was validated in multiple cohorts.The effect of the predictive model on drug selection was evaluated using the OncoPredict algorithm and relevant cellular biology experiments.Results:A high abundance of M2 macrophages in glioma tissues indicates poor prognosis,and cross-talk between macrophages and cancer cells plays a crucial role in shaping the tumor microenvironment.Eight genes involved in the cross-talk between macrophages and cancer cells were identified.Among them,periostin(POSTN),chitinase 3 like 1(CHI3L1),serum amyloid A1(SAA1),and matrix metallopeptidase 9(MMP9)were selected to construct a predictive model.The developed model demonstrated significant efficacy in distinguishing patient prognosis,recurrent cases,and characteristics of high inflammation,hypoxia,and immunosuppression.Furthermore,this model can serve as a valuable tool for guiding the use of trametinib.Conclusions:In summary,this study provides a comprehensive understanding of the interplay between M2 macrophages and cancer cells in glioma;utilizes a cross-talk gene signature to develop a predictive model that can predict the differentiation of patient prognosis,recurrence instances,and microenvironment characteristics;and aids in optimizing the application of trametinib in glioma patients.

Significance of Survivin and PTEN expression in full lymph node-examined gastric cancer

AIM： To study the relationship between Survivin and PTEN expression and lymph node metastasis, depth of invasion and prognosis of gastric cancer patients in China.METHODS： Specimens of gastric cancer tissue were collected from the Affiliated Hospital of Jianghan University. All the 140 patients had complete examination data. All lymph nodes were found by the fat-clearing method. The interrupted serial 4-micron sections, routine hematoxylin and eosin staining and immunohistochemical methods were used to detect the lymph node metastases. Gastric cancer tissue microarray was performed to test the expression of Survivin and PTEN （17A） in gastric cancer by immunohistochemical method. All data were processed using X^2 test, Fisher＇s exact test, Kaplan-Meyer Log-rank method and Cox multivariate analysis （SPSS 12.0 software）.RESULTS： One hundred and eighteen specimens were used in our tissue microarray （utilization rate was 82.4%）. A total of 7580 lymph nodes were found. Metastases were found in 90 specimens and 1618 lymph nodes were detected. The positive rate of Survivin and PTEN expression was 52.5% （62/118） and 76.2% （90/118）, respectively. A highly positive correlation was found between Survivin and PTEN expression （X^2=4.17, P=0.04）. Survivin expression was positively correlated with UICC N stage （X^2=8.69, P=0.03） and histological classification （X^2=4.41, P=0.04） by X^2 tests. PTEN expression was positively correlated with depth of invasion （P=0.02） and histological classification （X^2=5.47, P=0.02）. But Survivin and PTEN expressions were not related with prognosis of gastric cancer patients. A significant correlation between lymph node metastasis and prognosis was demonstrated by Cox multivariate analysis （X^2=4.85, P=0.028）.CONCLUSION： Survivin is positively correlated with PTEN expression in gastric cancer and is a molecular marker of lymph node metastasis while PTEN expression is a molecular marker of advanced gastric cancer. UICC N stage is the most important prognostic factor of gastric cancer in China.

Characterization of 500 Chinese patients with cervical esophageal cancer by clinicopathological and treatment outcomes

Objective: There are no comprehensive studies on survival outcomes and optimal treatment protocols for cervical esophageal cancer(CEC), due to its rare clinical prevalence. Our objective was to determine the relationship between pathological characteristics, treatment protocols, and survival outcomes in Chinese CEC patients.Methods: A total of 500 Chinese CEC patients were selected from our 500,000 esophageal and gastric cardia carcinoma database(1973–2018). There were two main groups: patients treated with surgery, and patients receiving non-surgical treatments(radiotherapy, radiochemotherapy, and chemotherapy). The Chi-square test and Kaplan–Meier method were used to compare the continuous variables and survival.Results: Among the 500 CEC patients, 278(55.6%) were male, and the median age was 60.9 ± 9.4 years. A total of 496 patients(99.2%) were diagnosed with squamous cell carcinoma. In 171(34.2%) patients who received surgery, 22(12.9%) had undergone laryngectomy. In 322(64.4%) patients who received non-surgical treatments, 245(76.1%) received radiotherapy. Stratified survival analysis showed that only T stage was related with survival outcomes for CEC patients in the surgical group, and the outcomes between laryngectomy and non-laryngectomy patients were similar. It was noteworthy that the 5-year survival rate was similar in CEC patients among the different groups treated with surgery, radiotherapy, chemotherapy, or radiochemotherapy(P = 0.244). Conclusions: The CEC patients had similar survival outcomes after curative esophagectomy and radiotherapy, including those with or without total laryngectomy. These findings suggest that radiotherapy could be the initial choice for treatment of Chinese CEC patients.

Identification of four novel prognosis biomarkers and potential therapeutic drugs for human colorectal cancer by bioinformatics analysis

Colorectal cancer(CRC)is one of the most deadly cancers in the world with few reliable biomarkers that have been selected into clinical guidelines for prognosis of CRC patients.In this study,mRNA microarray datasets GSE113513,GSE21510,GSE44076,and GSE32323 were obtained from the Gene Expression Omnibus(GEO)and analyzed with bioinformatics to identify hub genes in CRC development.Differentially expressed genes(DEGs)were analyzed using the GEO2 R tool.Gene ontology(GO)and KEGG analyses were performed through the DAVID database.STRING database and Cytoscape software were used to construct a protein-protein interaction(PPI)network and identify key modules and hub genes.Survival analyses of the DEGs were performed on GEPIA database.The Connectivity Map database was used to screen potential drugs.A total of 865 DEGs were identified,including 374 upregulated and 491 downregulated genes.These DEGs were mainly associated with metabolic pathways,pathways in cancer,cell cycle and so on.The PPI network was identified with 863 nodes and 5817 edges.Survival analysis revealed that HMMR,PAICS,ETFDH,and SCG2 were significantly associated with overall survival of CRC patients.And blebbistatin and sulconazole were identified as candidate drugs.In conclusion,our study found four hub genes involved in CRC,which may provide novel potential biomarkers for CRC prognosis,and two potential candidate drugs for CRC.

Effects of ATP-sensitive Potassium Channels on the Expression of P21, P27 and Leptin

This study investigated the effects of ATP-sensitive potassium channels on the expression of P21, P27 and leptin. The expression of receptor of ATP-sensitive potassium channels （sulphonylurea receptor, SUR） mRNA in the preadipocytes and leptin mRNA was detected by PCR after rat preadipocytes were treated with the opener （diazoxide） or inhibitor （glibenclamide） of ATP-sensitive potassium channels during the process of inducing differentiation. The expression of P21 and P27 in preadipocytes treated with diazoxide or glibenclamide was assayed by Western blot. The results showed that the expression of SUR2, not SUR1 was detected in adipose tissue, preadipocytes and adipocytes. Alter treatment of preadipocytes with diazoxide, the expression levels of P21 and P27 were obviously higher than glibenclamide-treat ed group those in control group, but the were lower than those in control expression levels of P21 and P27 in group. During the process of inducing differentiation, the expression of leptin mRNA in preadipocytes treated with diazoxide was increased greatly, but the expression of leptin mRNA in glibenclamide-treated group decreased obviously. It was concluded that ATP-sensitive potassium channels might be involved in the proliferation and differentiation of rat preadipocytes by changing the expression of P21, P27 and leptin.

Correlation between the expression of CyclinB1/CDK1 complex in esophageal cancer tissue and the contents of marker molecules in serum and lesion

Objective: To study the correlation between the expression of CyclinB1/CDK1 complex in esophageal cancer tissue and the contents of marker molecules in serum and lesion. Methods:Patients who were diagnosed with esophageal cancer in Mianyang Central Hospital between April 2014 and December 2016 were selected as esophageal cancer group, and the esophageal cancer lesion tissue, adjacent lesion tissue and serum samples were collected;healthy volunteers who received physical examination during the same period were selected as control group and serum samples were collected. The expression of CyclinB1/CDK1 complex, tumor suppressor genes and invasion genes in esophageal cancer lesion tissue and adjacent lesion tissue as well as the levels of tumor markers in serum were detected. Results: CyclinB1, CDK1 and E2F-1 mRNA expression in esophageal cancer lesion were significantly higher than those in adjacent lesion;serum tumor markers CYFRA21-1, SCC-Ag, CD44v5 and Stathmin levels of esophageal cancer group were significantly higher than those of control group and positively correlated with CyclinB1, CDK1 and E2F-1 mRNA expression in esophageal cancer lesion, PTEN, Spink8, p21 and p18 mRNA expression in esophageal cancer lesion were significantly lower than those in adjacent lesion and negatively correlated with CyclinB1, CDK1 and E2F-1 mRNA expression in esophageal cancer lesion, and nestin, β-catenin, Snail and Vimentin mRNA expression in esophageal cancer lesion were significantly higher than those in adjacent lesion and positively correlated with CyclinB1, CDK1 and E2F-1 mRNA expression in esophageal cancer lesion. Conclusion: The high expression of CyclinB1/CDK1 complex in esophageal cancer tissue can promote the proliferation and invasion of cancer cells.

P25/CDK5-mediated Tau Hyperphosphorylation in Both Ipsilateral and Contralateral Cerebra Contributes to Cognitive Deficits in Post-stroke Mice

Objective Post-stroke cognitive impairment(PSCI)develops in approximately one-third of stroke survivors and is associated with ingravescence.Nonetheless,the biochemical mechanisms underlying PSCI remain unclear.The study aimed to establish an ischemic mouse model by means of transient unilateral middle cerebral artery occlusions(MCAOs)and to explore the biochemical mechanisms of p25/cyclin-dependent kinase 5(CDK5)-mediated tau hyperphosphorylation on the PSCI behavior.Methods Cognitive behavior was investigated,followed by the detection of tau hyperphosphorylation,mobilization,activation of kinases and/or inhibition of phosphatases in the lateral and contralateral cerebrum of mice following ischemia in MACO mice.Finally,we treated HEK293/tau cells with oxygen-glucose deprivation(OGD)and a CDK5 inhibitor(Roscovitine)or a GSK3βinhibitor(LiCl)to the roles of CDK5 and GSK3βin mediating ischemia-reperfusion-induced tau phosphorylation.Results Ischemia induced cognitive impairments within 2 months,as well as causing tau hyperphosphorylation and its localization to neuronal somata in both ipsilateral and contralateral cerebra.Furthermore,p25 that promotes CDK5 hyperactivation had significantly higher expression in the mice with MCAO than in the shamoperation(control)group,while the expression levels of protein phosphatase 2(PP2A)and the phosphorylation level at Tyr307 were comparable between the two groups.In addition,the CDK5 inhibitor rescued tau from hyperphosphorylation induced by OGD.Conclusion These findings demonstrate that upregulation of CDK5 mediates tau hyperphosphorylation and localization in both ipsilateral and contralateral cerebra,contributing to the pathogenesis of PSCI.

Therapeutic Effect of Daphnetin on Mastitis Induced by Staphylococcus aureus in Mice

[Objectives]To observe the effects of daphnetin on mastitis induced by Staphylococcus aureus in mice.[Methods]18 postpartum ICR female mice were used to establish mastitis animal model,and were randomly divided into three groups(A,B,and C)with 6 mice in each group.Group A:blank control group;group B:S.Aureus model group;group C:S.Aureus model+daphnetin group.The experimental groups were injected 1 mL of 1.0×104 CFU/100μL of S.aureus of along the nipple catheter.The suspension was placed in the 3 rd and 4 th pairs of mammary glands,and the control group was injected with the same dose of normal saline.On the second day after infection,the rats in group A,B and C were given drugs by gavage,while the rats in group A and B were given normal saline and the rats in group C were given daphnetin once a day for 6 consecutive days.Blood samples were collected from living eyeballs,and blood cells were analyzed by automatic flow cytometer after anticoagulation.[Results]The NLR and Systemie Immune Inflammati-on Index(SII)in the blood of mastitis mice induced by S.aureus were significantly higher than those in the control group(P<0.01),suggesting that neutrophil to lymphocyte ratio(NLR)and SII can be used as diagnostic indicators of mastitis,and the levels of NLR and SII decreased significantly after daphnetin intervention.[Conclusions]NLR and SII showed high levels in mastitis mice,which are valuable for the diagnosis of mastitis and the evaluation of its prognosis.After the intervention of daphnetin,both of them decreased significantly,indicating that daphnetin has a good prognosis trend in mastitis mice induced by S.aureus.

Protective effect of lycopene on Parkinson's disease cell model based on endoplasmic reticulum stress

Objective:To evaluate the effect of lycopene on Parkinson's disease cell model and its possible mechanism.Methods:The SH-SY5Y cells were treated with 0.5μmol/L rotenone for 24 h to establish Parkinson's disease cell model.The experiments were randomly divided into the control group,the lycopene group,the rotenone group,the pretreatment groups of different concentrations lycopene(low,medium,high concentration).Cell viability was detected by CCK-8 assay,the morphological changes of cells were observed under an inverted microscope,Hoechst staining was used to observe cell apoptosis,the expression and distribution of endoplasmic reticulum stress marker proteins GRP78 and CHOP in each group were detected by Western blot and cell immunofluorescence.Results:The study found that compared with the control group,the cell viability in the rotenone group was significantly decreased with obvious apoptosis;compared with the rotenone group,the cell viability of the lycopene pretreatment group was improved,and the difference was statistically significant(P<0.05);The apoptosis in the lycopene pretreatment group was decreased.The expression of GRP78 and CHOP in the rotenone group was significantly higher than that in the control group(P<0.01),while the expression of both in the high concentration lycopene pretreatment group was lower than that in the rotenone group(P<0.05).Conclusion:Lycopene pretreatment had a significant protective effect on rotenone-induced SH-SY5Y cells,which may be related to the fact that lycopene pretreatment can effectively alleviate endoplasmic reticulum stress in SH-SY5Y cells damaged by rotenone.

Paris chinensis dioscin induces G2/M cell cycle arrest and apoptosis in human gastric cancer SGC-7901 cells

AIM:To investigate the anti-tumor effects of Paris chinensis dioscin(PCD)and mechanisms regarding cell cycle regulation and apoptosis in human gastric cancer SGC-7901 cells.METHODS:Cell viability was analyzed by the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay.Cell apoptosis was evaluated by flow cytometry and laser scanning confocal microscope(LSCM)using Annexin-V/propidium iodide(PI)staining,and the cell cycle was evaluated using PI staining with flow cytom-etry.Intracellular calcium ions were detected under fluorescence microscope.The expression of cell cycle and apoptosis-related proteins cyclin B1,CDK1,cytochrome C and caspase-3 was measured by immunohistochemical staining.RESULTS:PCD had an anti-proliferation effect on human gastric cancer SGC-7901 cells in a dose-and time-de-pendent manner.After treatment of SGC-7901 cells with PCD,apoptosis appeared in SGC-7901 cells.Morpho-logical changes typical of apoptosis were also observed with LSCM by Annexin V/PI staining,and the cell number of the G0/G1 phase was decreased,while the number of cells in the G2/M phase was increased.Cell cycle-related proteins,such as cyclin B1 and CDK1,were all down-regulated,but caspase-3 and cytochrome C were up-regulated.Moreover,intracellular calcium accumulation occurred in PCD-treated cells.CONCLUSION:G2/M phase arrest and apoptosis induced by PCD are associated with the inhibition of CDK-activating kinase activity and the activation of Ca2+-related mitochondrion pathway in SGC-7901 cells.

Extracellular histones stimulate collagen expression in vitro and promote liver fibrogenesis in a mouse model via the TLR4-MyD88 signaling pathway

BACKGROUND Liver fibrosis progressing to liver cirrhosis and hepatic carcinoma is very common and causes more than one million deaths annually.Fibrosis develops from recurrent liver injury but the molecular mechanisms are not fully understood.Recently,the TLR4-MyD88 signaling pathway has been reported to contribute to fibrosis.Extracellular histones are ligands of TLR4 but their roles in liver fibrosis have not been investigated.AIM To investigate the roles and potential mechanisms of extracellular histones in liver fibrosis.METHODS In vitro,LX2 human hepatic stellate cells(HSCs)were treated with histones in the presence or absence of non-anticoagulant heparin(NAHP)for neutralizing histones or TLR4-blocking antibody.The resultant cellular expression of collagen I was detected using western blotting and immunofluorescent staining.In vivo,the CCl4-induced liver fibrosis model was generated in male 6-week-old ICR mice and in TLR4 or MyD88 knockout and parental mice.Circulating histones were detected and the effect of NAHP was evaluated.RESULTS Extracellular histones strongly stimulated LX2 cells to produce collagen I.Histone-enhanced collagen expression was significantly reduced by NAHP and TLR4-blocking antibody.In CCl4-treated wild type mice,circulating histones were dramatically increased and maintained high levels during the duration of fibrosisinduction.Injection of NAHP not only reduced alanine aminotransferase and liver injury scores,but also significantly reduced fibrogenesis.Since the TLR4-blocking antibody reduced histone-enhanced collagen I production in HSC,the CCl4 model with TLR4 and MyD88 knockout mice was used to demonstrate the roles of the TLR4-MyD88 signaling pathway in CCl4-induced liver fibrosis.The levels of liver fibrosis were indeed significantly reduced in knockout mice compared to wild type parental mice.CONCLUSION Extracellular histones potentially enhance fibrogenesis via the TLR4–MyD88 signaling pathway and NAHP has therapeutic potential by detoxifying extracellular histones.

Three-dimensional-arterial spin labeling perfusion correlation with diabetes-associated cognitive dysfunction and vascular endothelial growth factor in type 2 diabetes mellitus rat

BACKGROUND Type 2 diabetes mellitus(T2DM) has been strongly associated with an increased risk of developing cognitive dysfunction and dementia.The mechanisms of diabetes-associated cognitive dysfunction(DACD) have not been fully elucidated to date.Some studies proved lower cerebral blood flow(CBF) in the hippocampus was associated with poor executive function and memory in T2DM.Increasing evidence showed that diabetes leads to abnormal vascular endothelial growth factor(VEGF) expression and CBF changes in humans and animal models.In this study,we hypothesized that DACD was correlated with CBF alteration as measured by three-dimensional(3D) arterial spin labeling(3D-ASL) and VEGF expression in the hippocampus.AIM To assess the correlation between CBF(measured by 3D-ASL and VEGF expression) and DACD in a rat model of T2DM.METHODS Forty Sprague-Dawley male rats were divided into control and T2DM groups.The T2DM group was established by feeding rats a high-fat diet and glucose to induce impaired glucose tolerance and then injecting them with streptozotocin to induce T2DM.Cognitive function was assessed using the Morris water maze experiment.The CBF changes were measured by 3D-ASL magnetic resonance imaging.VEGF expression was determined using immunofluorescence.RESULTS The escape latency time significantly reduced 15 wk after streptozotocin injection in the T2DM group.The total distance traveled was longer in the T2DM group;also,the platform was crossed fewer times.The percentage of distance in the target zone significantly decreased.CBF decreased in the bilateral hippocampus in the T2DM group.No difference was found between the right CBF value and the left CBF value in the T2DM group.The VEGF expression level in the hippocampus was lower in the T2DM group and correlated with the CBF value.The escape latency negatively correlated with the CBF value.The number of rats crossing the platform positively correlated with the CBF value.CONCLUSION Low CBF in the hippocampus and decreased VEGF expression might be crucial in DACD.CBF measured by 3D-ASL might serve as a noninvasive imaging biomarker for cognitive impairment associated with T2DM.

Dynamic changes in DNA demethylation in the tree shrew (Tupaia belangeri chinensis) brain during postnatal development and aging

Brain development and aging are associated with alterations in multiple epigenetic systems, including DNA methylation and demethylation patterns. Here, we observed that the levels of the 5- hydroxymethylcytosine （5hmC） ten-eleven transtocation （TET） enzyme-mediated active DNA demethylation products were dynamically changed and involved in postnatal brain development and aging in tree shrews （Tupaia belangeri chinensis）. The levels of 5hmC in multiple anatomic structures showed a gradual increase throughout postnatal development, whereas a significant decrease in 5hmC was found in several brain regions in aged tree shrews, including in the prefrontal cortex and hippocampus, but not the cerebellum. Active changes in Tet mRNA levels indicated that TET2 and TET3 predominantly contributed to the changes in 5hmC levels. Our findings provide new insight into the dynamic changes in 5hmC levels in tree shrew brains during postnatal development and aging processes.

Skin nerve regeneration and burn wound healing following spinal nerve root incision

Burn wounds were produced on two sides on the backs of Wistar rats, in addition to denervation on one side. The skin neural regeneration at the injury site and burn wound healing were evaluated following spinal nerve root incision. No nerve regeneration was observed in the burn wound region post-denervation, and the degree of epithelization was significantly less than the control group. With increasing time, expression of type I collagen, which plays a supporting role, and collagen III, which exhibits elastJc propertJes, were sJgnificantly increased Jn the two groups, but the expression was less in the denervation group compared with the control group, and the wound healing was faster in the control group. The ratio of type I collagen to type III collagen was significantly lower in the denervation group compared with the control group. The ratio gradually decreased with prolonged time in the denervation group, but remained unchanged in the control group. However, the elasticity of the tissues in the denervation group was better than the control group. During burn wound healing, innervations can promote wound healing, but denervation can improve the quality of wound remodeling.

STAT1 and Survivin Expression in Full Lymph Node Examined Gastric Cancer by Using Tissue Microarray Technique

Objective： To characterize the relationship between STAT1 and Survivin expression, and the relationship between them and lymph node metastasis, depth of invasion and prognosis in full lymph node examined gastric cancer patients of China. Methods： Specimens of curative dissection between 1988 and 2003 were collected from the affiliated hospital of Jianghan University. All 140 patients had complete examination data. All lymph nodes were found by clearing fat method. The interrupted serial 4 μm sections, routine hematoxylin and eosin staining and immunohistochemical methods were used to detect the lymph node metastases. Gastric cancer tissue microarray was formed and the expression of survivin and STAT1 in gastric cancer was detected by immunohistochemical method. All data were processed using Spearman rank correlation analysis, Kaplan-Meyer Log-rank method and Cox multivariate analysis （SPSS 12.0 software）. Results： Among 140 gastric cancer tissue microarrays constructed, 110 could be used （utilization rate was 78.6%）. 7079 lymph nodes were found in 110 cases （64.4/case）. Metastases were found in 89 cases and 1679 lymph nodes. Positive expression rate of survivin and STAT1 was 52.7% （58/110） and 40% （44/110） respectively. There was a significant negative correlation between STAT1 expression and survivin expression （r=-0.19, P=0.04）. STAT1 expression had a negative correlation with depth of invasion （r=0.21, P=0.04）. Survivin expression had a negative correlation with UICC N stage （r=-0.24, P=0.01） and histological classification （r=-0.21, P=0.03） by Spearman rank correlation analysis. But survivin and STAT1 expression was not related with prognosis. A significant correlation between lymph node metastasis and prognosis was demonstrated by Cox multivariate analysis （X^2=4.85, P=0.028）. Conclusion： STAT1 has a negative correlation with survivin expression in gastric cancer. Both of them have no correlation with prognosis in gastric cancer. STAT1 expression can be a molecular marker to predict advanced gastric cancer and survivin a molecular marker of lymph node metastasis in gastric cancer. UICC N stage is the most important prognostic factor in gastric caner in China.

O6-methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

BACKGROUND O_(6)-methylguanine-DNA methyltransferase(MGMT)is a suicide enzyme that repairs the mispairing base O_(6)-methyl-guanine induced by environmental and experimental carcinogens.It can transfer the alkyl group to a cysteine residue in its active site and became inactive.The chemical carcinogen N-nitroso compounds(NOCs)can directly bind to the DNA and induce the O_(6)-methylguanine adducts,which is an important cause of gene mutation and tumorigenesis.However,the underlying regulatory mechanism of MGMT involved in NOCs-induced tumorigenesis,especially in the initiation phase,remains largely unclear.AIM To investigate the molecular regulatory mechanism of MGMT in NOCs-induced gastric cell malignant transformation and tumorigenesis.METHODS We established a gastric epithelial cell malignant transformation model induced by N-methyl-N’-nitro-N-nitrosoguanidine(MNNG)or N-methyl-N-nitroso-urea(MNU)treatment.Cell proliferation,colony formation,soft agar,cell migration,and xenograft assays were used to verify the malignant phenotype.By using quantitative real-time polymerase chain reaction(qPCR)and Western blot analysis,we detected the MGMT expression in malignant transformed cells.We also confirmed the MGMT expression in early stage gastric tumor tissues by qPCR and immunohistochemistry.MGMT gene promoter DNA methylation level was analyzed by methylation-specific PCR and bisulfite sequencing PCR.The role of MGMT in cell malignant transformation was analyzed by colony formation and soft agar assays.RESULTS We observed a constant increase in MGMT mRNA and protein expression in gastric epithelial cell malignant transformation induced by MNNG or MNU treatment.Moreover,we found a reduction of MGMT gene promoter methylation level by methylation-specific PCR and bisulfite sequencing PCR in MNNG/MNU-treated cells.Inhibition of the MGMT expression by O_(6)-benzylguanine promoted the MNNG/MNU-induced malignant phenotypes.Overexpression of MGMT partially reversed the cell malignant transformation process induced by MNNG/MNU.Clinical gastric tissue analysis showed that MGMT was upregulated in the precancerous lesions and metaplasia tissues,but downregulated in the gastric cancer tissues.CONCLUSION Our finding indicated that MGMT upregulation is induced via its DNA promoter hypomethylation.The highly expressed MGMT prevents the NOCs-induced cell malignant transformation and tumorigenesis,which suggests a potential novel approach for chemical carcinogenesis intervention by regulating aberrant epigenetic mechanisms.

N-myc downstream regulated gene 1 inhibition of tumor progression in Caco2 cells

BACKGROUND Invasion and migration are the irreversible stages of colorectal cancer(CRC).The key is to find a sensitive,reliable molecular marker that can predict the migration of CRC at an early stage.N-myc downstream regulated gene 1(NDRG1)is a multifunctional gene that has been tentatively reported to have a strong relationship with tumor invasion and migration,however the current molecular role of NDRG1 in CRC remains unknown.AIM To explore the role of NDRG1 in the development of CRC.METHODS NDRG1 stably over-expressed Caco2 cell line was established by lentiviral infection and NDRG1 knock-out Caco2 cell line was established by CRISPR/Cas9.Furthermore,the mRNA and protein levels of NDRG1 in Caco2 cells after NDRG1 over-expression and knockout were detected by real-time polymerase chain reaction and western blot.The cell proliferation rate was measured by the cell counting kit-8 method;cell cycle and apoptosis were detected by flow cytometry;invasion and migration ability were detected by the 24-transwell method.RESULTS NDRG1 over-expression inhibited Caco2 proliferation and the cell cycle could be arrested at the G1/S phase when NDRG1 was over-expressed,while the number of cells in the G2 phase was significantly increased when NDRG1 was knocked out.This suggests that NDRG1 inhibited the proliferation of Caco2 cells by arresting the cell cycle in the G1/S phase.Our data also demonstrated that NDRG1 promotes early cell apoptosis.Invasion and migration of cells were extensively inhibited when NDRG1 was over-expressed.CONCLUSION NDRG1 inhibits tumor progression in Caco2 cells which may represent a potential novel therapeutic strategy for the treatment of CRC.

Effects of Benzo(a)pyrene on the Contractile Function of the Thoracic Aorta of Sprague-dawley Rats

Abstract Objective To evaluate the possible vascular effects of an environment carcinogen benzo（a）pyrene （BaP）. Methods The cytotoxicit of BaP and rat liver S9 （0.25 mg/mL）-activated BaP were examined by MTT assay. Thoracic aortic rings were dissected from Sprague-Dawley rats. Contraction of aortic rings was induced by 60 mmol/L KCl or 10-6 mol/L phenylephrine （PE） in an ex-vivo perfusion system after BaP （100 tlmol/L） incubation for 6 h. [Ca^2＋]i was measured using Fluo-4/AM. For in-vivo treatment, rats were injected with BaP for 4 weeks （10 mg/kg, weekly, i.p.）. Results BaP （1-500 μm） did not significantly affect cell viability; S9-activated BaP stimulated cell proliferation. BaP did not affect the contractile function of endothelium-intact or -denuded aortic rings. BaP did not affect ATP-induced （[Ca2＋]i） increases in human umbilical vein endothelial cells. In BaP-treated rats, heart rate and the number of circulating inflammatory cells were not affected. Body weight decreased while blood pressure increased significantly. The maximum aortic contractile responses to PE and KCI and the maximum aortic relaxation response to acetylcholine were significantly decreased by 25.0%, 34.2%, and 10.4%, respectively. Conclusion These results suggest, in accordance with its DNA-damaging properties, that metabolic activation is a prerequisite for BaP-induced cardiovascular toxicity.