| Gastric cancer is one of the most common malignant tumors in China.However,the prognosis is still poor,despite the continuous improvement of surgical and postoperative adjuvant therapy.Recurrence and metastasis is the main cause of the poor prognosis.The recurrence and metastasis of tumor are the result of interaction of many factors and many genes.Angiogenesis,a multi-step and multifactorial process,is the key step of tumor development and metastasis.The putative receptor protein related to angiotensin type Ⅱ-1 receptor(APJ)is one of G protein coupled receptors(GPCR).Apelin,the endogenous ligand of APJ,was identified from bovine stomach extracts by Tatemoto et al.in 1998.Recently,the role of Apelin in tumor angiogenesis and tumor development has attracted more and more attention.It has been found that Apelin involved in the occurrence and development of a variety of malignant tumors through autocrine and paracrine.Through activing mitogen-activated protein kinases/extrallular signal regulatedprotein kinase(MAPK/ERK)and phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT)signaling pathways,Apelin can promote angiogenesis and lymphangiogenesis,proliferation,invasion and inhibit apoptosis in malignant tumor cell.It also has been confirmed that Apelin can promote angiogenesis and tumor proliferation in vivo experiments.In previous research,we have found that APJ can be used to predict the therapy response and prognosis in locally advanced gastric cancer patients receiving concurrent chemoradiotherapy+Endostar therapy.However,the expression level of Apelin in gastric cancer and its internal regulation mechanism have not been reported.On the basis of existing studies,firstly,our study focused on the expression of Apelin and its role in the occurrence,development and prognosis of gastric cancer.Then,the effect of Apelin on malignant biological behavior of gastric cancer cells and its molecular mechanism were investigated.Finally,the effect of Apelin expression on the proliferation of gastric cancer and its relationship with angiogenesis were further investigated by in vivo experiments.To lay the foundation for the study of Apelin as a prognostic marker and potential.target for molecular targeted therapy of gastric cancer.Part one The expression of Apelin in gastric cancer and its clinical significanceObjective1.To investigate the expression levels of Apelin and vascular endothelial growth factor(VEGF)in gastric caner.2.To analyze the correlation between the expression of Apelin and VEGF and microvessel density(MVD).3.To study the correlation between the expressions of Apelin and the clinicopathological factors and prognosis in gastric cancer patients.Methods1.A retrospective analysis of 178 patients with gastric cancer who underwent radical resection(D2 and Ro)and completed adjuvant treatment from January 2009 to December 2011 in Binzhou Medical University Hospital was carried out.We collected 178 pairs of cancer tissues and adjacent tissues.2.Immunohistochemical(IHC)Envision method was used to detect the expression levels of Apelin and VEGF proteins in gastric cancer tissues and adjacent tissues.According to the expression levels of Apelin and VEGF,they were divided into positive expression group and negative expression group.The CD34 mark is used to calculate the MVD.3.Chi-square test was used to analyze the relationship between the expression levels of Apelin and clinical pathological features.4.T test was used to analyze the relationship between the expression of Apelin and VEGF and MVD.5.Spearman rank correlation test was used to analyze the correlation between expression of Apelin and VEGF.6.Kaplan-Meier and COX proportional hazards model were used to analyze the role of Apelin expression in prognosis of gastric cancer.Results1.The Apelin was diffusely expressed in cytoplasm,the positive rate of cancer tissue was higher than that of adjacent tissues(49.44%vs 29.78%,P<0.01).The VEGF was also diffusely expressed in cytoplasm,the positive rate of cancer tissue was higher than that of adjacent tissues(49.44%vs 29.78%,P<0.01).2.The expression of Apelin was correlated with the vascular invasion,T stages,N stages,pathological type and nerve invasion(P<0.05),but was not related to gender,age and location of the disease(P>0.05).The expression of VEGF was correlated with the T stages,vascular invasion and N stages(P<0.05),but was not related to gender,age,location of the disease,pathological type and nerve invasion(P>0.05).3.The MVD of Apelin positive group is 33.086 ±7.862.The MVD of Apelin negative group is 21.071 ±6.320.The difference was statistically significant(P<0.05).The MVD of VEGF positive group is 29.075 ± 8.193.The MVD of VEGF negative group is 21.071 ±6.320.The difference was statistically significant(P<0.05).4.In gastric cancer tissue,the expression of Apelin and VEGF protein was positively correlated(rs=0.856,P<0.01).5.In Apelin negative group,the 1,3 and 5 year overall survival rates(OS)were 97.78%,76.67%and 53.33%,respectively.The 1,3 and 5 year OS of Apelin positive group were 97.73%,62.50%and 28.41%,respectively.The median survival time was 60.2 months vs 40.7 months.The difference was statistically significant(P<0.01).The 1,3 and 5 year progression free survival rates(PFS)in the Apelin negative group were 94.44%,65.56%and 40%,respectively.The 1,3 and 5 years PFS in the Apelin positive group were 96.60%,44.32%and 19.32%,respectively.The median progression time were 49.8months vs 30.5months.The difference was also statistically significant(P<0.01).6.COX single factor analysis showed Apelin,VEGF,T stages,N stages,vascular invasion,pathological type are correlated with prognosis(P<0.05),and age,gender,location of the disease and invasion of nerve are not correlated with prognosis(P>0.05).COX multivariate analysis showed that Apelin,VEGF,and vascular invasion were not independent poor prognostic factors,but N stages,T stages and pathological type were independent poor prognostic factors in this patiens.ConclusionsThe expression of Apelin in gastric cancer tissues is higher than that in adjacent tissues,and the expression of Apelin is related to vascular invasion,lymph node metastasis,T stages and expression of VEGF.Apelin may contribute to the growth and metastasis of gastric cancer by promoting angiogenesis,which results in poor prognosis.Part Two The effect of Apelin expression on malignant biological behaviors and its molecular mechanism in gastric cancer cells.ObjectiveTo clarify the expression of Apelin in gastric cancer cell lines.To investigate the effect of Apelin on malignant biological behavior in gastric cancer cells and its molecular mechanism.Methods1.The gastric cancer cell lines MGC-803,HGC-27 and SGC-7901 were cultured.2.The levels of Apelin mRNA,Apelin protein and secreted Apelin were detected by quantitative real-time reverse transcription polymerase chain reaction(Real time RT-PCR),Western blot and enzyme linked immunosorbent assay(ELISA),respectively.3.Construction of pcDNA3.1-Apelin and transiently transfected into gastric cancer cell line MGC-803 with liposome.4.MTT,Transwell and Annexin V-FITC/PI flow cytometry were used to detect the proliferation,invasion and apoptosis of gastric cell line MGC-803 before and after Apelin overexpression and Apelin-13(the major subtypes of Apelin)induction.5.Western blot was used to detect the expression of pERK,pAKT,Cyclin D1,and MMP-9 proteins in MGC-803 cell lines before and after Apelin overexpression and Apelin-13 induction.Results1.The relative expression level of Apelin mRNA in MGC-803,SGC-7901 and HGC-27 cell line was 1.000010.344,14.596±2.928 and 29.755±5.067,respectively.The difference of Apelin mRNA expression levels in the three cell lines were statistically significant(P<0.05).2.The relative expression level of Apelin protein in MGC-803,SGC-7901 and HGC-27 cell line was 0.150±0.067,0.420±0.102 and 0.873±0.114,respectively.The difference of Apelin protein expression levels in the three cell lines were statistically significant(P<0.05).3.The secreted Apelin in MGC-803,SGC-7901 and HGC-27 cell line was 132.000±31.97pg/ml,492.33±64.21pg/ml and 761.00±45.52 pg/ml,respectively.The difference of secreted Apelin in the three cell lines were statistically significant(P<0.05).4.The eukaryotic expression vector pcDNA3.1-Apelin of Apelin gene was successfully constructed by gene cloning technique and transfected into gastric cancer cell line MGC-803.5.Apelin-13 induction promoted the proliferation of gastric cancer cell line MGC-803,and in which the 2.5μmol/L was the best concentration.After induction,the difference of cell proliferation ability started from 12h and lasted to 48h.6.Apelin overexpression promoted the proliferation of gastric cancer cell line MGC-803.The difference of cell proliferation ability started from 24h and lasted to 48h after overexpression of Apelin.7.The number of transmembrane cells in untreated cells and Apelin-13 induced cells was 102.00±24.59 and 406.33±37.62,respectively.The difference was statistically significant(P<0.01).The number of transmembrane cells in pcDNA3.1 transfected cells and pcDNA3.1-Apelin transfected cells was 111.00±33.30 and 302.33±47.23,respectively.The difference was statistically significant(P<0.01).8.The apoptosis rate in untreated cells,suberoylanilide hydroxamic acid(SAHA)induced cells,SAHA+Apelin-13 induced cells,SAHA induced cells that transfected by pcDNA3.1 and SAHA induced cells that transfected by pcDNA3.1-Apelin were(7.300±1.745)%,(15.100±3.997)%,(14.000±3.899)%,(17.275±4158)%and(14.425±4.393)%,respectively.The apoptosis rate of MGC-803 was statistical difference before and after induction by SAHA(P<0.05),but there are not statistical difference before and after overexpression of Apelin or Apelin-13 induction(P>0.05).9.Apelin overexpression and Apelin-13 induction could result in up regulation of pERK,Cyclin D1 and MMP-9 in MGC-803 cells,but there was no significant change in the expression of pAKT.Conclusions1.The expression levels of Apelin in gastric cancer cell line MGC-803,HGC-27 and SGC-7901 were different.Apelin mRNA,protein and secreted Apelin in MGC-803 were the lowest among the three cell lines.2.Overexpression of Apelin and Apelin-13 induction promoted proliferation and invasion of gastric cancer cell line MGC-803.3.SAHA could induce apoptosis in gastric cancer cell line MGC-803,but overexpression of Apelin and Apelin-13 induction did not induce apoptotic resistance in this process.4.Apelin may work by activating the MAPK/ERK signaling pathway and up regulating the expression of Cyclin D1 and MMP-9.Part Three The effect of Apelin overexpression on the growth of gastric cancer xenografts in nude mice and its relationship with angiogenesisObjectiveTo investigate the effect of Apelin overexpression on the growth of gastric cancer xenografts in nude mice and to analyze its relationship with angiogenesis.Methods1.PLV-puro-Apelin stably transfected MGC-803 cell line and pLV-puro-NC stably transfected MGC-803 cell line were established by lentivirus mediated and puromycin screening.2.To establish gastric cancer xenografts model in nude mice by PLV-puro-Apelin stably transfected MGC-803 and pLV-puro-NC stably transfected MGC-803 cell line.3.The growth of gastric cancer xenografts in two groups nude mice was observed and the volume growth curve was plotted.4.After the mice were killed,the weight of gastric cancer xenografts was measured,and the proliferation rate was calculated.5.Immunohistochemical Envision method with CD34 marking was used to detect MVD in gastric cancer xenografts of nude mice.Results1.PLV-puro-Apelin stably transfected MGC-803 cell line and pLV-puro-NC stably transfected MGC-803 cell line were successfully established.2.14 nude mice were randomly divided into two groups:the control group and the Apelin overexpression group.PLV-puro-NC-MGC-803 and pLV-puro-Apelin-MGC-803 cells were inoculated respectively.The tumor formation rate was 85.7%(6/7)in the two groups.The nude mice showed decreased activity and poor eating condition in the Apelin overexpression group after 24 days,and the observation was terminated.3.At 7,10,14,17,20 and 24 days,the tumor volume(mm3)in the control group and the Apelin overexpression group were 22.656±8.702vs26,775±11.745(P>0.05),61.517±23.151vs80.836±26.352(P>0.05),106.115±36.515vs±96.010±66.854(P<0.05),306.789±105.315vs567.346±198.854(P<0.05),748.919±214.059vs1399.063±499.872(P<0.05),1367.348±444.701vs2557.645±684.409(P<0.01),respectively.4.The tumor weight in the control group and Apelin overexpression group was 1.70±0.43g and 2.96±0.61g,respectively.The difference was statistically significant(P<0.01).The proliferation rate was 42.5%.5.The MVD of gastric cancer xenografts in nude mice in control group and Apelin.overexpression group was 112.333±29.859 and 168.833±35.078,respectively.The difference was statistically significant(P<0.05).ConclusionsApelin overexpression can promote the proliferation of gastric cancer xenografts in nude mice,which may be related to the increase of MVD. |
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