The Studies Of PBX3 Effects In Gastric Cancer And Its Molecular Mechanisms

It's urgent to protect human health by preventing tumor progression,because malignant tumors are the major diseases that threaten human health in the worldwide.And gastric cancer is one of the most malignant tumors in the world.The morbidity of gastric cancer is next only to morbidity of lung carcinoma in our country and the morbidity of gastric cancer is in the second place,and gastric cancer is the primary cause in cancer-related death in our country.The morbidity of gastric cancer stands first on the list in the East Asia,especially in China,Mongolia,Japan and Korean peninsula.Therefore the study of occurrence and development of gastric cancer carries important significance for diagnosis and treatment of gastric cancer.The pre-leukemia transcription factor 3(PBX3)is a member of the PBX family of transcription factors,which is known to encode transcription PBX3 protein,and wide expressed in multiple tissues.HOX genes play a normal biological function role in evolution of cells and are indispensable in the growth of body.Howerve,when these genes are abnormal,such as bases deletion,bases insertion and bases translocation,tumours can be made occur and be promoted.PBX proteins can interact with HOX proteins leading to increase HOX/DNA binding ability,thereby enhance the transcription of the downstream target genes,regulating biological behaviors of cells.These effects carry important functions in regulating occurrence and development of tumors.In our study,we investigated the effects of PBX3 protein in gastric cancer and its underlying potential molecular mechanisms.In this study,we used real time quantification polymerasechain reaction(q RT-PCR)and immunohistochemistry assays to examine the expression levels of PBX3 protein in gastric cancer tissues and the results indicated that PBX3 protein was overexpressed in gastric cancer tissues compared with paired adjacent non-tumorous tissues.And the expression of PBX3 gene is positively associated with depth of invasion and lymph node metastasis.To screen out high expression level and low expression level of PBX3 protein in cancer cells,western blott assay was taken to determine.Then we constructed overexpressed PBX3 vector and down-regulated PBX3 vector,and transfected them to gastric cancer cells.Overexpressed PBX3 gene gastric cancer cells SGC-7901-PBX3 and down-regulated PBX3 MKN-45-sh/PBX3 gastric cancer cells were obtained for followed tests.CCK-8,colony formation,transwell,tube formation,and gelatin zymography assays were performed to examine the effects of PBX3 gene in gastric cancer,and the results showed that gene PBX3 promoted gastric cancer migration and invasion,and tube formation of human umbilical vein endothelial cells,and stimulated the proliferation of gastric cancer cells.Thus PBX3 gene promoted development of gastric cancer.We then found that PBX3 gene promoted peritoneal spreading.Abdominal cavity metastasis models of nude mice were made in vivo assay,and mice were euthanized after 4 weeks.Furthermore,western blott result showed that PBX3 gene enhanced phosphorylation of AKT 473 and induced gastric cacner EMT.Taken together,PBX3 protein is upregulated in gastric cancer and indicates poor prognosis of gastric cancer patients.The tube formation,migration,invasion and peritoneal spreading ability were elevated by PBX3 protein in gastric cancer.The potential molecular mechanisms of PBX3 gene promoting gastric cancer metastasis maybe that PBX3 gene regulates gastric cancer metastasis by inducing EMT via AKT signal pathway.Thus PBX3 gene is able to be a potential neobiomarker and prognostic indicator in gastric cancer targeted treatment.Chapter? The expression level of PBX3 in gastric cancer tissues and its expression clinical significance Objectives:The goal of this study was to definite the expression level of PBX3 protein in gastric cancer,and analyze the relationship between PBX3 protein expression and clinicopathological parameters of gastric cancer patients.Methods:A number of 60 cases samples were gathered from postoperative gastric cancer patients,including gastric cancer tissues and adjacent non-tumorous tissues.One of them was used to be paraffin embedded and others were taken to extract RNA of tissues.Then immunohistochemical assay and q RT-PCR assay were performed to determine the levels of PBX3 protein in gastric cancer tissues and adjacent non-tumorous tissues.Next,statistical analysis was used to analyze the relationship between PBX3 protein expression and clinicopathological parameters.Results:PBX3 positive rate was 41.67%(25 / 60)in tumorous tissues,and the positive rate was 16.67%(10 / 60)in non-tumorous tissues.The difference was statistically significant(P<0.05).Real time quantitative PCR experiments showed that the expression level of PBX3 in gastric cancer was 0.7955±0.05261,and the expression level of adjacent tissues was 0.3760±0.03008(n=20,P<0.0001).The expression of PBX3 protein was significantly different(P<0.05)from the depth of tumor invasion and lymph node metastasis in patients with gastric cancer,but there was no significant difference in age,sex,tumor location and tumor size between the patients(P>0.05).Conclusions:PBX3 was overexpressed in gastric cancer compared with adjacent non-tumorous tissues,and the expression of PBX3 protein is positively associated with the depth of invasion and lymph node metastasis and indicated poor prognosis of gastric cancer patients.Chapter ? Overexpressed and down-regualated PBX3 gastric cancer cell vectors construction Objectives:To better perform continuous tests,stable gastric cancer cells transfected with overexpressed PBX3 gene and downregulated PBX3 gene vectors were constructed.Methods:RNA and proteins were extracted from gastric cancer cells,and then were used to reverse transcription,semi-quantitive PCR and western blot for determining the levels of PBX3 in gastric cancer cells.PBX3 protein targeted gene was amplified andsequenced correct.Then targeted gene PBX3 protein was inserted into p CMV6-EGFP vector and p CMV6-EGFP/PBX3 vector plasmids were obtained.This plasmid was transfected into SGC-7901 cells which expressed low level PBX3 protein and G 418 was used to stimulate gastric cancer cells to screen out gastric cancer cells with stable overexpressing PBX3 protein.Western blot assay was used to examine the effect of transfection.Meanwhile,based on the PBX3 gene information,sh RNA sequences were designed and packaged into lentivirus p L/sh RNA/PBX3.MKN-45 gastric cancer cells which expressed high level PBX3 protein were transfected with lentivirus p L/sh RNA/PBX3.Puromycin was used to stimulate gastric cancer cells to screen out cells with stable downregulating PBX3 protein and then western blott assay was used to examine the effect of transfection.Results:PBX3 protein was in a high expression level in MKN-45 and a low expression level in SGC-7901 gastric cancer cells.SGC-7901 was transfected with p CMV6-EGFP/PBX3 vector and MKN-45 was transfected with lentivirus p L/sh RNA/PBX3.Western blot assays showed that the content of PBX3 protein was increased in p CMV6-EGFP/PBX3 gastric cancer cells,while the content was decreased in p L/sh RNA/PBX3 gastric cancer cells.Conclusions:SGC-7901-PBX3 overexpressed PBX3 protein gastric cancer cells and MKN-45-sh/PBX3 downregulated PBX3 protein gastric cancer cells were succeed constructed.Chapter ? Effects of PBX3 on proliferation,migration,invasion and angiogenesis of gastric cancer in vitro and in vivo study Objectives: To investigate the functions of PBX3 gene in gastric cancer,and to determine the effects of PBX3 gene on proliferation,colony formation,migration and invasion and tube formation in gastric cancer.Methods: SGC-7901-PBX3 and MKN-45-sh/PBX3 gastric cancer cells were used inTranswell,CCK-8,colony formation and endothelial cells tube formation assays to examine the biological functions of PBX3 gene in gastric cancer.And to test the effect of PBX3 gene on peritoneal metastasis nodules,abdominal cavity metastasis models of nude mice were made in vivo assay.Results: CCK-8 cell proliferation assay showed that the number of cells in SGC-7901-PBX3 gastric cancer cells was higher than that in the control group(P=0.0476),and the number of cells in MKN-45-sh/PBX3 gastric cancer cells was lower than that in the control group(P=0.0277).Colony formation,transwell and endothelial cells tube formation assays showed the same situation.In vivo assay,the number of metastatic nodule was higher than that in the control group.Conclusions: PBX3 gene promoted gastric cancer migration and invasion,and tube formation of human umbilical vein endothelial cells,and stimulated the proliferation and colony formation of gastric cancer cells.Furthermore,PBX3 gene promoted peritoneal metastasis spreading in vivo assay.Chapter ? The study of molecular mechanisms of PBX3 effects on metastasis and angiogenesis in gastric cancer Objectives: To explore potential molecular mechanisms of PBX3 promoting gastric cancer,providing novel ideas and basic studys for gastric cancer diagnosis and treatment.And exploring valid tumor biomarker and targeted therapy.Methods: Proteins were extracted from gastric cancer cells using RIPA with phosphatase inhibitors and next western blot assay was performed to examine phosphorylation of AKT 473 induced by PBX3 gene.To investigate the effect of PBX3 protein on EMT,E-cadherin,N-cadherin and Vimentin were determined at protein level and RNA level using western blot and q RT-PCR assays.To explore the mechanisms of tube formation and gastric cancer metastasis,we used gelatin zymography assay toexamine the activity of MMP-9 in tumor supernatant.Results: Compared with control groups,PBX3 gene promoted AKT 473 phosphorylation and reduced E-cadherin expression and increased N-cadherin,Vimentin expression in gastric cancer cells.Thus PBX3 gene induced gastric cancer cells EMT.The result of gelatin zymography assay showed that PBX3 gene increased the activity of MMP-9 in tumor supernatant of gastric cancer cells.Conclusions: PBX3 gene regulated EMT process may through PI3K/AKT signal pathway in gastric cancer.