Roles And Mechanisms Of Transcription Factor HMBOX1 In The Occurrence And Development Of Human Gastric Cancer

ObjectsGastric cancer is one of the most common cancer and leading cause of cancer death worldwide.The morbidity and mortality of gastric cancer both situated the second of all the tumors in China.Most patients were diagnosed at advanced stage owing to early-stage gastric cancer is often asymptomatic.Mestastatic is associated with the poor survival of advanced gastric carcinoma patients.It is urgent necessary to study the mechanisms of the occurrence and development of gastric cancer,such as proliferation and migration.HMBOX1(homeobox containing 1)was identified and isolated from a pancreatic cDNA library by Chen and his colleagues in 2006,and they proposed HMBOX1 to be a repressive transcriptional factor.HMBOX1 was one member of Homeobox family,which has been reported to be involved in the occurrence and progression of many tumors.However,the relationship between HMBOX1 and tumors has rarely been studied.Many Homeobox genes have been found to modulate cancer cell functions,such as cell proliferation and metastasis.Hox genes,a highly conserved subgroup of Homeobox family,have crucial roles in development,apoptosis and motility of cancer cells.HOX genes are also thought to be involved in maintenance and regulation of cancer stem cell function.Our study aimed to explore the roles and mechanisms of HMBOX1 on the proliferation,migration and cell cycel of gastric cancer cells,and to discuss the clinical application of HMBOX1 on gastric diagnosis and treatment.Methods1.IHC was used to investigate the HMBOX1 expression pattern in normal gastric gland epithelium and neoplastic tissues.The relationship between survival rates of patients with gastric cancer patients and HMBOX1 expression level was analyzed.2.Q-PCR and WB were used to investigate the mRNA and protein expression of different molecules.3.Immunofluorescence was conducted to study the localization and districbution of HMBOX1 in normal gastric epithelium cell line GES-1 and gastric cancer cell lines(N87,SGC-7901 and HGC-27).4.The expression of HMBOX1 mRNA and protein was negatively correlated with the differentiation degree of gastric cancer cells.5.MTT assay and transwell assay were performed to explore the proliferation and migration abilities of gastric cancer cell lines.6.HMBOX1 in SGC-7901 was upregulated with lentivirus system and down-regulated with adenovirus system.7.Cell cycle distribution was investigated by flow cytometry methods.8.The data were analyzed statistically with GraphPad software with suitable statistical methods.Results1.HMBOX1 expression pattern in normal gastric epithelium and neoplastic tissues(IHC):The HMBOX1 was mainly expressed in parietal cells of normal gastric epithelium.While in neoplastic tissue,HMBOX1 was maily expressed in nucleus of the cancer cells.The nucleus positive rate of neoplastic tissues(58.00%)was significantly higher than that of normal gastric glands(23.8%).2.The correlation between HMBOX1 expression and clinical manifestation:HMBOX1 nucleus positive expression was positively correlated with the clinical stage and lymph node metastases and negatively correlated with the survival rates.3.HMBOX1 expression and location in normal gastric epithelial cell line GES-1 and gastric cancer cell lines(N87,SGC-7901 and HGC-27):HMBOX1 expression in gastric cancer cell lines was higher than that in normal gastric epithelial cell line.Immunofluorescence results showed that HMBOX1 positive expressionwas maily in cytoplasm and nucleus of GES-1,N87 and HGC-27,while was in nucleus of SGC,4.The gastric cancer cell lines with high level expression of HMBOX1 had stronger cell proliferation and migration ability compared with that with low level expression of HMBOX1.5.The effect of HMBOX1 on proliferation and migration ability by HMBOX1 up-regulation and down-regulation:HMBOX1 up-regulation accelerated cell proliferation and migration ability and promoted the cell cycle from G0/G1 phase to G2/M phase.6.HMBOX1 upregulation was positive correlated with higher expression of VEGFR,TGF-β,CD133,especially CD133.7.The CD133 expression was correlated with HMBOX1 in gastric cancer.HMBOX1/CD133 double positive patients had worse prognosis,especially for patients in advanced TNM Stage Ⅲ or Ⅳ.ConclusionHMBOX1 positive expression in normal gastric epithelium was mainly in parietal cells.The HMBOX1 positive rate of normal gastric epithelium and normal gastric epithelial cell line was higher than that of neoplastic tissue and gastri cancer cell lines.HMBOX1 promoted the clinical stage and lymph node metastases and reduced the survival of gastric cancer patients.HMBOX1 accelerated cell proliferation and migration ability and promoted the cells from G0/G1 phase to G2/M phase by upregulating the expression of TGF-β,VEGFR,CD133.The survival of HMBOX1/CD133 double positive patients was lower than that of HMBOX1/CD133 negative patients.HMBOX1 might be a potential molecular target of diagnosis and treatment.