| Background and ObjectiveThe transcription factor HOXD9 is one of the members of the HOX family,which is correlated with the regulation of many cellular processes such as cell proliferation,apoptosis,cell morphology and cell migration.It has been found that HOXD9 was associated with several cancers.However,the role of HOXD9 in the progression of gastric cancer(GC)remains to be elucidated.The RUN and FYVE domain-containing protein 3(RUFY3)plays an important role in multiple Ras-like GTPase tumor pathways.Our previous study has implicated RUFY3 as an oncogenic factor which induced the invasion and metastasis of colorectal cancer.We analyzed the RUFY3 interaction with the proximal promoter using Promo software and have found a HOXD9 sequence-specific binding site.In this study,we sought to determine the role of HOXD9 in the growth,migration and invasion of GC cells.We investigate whether HOXD9 directly regulates RUFY3 expression in GC cells,and the molecular mechanism of HOXD9 in the proliferation and metastasis of GC.MethodsFirst of all,the expression of the HOXD9 protein level in gastric cancer cells and gastric cancerous tissues were tested by Western Blot.We investigated the expression of HOXD9 protein with a 90-primary GC tissue microarray by using immunohistochemistry(IHC),and analyzed the correlation between HOXD9 expression and the pathologic features of GC.Moreover,we assessed the prognostic effect of HOXD9 on overall survival by comparing the overall survival of GC patients with high or low HOXD9 protein levels.Secondly,by transferring lentivirus and plasmid with HOXD9 and then stable transfectants were constructed.Given the increased expression of HOXD9 in the GC cells,colony forming assay,Cell Counting Kit-8(CCK-8)assay,EdU incorporation assay,Transwell invasion experiment,wound healing experiment and gelatin zymography were used to evaluated the functional effect of HOXD9 overexpression on cell behaviors in vitro.The dual-luciferase reporter assay and chromatin Immunoprecipitation assay(ChIP)were used to detect whether the transcription factor HOXD9 could directly bind to the RUFY3 promoter.Spearman’s correlation analysis was used to determine the relationship between HOXD9 and RUFY3 protein expression by evaluating consecutive primary GC sections.By building mouse tumorigenesis model and orthotopic mouse metastatic model,H&E,IHC and qRT-PCR were used to clarify the molecular mechanism of HOXD9-RUFY3 on gastric cancer in vivo.Results1.HOXD9 is markedly overexpressed in GC cell lines and gastric cancer tissues.2.The high expression of HOXD9 is correlated with poor survival in GC patients and HOXD9 expression is associated with differentiation,lymph node metastasis,tumor size,AJCC stage and TNM stage,but no significant association was observed between HOXD9 expression and age and gender.3.Functionally,overexpression of HOXD9 significantly promotes the proliferation,invasion and migration of GC cells.4.Mechanically,RUFY3 is a direct target of transcriptional activation by HOXD9.The dual-luciferase reporter and ChIP assay shows that HOXD9 transactivates the RUFY3 promoter and increases the transcriptional activity of RUFY3.5.Moreover,both HOXD9 and RUFY3 are highly expressed in gastric cancer tissues but not in normal gastric tissues,with their expressions being positively correlated.6.Inhibition of RUFY3 attenuates the proliferation,migration and invasiveness of HOXD9-overexpressing GC cells in vitro and in vivo.ConclusionsHOXD9 is overexpressed in GC cells and tissues.We presented a novel molecular basis for the role of HOXD9 in GC carcinogenesis,invasion and metastasis.Kaplan-Meier analysis of the survival curves showed a significantly worse overall survival for patients whose tumor had high HOXD9 levels.The HOXD9 protein could enhance the malignant properties of tumor cells via its transactivation of the RUFY3.The evidence presented here suggests that the HOXD9-RUFY3 axis promotes the development and progression of human GC. |
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