| Stomach adenocarcinoma is a common malignant tumor of the digestive tract.The incidence rate of gastric cancer(GC)is relatively higher in China,and the number of new cases is about 400,000 each year.The major reasons are tumor metastasis and recurrence.Despite the continuous improvement in surgical techniques and various radiotherapy and chemotherapy techniques,the prognosis of GC is still poor,and the mortality rate is still the second in all kinds of cancer mortality.This situation brings heavy burden to society and family.In recent years,more and more studies have found that sympathetic nerve system(SNS)plays an important role in the development and metastasis of tumors.It is known that sympathetic nerve regulates gene expression and cellular function of the nervous,endocrine,cardiovascular,digestive,respiratory,reproductive and immune systems by releasing its neurotransmitter norepinephrine(NE).Studies suggest that stress-related psychosocial pressor may be an important factor in the progress of cancer.Adrenergic receptors are highly expressed in variant cancer cells,such as esophageal cancer,breast cancer,nasopharyngeal cancer and colon cancer.If sympathetic nerve infiltrates into the cancer tissues,its released NE binds to these adrenergic receptors in cancer cells and thus may affect the behaviors of these cancer cells via the β-adrenergic receptors(β-ARs)and the downstream signaling,resulting in cancer cell proliferation or apoptosis,angiogenesis and metastasis.However,it has been rarely reported about whether and how sympathetic nerve affects gastric cancer.We hypothesized that there is certain correlation between sympathetic nerve and GC progression,metastasis and prognosis.In this study,we first screened out highly expressed genes in GC using some online tools(UALCAN,GEPIA and Timer,etc.),and made relevant analyses on the correlation between β-AR signaling and GC prognosis.We then collected human stomach adenocarcinoma(STAD)tissue samples of 46 cases during GC radical gastrectomy,observed the morphology of sympathetic distribution and its function in the GC tissue,including sympathetic nerve sprouting and infiltration related proteins,neurotransmitter NE,β-ARs,downstream signaling molecules,and possible effector molecules for GS progression.We further investigated the effects of isoprenaline(ISO)on the proliferation,migration and invasion of GC cell lines and the underlying mechanism.Part 1: Bioinformatics analysis on the expressions of β-ARs signaling in STAD and the correlations of between genes and GC prognosis Objective:To identify overexpressed genes from online database and to analyze the correlations between expressions of adrenergic signaling genes and the prognosis of STAD.Methods:We used online network databases(UALCAN,GEPIA,Timer,etc.)to perform TGCA data extraction and analysis.These online network resources were comprehensive,easy-touse resource for analyzing cancer omics data,which may provide: a)easy access to cancer omics data(TCGA MET500);b)cancer biomarkers or validate certain genes of potential interest;c)graphs based on gene expression,correlations analysis of two or more genes and survival analysis.These bioinformatic analyses provided valuable information for further studies.Spearman correlation analysis was used to evaluate the correlation between pair-wise genes and to calculate Spearman’s rank correlation coefficient(R).P value less than 0.05 was considered statistically significant.Results:Among the top 50 highly expressed genes in patients with STAD,CHI3L1 was the 28 th highly expressed gene.At the RNA level,the expression of CHI3L1 in STAD tissues was significantly higher than that in the adjacent tissues(P < 0.01),especially in stage Ⅰ of STAD(P < 0.01).The overall survival was significantly reduced in patients with high levels of NGF(P = 0.008)and macrophage infiltration(P = 0.004).There was no statistically significant correlation between ADRB1 or ADRB3 expression levels and STAD survival rate(P = 0.72,P = 0.63),but high expression of ADRB2 significantly correlated with lower survival rate of STAD patients(P = 0.02).There were no significant correlation between CHI3L1 and ADRB1(Cor = 0.096,P = 0.051),while significant positive correlation were observed between CHI3L1 and ADRB2 or ADRB3(P = 0.00197,Cor = 0.152;P = 0.0346,Cor = 0.104,respectively).CHI3L1 was all positively correlated with STAT3(R = 0.48,P < 0.001),MAPK1(R = 0.41,P < 0.001),MAPK3(R = 0.35,P < 0.001),MMP9(R = 0.66,P < 0.001),and SDC1(R = 0.37,P < 0.001).The overall survival rate was significantly reduced in the high SDC1 expression group(P = 0.031).Conclusion:β-AR signaling promotes the progression of STAD via the ERK-STAT3-YKL-40 signaling,and MMP9 and syndecan-1 are the downstream effectors in the progression of STAD.Part 2: Evidence of sympathetic nerve infiltration in STAD tissues and the STAD promoting effects of β-adrenergic signaling Objective:To investigate the status of sympathetic nerve infiltration,sympathetic neurotransmitter release,and the regulation of β-AR on YKL-40 and the downstream effectors MMP9 and syndecan-1,and the underlying and mechanism of STAD progression.Methods:We collected 46 human STAD tissue samples during radical gastrectomy.These tissue samples were divided into STAD tissue group and adjacent tissue group.Using immunohistochemistry,HPLC and western blotting,we examined sympathetic infiltration(using TH as a marker),neurotransmitter NE,the expression levels of NGF,TrkA,GAP-43,TH,CD206,CHI3L1,MMP9 and Syndecan-1 both in the STAD tissues and adjacent tissue.SPSS22.0 software was used to perform statistical analysis.All data were expressed as mean ± standard deviation(SD).Differences between groups were determined by ANOVA and ttest.P value less than 0.05 was considered statistically significant.Results:Immunohistochemical results showed that proteins associated with sympathetic nerve sprouting and infiltration,including NGF,TrkA(high-affinity NGF receptor)GAP-43 and S100,were all highly expressed in the interstitial areas of STAD tissues compared to the adjacent tissues.Sympathetic nerves mainly distributed on the edge of STAD nests and some in the interstitial areas between STAD nests.β2-AR,but not ?1-AR and ?3-AR,was strongly expressed in the STAD tissues compared to the adjacent tissues.Amplified amounts YKL-40,CD206 and MMP9 were mainly found in the interstitial areas of STAD tissues,while CD31 and Syndecan-1 were expressed mainly in the vascular beds.Western blotting results showed that the expression levels of NGF,TrkA,GAP43,TH,S100,β2-AR,YKL-40,syndecan-1,MMP9,CD206 and CD31 were all significantly higher in the STAD tissues than in the adjacent tissues(P < 0.005).HPLC-ED detected significantly high NE level in the STAD tissues compared to the adjacent tissues.Conclusion:Sympathetic nerves highly infiltrated into human STAD tissues as a result of high NGF and TrkA expressions.The infiltrated sympathetic nerves in the STAD tissues are likely affect the progression of STAD via the NE-β2-AR-YKL-40-MMP9/Syndecan-1 signaling.Part 3.Effects of β-AR signaling on the proliferation,migration and invasion of GC cells in vitro Objective:To explore the effects of β-AR activation on the proliferation,migration and invasion of GC cells in vitro and the underlying signaling.Methods:Two gastric cancer cell lines SGC-7901 and AGS were cultured in a six-well plate and divided into three groups: the control group,the ISO group(10 mol/L ISO)and the ISO+ICI group(10 μmol/L ISO and 25μmol/L ICI118,551(a selective β2-AR antagonist)).Cell expressions of NGF,STAT3,pSTAT3,ERK,pERK CHI3L1,MMP9,and syndecan-1 were examined by western blotting.SPSS22.0 software was used for statistical analysis,data were expressed as mean ± standard deviation(SD).Differences between groups were determined by ANOVA and t-test.P < 0.05 was considered statistically significant.Results:Compared with the control group,the proliferation,migration and invasion abilities of the two GC cell lines in the ISO group were significantly increased(P < 0.05).The expressions of NGF,pSTAT3,pERK,YKL-40,MMP9,and syndecan-1 were significantly increased in the ISO group.These effects of ISO were almost totally abolished by ICI118,551.Conclusion:β2-AR activation promotes the proliferation,migration and invasion of GC cells via the ERK/STAT3-YKL-40 signaling in vitro.GC cell is a also a source of MMP9 and syndecan-1 which promote matrix degradation,angiogenesis and GC metastasis.Summary of the study1.Dramatic sympathetic nerve infiltration occurred in the STAD tissues as a result of NGF,GAP-43 and TrkA high expression.2.The infiltrated sympathetic nerves released a lot of NE which activated β2-AR and its downstream signaling PKA-ERK/STAT3 and promoted the expression of YKL-40.YKL-40 then promoted the expression of MMP9 and syndecan-1,leading to matrix degradation,GC cell metastasis,angiogenesis and STAD progression.3.We were the first to use the gold-standard HPLC-ED to determine sympathetic neurotransmitter NE in the STAD tissues.4.At the GC cell level,the above signaling was also verified,and blocking β2-AR inhibited β2-AR activation-induced GC cell proliferation,migration and invasion,suggesting that β2-AR antagonism is a promising strategy in the treatment of STAD.5.The signaling by which sympathetic nerve infiltration promotes STAD may be: NE-β2-AR-PKA-ERK/STAT3-YKL-40-MMP9/syndecan-1. |
