| Objectives:Death domain-associated protein(DAXX)plays an important role as an apoptotic regulator and transcriptional regulator in a variety of physiological and pathological processes.Early studies of DAXX was mainly focused on its effect on apoptosis and recent studies found that DAXX is also involved in the development of many different types of cancers.However,the role of this molecule in gastric cancer remains unknown.Method:(1)In order to explore the clinical significance of DAXX,we analyzed the expression level of DAXX in 83 pairs of gastric cancer samples by Western blot and classified the patients according to the clinical stage.(2)To examine the effect of DAXX on gastric cancer cells,we examined the expression of DAXX in three human gastric cancer cell lines:MKN45,N87,and AGS.In addition,we used lentivirus to overexpress or knock down DAXX in human gastric cancer cell lines to determine the effect of DAXX on the expression of cancer stem cell markers CD44,CD24,and OCT4.(3)We overexpressed and knocked down DAXX in human gastric cancer cell lines to examine the effect of DAXX modulation on the migration and invasion ability of gastric cancer cells.We also examined the impact of DAXX on the ability of cancer cells undergoing epithelial-to-mesenchymal transition(EMT).(4)To explore how DAXX affects EMT in gastric cancer cells,we examined the changes in the expression of EMT-related factors such as SNAI,ZEB,and GRHL2 in gastric cancer cells with DAXX overexpression or silencing.(5)We used IP,ChIP,immunofluorescence and other techniques to further explore the molecular mechanisms by which DAXX affects EMT-related factors in human gastric cancer cells.(6)We performed mouse subcutaneous tumor formation assay,soft agar assay,and drug resistance assay to investigate the effect of DAXX overexpression or silencing on the tumorigenicity of gastric cancer cells as well as stem cell characteristics.Results:(1)By detecting the expression of DAXX in 83 pairs of clinical samples,we found that the relative expression of DAXX in patients with different stages of gastric cancer was different,and showed a downward trend from stage 1 to stage 4.(2)q-PCR results showed that the expression of DAXX is negatively correlated with the expression of cancer stem cell markers CD44 and OCT4 in the human gastric cancer cell lines MKN45,N87 and AGS.Overexpression of DAXX inhibited the expression of CD44 and OCT4,promoted the expression of CD24,and significantly decreased the proportion of CD44high/CD24low cells,while knockdown induced the opposite effect.(3)DAXX overexpression not only inhibited the migration and invasion of gastric cancer cells,but also inhibited the expression of EMT markers.(4)DAXX inhibited EMT promoting factors such as SNAI,and promoted the expression of EMT inhibiting factors such as GRHL2.(5)DAXX forms a complex with HDAC1 and recruits HDAC1 to the nucleus,where HDAC1 inhibits the transcription of SNAI3 by deacetylating histone H3K14 sites,thereby inhibiting EMT in gastric cancer cells.(6)DAXX inhibits the colony-forming ability of gastric cancer cells and increases their drug sensitivity.Subcutaneous tumor formation experiments in mice showed that DAXX overexpression inhibited the tumor formation ability of MKN45.While wild type AGS cells failed to form tumors in nude mice,did not have tumor formation ability,AGS cells with DAXX knockdown were able to form small but visible tumor.Conclusion:The expression of DAXX was down-regulated in advanced gastric cancer samples and was negatively correlated with stem cell markers of gastric cancer cells.DAXX inhibits the migration,invasion,self-renewal capacity and potential tumorigenicity of gastric cancer cells,while reducing the tolerance of gastric cancer cells to chemotherapeutic agents.DAXX forms a complex with HDAC1 and recruits HDAC1 to the nucleus,where HDAC1 inhibits the transcription of SNAI3 by deacetylating histone H3K14 sites,thereby inhibiting EMT in gastric cancer cells.Taken together,DAXX acts as a tumor suppressor and inhibits the potential tumorigenicity and metastatic ability of gastric cancer. |
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