| Background:Gastric cancer is the fifth most common cancer and the third most common cause of cancer death in the world.It is a highly heterogeneous tumor originating from gastric mucosal epithelial cells,with insidious onset and no obvious early symptoms.Excavating the underlying mechanism of GC,identifying potential biomarkers,and predicting the risk and prognosis are of great significance for the early detection of GC,which would finally improve the prognosis and find potential therapeutic targets.Tumor cells immune cells and tumor-associated stromal cells in the tumor microenvironment can secrete a variety of cytokines,which play an important role in the survival,invasion and metastasis of tumor cells,drug resistance to chemotherapy,and the maintenance of survival signals.Chemokines are a subfamily of cytokines,which are mainly responsible for the development of lymphoid tissues and the transport of immune cells.Based on the position of the first two cysteine residues in the major protein structure,they are classified into four classes,namely C,CC,CXC,and CX3 C chemokines.In addition,chemokines can act on non-immune cells,including tumor cells,stromal cells and vascular endothelial cells,and participate in a variety of cancer development processes,such as angiogenesis,tumor cell proliferation,invasion and metastasis,etc.It is a key factor for disease progression,and has a significant impact on patient prognosis and treatment response.CXCL13,a member of the CXC chemokine family,plays an important role in many pathological processes such as infection,inflammation and immune response.In recent years,CXCL13 has also been found to be closely related to the occurrence and development of tumors,but there are few studies on the relationship between CXCL13 and GC,and research on its clinical significance and mechanism on GC are rare till now.The purpose of this study is to clarify the clinical significance,biological function and mechanism of CXCL13 expression in gastric cancer,and to provide experimental basis and theoretical reference for finding new gastric cancer risk/prognostic markers or potential therapeutic targets for gastric cancer.Objectives:To clarify the relationship between CXCL13 and gastric cancer and its clinical significance as well as its effect on biological function of gastric cancer cells and its mechanisms.Materials and Methods:Part Ⅰ:The expression characteristics of CXCL13 in pan-cancer and the clinical significance of epithelial/stromal expression in gastric cancer1.The expression characteristics of CXCL13 in pan-cancer1.1 RNA-seq gene expression data and clinical and prognostic information of 33 different tumor samples were downloaded from TCGA database,and RNA-seq gene expression data of 31 normal human tissues(7858 samples)were downloaded from GTEx database.CXCL13 expression data in 24 different tumor cell lines were obtained from CCLE database,and the data of each dataset was standardized.1.2 Kruskal-Wallis test was used to analyze the expression of CXCL13 in 31 normal tissues and 24 tumor cell lines.Wilcox test was used to analyze the expression difference of CXCL13 in pan-cancer between para-cancerous tissues and normal tissues.Based on TCGA data,the correlation between CXCL13 expression and age,sex,pathological stage and other clinicopathological features of 33 kinds of tumors was analyzed.Kaplan-Meier method and univariate cox regression were used to analyze the relationship between CXCL13 expression and prognosis of 33 kinds of tumors,including overall survival(OS),disease-specific survival(DSS),progression-free interval(PFI)and disease-free interval(DFI).2.The correlation between the expression of CXCL13 epithelium / stroma and the risk and prognosis of gastric cancer2.1 A total of 431 gastric antrum biopsy and ESD specimens and gastric cancer and adjacent normal tissue specimens from patients who underwent gastroscopy at the Digestive Endoscopy Center of the First Hospital of China Medical University from 2012 to 2019 were collected,including 62 cases of superficial gastritis,94 cases of atrophic gastritis,129 cases of early gastric cancer and 146 cases of advanced gastric cancer.The collected tissues were embedded in paraffin and pathological sections were prepared.All tissue sections were histopathologically diagnosed by two experts in gastrointestinal pathology independently.Histopathological diagnosis was made according to the latest Sydney classification of gastritis and WHO classification of tumors of the digestive system(4th edition,2010).TNM staging of gastric cancer patients was determined according to the 8th edition of the AJCC Cancer staging manual.The clinicopathological parameters of postoperative gastric cancer patients were collected and followed up.In addition,25 fresh samples of gastric cancer and paired adjacent normal tissues resected at the Department of Surgery,the First Hospital of China Medical University in 2019 were collected.This study was approved by the Research Medical Ethics Committee of the First Hospital of China Medical University,and informed consent was signed.2.2 The expression of CXCL13 m RNA was detected by Real-time reverse transcription PCR(RT-q PCR),and the expression of CXCL13 protein was detected by immunohistochemistry.2.3 The expression of CXCL13 protein was detected by immunohistochemistry.2.4 Paired T test was used to investigate the difference of CXCL13 m RNA expression between gastric cancer and adjacent normal tissues.Mann-Whiney U non-parametric test was used to analyze the expression of CXCL13 in epithelium and stroma of different gastric disease groups.Paired nonparametric test was used to analyze the expression difference of CXCL13 in epithelium and stroma between gastric cancer and paracancerous tissues.Chi-square test was used to analyze the correlation between the expression of CXCL13 in epithelium and stroma and clinicopathological features of patients with gastric cancer.Correlation between expression of CXCL13 in epithelium and stroma and prognosis of patients with gastric cancer was analyzed by Kaplan-Meier,univariate and multivariate Cox regression.3.Evaluation of CXCL13 expression as a risk marker for gastric cancerThe diagnostic efficacy of CXCL13 epithelial expression,CXCL13 interstitial expression and epithelial-mesenchymal co-expression in gastric cancer was evaluated by receiver operating characteristic(ROC)curve.4.Construction of nomogram model based on CXCL13 expression in gastric cancer patientsIndependent prognostic indexes were screened by multivariate Cox regression analysis,the factors such as gastric cancer epithelial CXCL13 expression,gastric cancer stromal CXCL13 expression,extra-nodal tumor implantation,lymph node metastasis,p TNM stage and tissue classification were included to construct the nomogram of gastric cancer.The time-dependent area under ROC curve(AUC)was used to test whether the prognostic nomogram model could accurately predict the prognosis and survival of patients with gastric cancer.Calibration curve(calibration curve)and consistency index(C index)were used to evaluate the effectiveness of the model.Part Ⅱ.The role of CXCL13 expression in promoting the malignant biological behaviors of gastric cancer cells1.Screening of cell lines and construction of plasmidsCXCL13 overexpression and negative control plasmids were constructed,and plasmids were then extracted and transiently transfected into gastric cancer cells(AGS and HGC27 cells bought from the Cell Bank of Chinese Academy of Medical Sciences)to construct gastric cancer cell lines with CXCL13 overexpression.The transfection efficiency was observed under inverted fluorescence microscope.RT-q PCR was used to confirm its overexpression at m RNA level and Western Blot at intracellular protein level.2.The effect of CXCL13 expression on the proliferation of gastric cancer cellsThe effect of CXCL13 on the activity of gastric cancer cells was observed by CCK-8test and the effect of CXCL13 on the proliferation of gastric cancer cells was observed by Ed U staining experiment.3.The effect of CXCL13 expression on apoptosis of gastric cancer cellsThe effect of CXCL13 on apoptosis of gastric cancer cells was detected by flow cytometry after hochest staining and Annexin-V/PI staining,and the effect of CXCL13 on the expression of pro-/ anti-apoptotic proteins was observed by Western Blot experiment.4.The effect of CXCL13 expression on the migration and invasion of gastric cancer cellsThe migration ability of gastric cancer cells was detected by scratch healing test and transwell migration test,and the invasive ability of gastric cancer cells was detected by transwell invasion test.5.The effect of CXCL13 expression on EMT expression in gastric cancer cellsWestern Blot experiments was used to observe the effect of CXCL13 on the expression of EMT(Epithelial-mesenchymal transformation)-related molecular markers.6.The effect of CXCL13 expression on the vasculogenic mimicry ability of gastric cancer cellsImmunohistochemistry and CD31/PAS double staining were used to detect the expression of CXCL13 and VM in 31 cases of gastric cancer tissues.The correlation between CXCL13 expression and VM was analyzed.The effect of CXCL13 on the vasculogenic mimicry ability of gastric cancer cells was observed by Matrigel three-dimensional culture experiment,including the number of formed tubes,the total length of tubes and the number of branches.Part Ⅲ.The mechanisms of CXCL13 expression promoting the malignant biological behaviors of gastric cancer cells1.Bioinformatics analysis of CXCL13 enrichment pathway in gastric cancerTCGA and GEPIA databases were used to analyze and screen the key genes related to the CXCL13 and differentially expressed in gastric cancer tissues,and KEGG method was used to enrich the downstream signaling pathways.GEPIA database was used to analyze the correlation between CXCL13 and the enriched downstream signaling pathway key genes.The pathways that may be regulated by CXCL13 and affect the malignant biological behavior of gastric cancer cells were screened for subsequent verification.2.Validation of the CXCL13 related pathwaysGastric cancer cell lines transfected with CXCL13 overexpression plasmid and negative control cell lines were used.Western Blot was used to detect the expression of PI3 K,p-PI3 K,AKT,p-AKT and NFKB/p65,p-NFKB/p65,IKBα and p-IKBα in the downstream signaling pathway of PI3K-AKT.3.The mechanism of CXCL13 promoting the proliferation of gastric cancer cellsand the formation of vascular mimicryCXCL13 overexpression plasmid was transfected into gastric cancer cell lines and negative control cell lines,and PI3K-AKT signaling pathway inhibitor LY294002 was used for intervention.Western Blot was used to detect the changes in pathline-related proteins.Ed U and Matrigel three-dimensional culture experiments were used to detect the proliferation and vasculogenic mimicry formation ability of gastric cancer cells to determine whether LY294002 could reverse the promoting effect of CXCL13 on the proliferation and vasculogenic mimicry of gastric cancer cells.4.The mechanism of CXCL13 promoting the migration and invasion of gastric cancer cellsCXCL13 overexpression plasmid was transfected into gastric cancer cell lines and negative control cell lines,and NFKB signaling pathway inhibitor QNZ was used for intervention.Western Blot was used to detect the changes of pathway-related proteins.The migration and invasion abilities of gastric cancer cells were detected by wound healing assay,transwell migration and invasion assay.To determine whether QNZ could reverse the inhibitory effect of CXCL13 on gastric cancer cell migration and invasion.ResultsPart Ⅰ:The expression characteristics of CXCL13 in pan-cancer and the clinical significance of epithelial/stromal expression in gastric cancer1.The expression characteristics of CXCL13 in pan-cancer1.1 The results of pan-cancer expression analysis showed that the expression of CXCL13 was at a low level in most normal tissues,and the relative expression level of CXCL13 in most cell lines was at a medium-to-low level.The expression of CXCL13 in24 kinds of tumors was significantly different from that in normal tissues.The CXCL13 expression levels of 22 cancer types(squamous cell carcinoma of the head and neck,cutaneous melanoma,low-grade glioma of the brain,glioblastoma multiforme,adenocarcinoma of the lung,squamous cell carcinoma of the lung,thyroid carcinoma,adrenocortical carcinoma,clear cell carcinoma of the kidney,invasive breast cancer,squamous cell carcinoma and adenocarcinoma of the cervix,serous cystadenocarcinoma of the ovary,endometrial carcinoma and carcinosarcoma of the uterus,germ cell tumor of the testis,acute myeloid leukemia,cholangiocarcinoma,hepatocellular carcinoma,esophageal carcinoma,pancreatic carcinoma,colon adenocarcinoma,gastric adenocarcinoma)increased significantly(P<0.05),while the expression levels of two cancer types(Prostate cancer and rectal adenocarcinoma)decreased significantly(P<0.05).1.2 CXCL13 and pan-cancer clinicopathological features analysis showed that CXCL13 was correlated with age of patients with four types of cancer(P<0.05);Among them,the expression of CXCL13 in bladder urothelial carcinoma and renal chromophobe cell carcinoma in the older group was significantly higher than that in the younger group,while the expression of CXCL13 in invasive breast cancer and thyroid cancer in the older group was significantly lower than that in the younger group.CXCL13 was correlated with the gender of patients with three types of cancer(P<0.05);The expression of CXCL13 was significantly higher in male lung adenocarcinoma patients than in female lung adenocarcinoma patients,and was significantly higher in female pheochromocytoma and paraganglioma than in male lung adenocarcinoma patients.CXCL13 was mainly correlated with TNM stage of five cancers(P<0.05),including thyroid carcinoma,renal clear cell carcinoma,renal papillary cell carcinoma,cutaneous melanoma,and gastric adenocarcinoma.1.3 Pan-cancer prognostic analysis of CXCL13 showed that CXCL13 was a protective factor for the prognosis of invasive breast cancer,endometrial cancer,head and neck squamous cell carcinoma,cutaneous melanoma,ovarian serous cystadenocarcinoma,bladder urothelial carcinoma,cervical squamous cell carcinoma,cervical adenocarcinoma and lung squamous cell carcinoma(P<0.05,HR<1),but it is a risk factor for the prognosis of renal papillary cell carcinoma,renal clear cell carcinoma,glioblastoma multiforme,thymoma,and uveal melanoma(P<0.05,HR>1),.2.The correlation between the expression of CXCL13 epithelium/stroma and the risk and prognosis of gastric cancer2.1 RT-q PCR results showed that the expression of CXCL13 m RNA in gastric cancer tissues was significantly higher than that in paired adjacent normal tissues(P<0.01).2.2 Immunohistochemical staining results showed that CXCL13 was expressed in the epithelial and stromal tissues of different gastric diseases.There was a gradual upward trend from non-atrophic gastritis to atrophic gastritis to gastric cancer(non-atrophic gastritis vs.Atrophic gastritis: P<0.001;Atrophic gastritis vs.gastric cancer: P<0.001;Nonatrophic gastritis vs.gastric cancer: P<0.001),and its expression in gastric cancer tissues was significantly higher than that in adjacent tissues(P<0.001).2.3 Epithelial CXCL13 expression was significantly correlated with TNM stage(P=0.029)and lymph node metastasis(P=0.012).Stromal CXCL13 expression was significantly correlated with tumor diameter(P=0.030),depth of invasion(P=0.023),TNM stage(P=0.020)and lymph node metastasis(P=0.002).2.3 K-M analysis showed that high CXCL13 epithelial/stromal expression were significantly associated with poor prognosis of gastric cancer.Univariate and multivariate cox analysis showed that CXCL13 was epithelial(P=0.002),stromal(P<0.001)were independent risk factors for poor prognosis of gastric cancer.3.Evaluation of CXCL13 as a risk marker for gastric cancerThe area under the ROC diagnostic curve(area under curve,AUC)showed that compared with the expression of CXCL13 in stroma,the expression of CXCL13 in epithelium had better diagnostic efficacy in gastric cancer(epithelial CXCL13-AUC=0.908,stromal CXCL13-AUC=0.702).4.Construction of a nomogram model based on CXCL13 expression in gastric cancer patientsThe prognostic nomogram model of gastric cancer was constructed based on epithelial CXCL13 expression,stromal CXCL13 expression,extra-nodal tumor implantation,lymph node metastasis,p TNM and histological type.The time-dependent ROC curve of OS was used to test whether the nomogram model could accurately predict the prognosis of GC patients.Calibration curve and consistency index(C index)were used to evaluate the effectiveness of the model.The AUC values of 1-year,3-year and 5-year overall survival rates were 0.863,0.897 and 0.916,respectively,indicating that this model can accurately predict the prognosis of patients with gastric cancer.The C index was 0.835(95%CI=0.782-0.888),and the calibration curve did not deviate from the reference line,indicating that the prediction results of the model were in good agreement with the actual observation results,and the prediction accuracy of the model was good.Part Ⅱ.The role of CXCL13 expression in promoting the malignant biological behaviors of gastric cancer cells1.Screening of cell lines and construction of plasmidsCXCL13 overexpressed plasmid was constructed and plasmid transfection and follow-up function experiments were carried out with AGS cells and HGC27 cells.RTq PCR and Western Blot confirmed that it was successfully overexpressed at m RNA level and intracellular protein level.2.CXCL13 can promote the proliferation of gastric cancer cellsIn AGS and HGC27 cells,the effect of CXCL13 on the proliferation and activity of gastric cancer cells was observed by CCK-8 assay and Ed U staining,respectively.The results of CCK-8 assay revealed that CXCL13 did not affect the activity of gastric cancer cells.The results of Ed U assay confirmed that CXCL13 could significantly promote the proliferation of gastric cancer cells(AGS,OE-con vs.OE-CXCL13,P<0.05;HGC27,OEcon vs.OE-CXCL13,P<0.001).3.CXCL13 can inhibit the apoptosis of gastric cancer cells to a certain extentIn AGS and HGC27 cells,flow cytometry analysis was performed on the experimental group and the control group after Annexin-V/PI staining,The results showed that the number of apoptotic cells decreased to a certain extent after CXCL13 overexpression,but no significant difference was observed.Hoechst staining analysis also showed that CXCL13 overexpression reduced the apoptotic cell rate to some extent,but it did not reach significant difference.The results of Western Blot assay suggested that CXCL13 could inhibit the expression of pro-apoptotic proteins Cleaved-caspase3(P<0.05)and Bad(P<0.05)in gastric cancer cells.4.CXCL13 can promote the migration and invasion of gastric cancer cellsIn AGS and HGC27 cells,the results of scratch assay showed that CXCL13 could significantly promote the migration rate of gastric cancer cells.Transwell experiment found that CXCL13 could significantly promote the migration(AGS:P<0.01;HGC27:P<0.01)and invasion(AGS:P<0.01;HGC27:P<0.01)of gastric cancer cells.5.CXCL13 can promote EMT of gastric cancer cellsWestern Blot was used to detect the protein expression levels of epithelialmesenchymal transition(EMT)related markers(E-cadherin,N-cadherin,MMP2,Vimentin,Snail,Slug).The results showed that CXCL13 could up-regulate the expression of MMP2(P<0.01),Vimentin(P<0.05)and Snail(P<0.01),down-regulate the expression of E-cadherin(P<0.01)in HGC27 cells.CXCL13 could up-regulate Snail(P<0.001)expression,down-regulate E-cadherin(P<0.001)expression in AGS cells.6.CXCL13 can promote vasculogenic mimicry formation of gastric cancer cellsIn 31 cases of gastric cancer,VM structure was found in 15 cases by CD31/PAS double staining,and the positive rate was 48%.CXCL13 epithelial expression was correlated with VM(r=0.49,P=0.011).The positive rate of mimicry formation in the CXCL13 epithelial high expression group was higher than that in the CXCL13 epithelial low expression group.There was no significant correlation between the stromal expression of CXCL13 and VM(r=0.23,P=0.285).In both AGS and HGC27 cells,matrigel three-dimensional culture experiment showed that the number of formed tubes(AGS:P<0.01;HGC27:P<0.001),the total length of tubes(AGS:P<0.01;HGC27:P<0.001)and the number of branches(AGS:P<0.01;HGC27:P<0.01)increased significantly after CXCL13 overexpression.Western Blot result showed CXCL13 can up-regulate the expression of VE-cadherin(P<0.001).Part Ⅲ.The mechanisms of CXCL13 expression promoting the malignant biological behaviors of gastric cancer cells1.The related and differentially expressed genes of CXCL13 in gastric cancer were screened by TCGA database,and GEPIA database.KEGG enrichment analysis was carried out on the genes obtained after intersection.The results suggested that these genes mainly enriched in cytokine-cytokine receptor pathway,chemokine pathway,NF-Kappa B signal pathway,EB virus infection pathway,PI3K-AKT signal pathway,PD-L1-PD1 signal pathway and so on.Furthermore,we analyzed the correlation between CXCL13 and the key genes of the enriched downstream signal pathway in gastric cancer by GEPIA database,and found that CXCL13 had a strong correlation with PI3K-,AKT-and NF-KB related genes.2.CXCL13 up-regulates the expression of proteins related to PI3K-AKT pathway and NF-Kappa B pathway2.1 The expression of markers of PI3K-AKT signaling pathway including PI3 K,pPI3K,AKT,p-AKT were detected by Western Blot.The results suggested that the expression of p-PI3 K and p-AKT increased in AGS and HGC27 cells after overexpression of CXCL13(P<0.001).2.2 The expression of NF-KB pathway markers including NF-KB/p65,p-NF-KB/p65,IKB α and p-IKB α was detected by Western Blot.The results showed that the expression of p-NFKB/p65 and p-IKB α in AGS and HGC27 cells increased after overexpression of CXCL13(P<0.05).3.CXCL13 can significantly promote the proliferation and vasculogenic mimicry ability of gastric cancer cells by activating PI3K-AKT pathway.3.1 The optimal concentration of PI3K-AKT pathway inhibitor LY294002 was detected by Western BlotAfter overexpression of CXCL13,gastric cancer cells were added with concentration gradient LY294002(0μM,10μM 30μM and 50μM).Western Blot was used to detect the expression of key proteins in PI3K-AKT pathway.The results showed that after addition of 30μM and 50μM LY294002,The expressions of p-PI3 K and p-AKT were significantly decreased(P<0.05).The concentration of 30μM was selected for the follow-up experiment.3.2 The effects of CXCL13 on the proliferation and vascular mimicry of gastric cancer cells could be reversed by the PI3K-AKT pathway inhibitor LY294002Gastric cancer cells overexpressing CXCL13 were treated with 30μM PI3K-AKT pathway inhibitor LY294002.Ed U staining and matrigel three-dimensional culture experiments were used to detect the proliferation and vasculogenic mimicry formation ability of gastric cancer cells.The results showed that treatment with LY294002,a protein kinase inhibitor of PI3K-AKT signaling pathway,reversed the effect of CXCL13 on gastric cancer cell proliferation and vasculogenic mimicry(P<0.05).4.CXCL13 can significantly promote the migration/invasion of gastric cancer cells by activating NF-KB pathway4.1 Western Blot detection of the optimal inhibition concentration of NFKB inhibitor QNZGastric cancer cells overexpressing CXCL13 were treated with 0n M QNZ,10 n M QNZ and 20 n M QNZ.Western Blot analysis showed that after 10 n M and 20 n M QNZ were added,the protein expressions of p-NFKB/p65 and p-IKBα in AGS and HGC27 cells were significantly inhibited(P<0.05).The 10 n M concentration was selected for subsequent experiments.4.2 CXCL13 can significantly enhance the migration and invasion ability of gastric cancer cells by NFKB inhibitor QNZAfter CXCL13 overexpression,gastric cancer cells were treated with 10 n M NFKB inhibitor QNZ.The migration and invasion ability of gastric cancer cells were detected by scratch healing assay and transwell assay.The results showed that the enhancement of migration and invasion of gastric cancer cells by CXCL13 was reversed after the use of NFKB inhibitor QNZ.Conclusions:1.The pan-cancer expression profile of CXCL13 was mapped in this study.Panorama showed that CXCL13 was highly expressed in most tumors and was closely related to cancer risk.The effect of CXCL13 on the prognosis of cancer patients has a "double-edged sword" effect,and it may play opposite roles in different tumors.2.CXCL13 was expressed in both epithelial and mesenchymal sites in different gastric diseases.The expression of CXCL13 in gastric cancer tissues was significantly higher than that in adjacent tissues.The results of ROC curve evaluation confirmed that CXCL13 epithelial expression had better diagnostic efficacy for gastric cancer,and CXCL13 had potential value as a risk marker for gastric cancer.3.CXCL13 expression was positively correlated with TNM stage and lymph node metastasis of gastric cancer patients,and was significantly correlated with poor prognosis of gastric cancer,which was an independent risk factor for the prognosis of gastric cancer.The prognostic nomogram model of gastric cancer based on epithelial CXCL13epithelial/stromal expression and related clinicopathological parameters had good predictive performance.4.CXCL13 expression could promote the proliferation,migration and invasion of gastric cancer cells,and could promote EMT and vasculogenic mimicry formation of gastric cancer cells.CXCL13 played a role in promoting the malignant biological behavior of gastric cancer cells.5.CXCL13 could promote the proliferation and vasculogenic mimicry formation of gastric cancer cells by activating PI3K-AKT pathway.CXCL13 could promote the migration and invasion of gastric cancer cells by activating the NF-kappa B pathway. |
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