The Mechanisms Of DNMT1/SOX7/NANOG Positive Feedback Loop Promotes Progression And Stemness Maintenance In Gastric Cancer

Background and objective: Gastric cancer is one of the most common malignant tumors worldwide and the most frequent cause of cancer-related death in China.Although its early diagnosis and more effective treatments have improved the prognosis of patients with gastric cancer,a lack of significant early symptoms means that many patients have advanced-stage disease at diagnosis,and the 5-year survival and disease-free survival rates remain low.The occurrence and development of gastric cancer are affected by a combination of factors,including dietary,environmental,and genetic and epigenetic factors.Epigenetics has been a recent research hotspot,with DNA methylation being the most common epigenetic modification associated with carcinogenesis in humans.Aberrant methylation of Cp G islands leads to loss of tumor suppressor gene expression and plays an important role in tumorigenesis and tumor progression.There is thus a need to discover new epigenetic biomarkers and to identify novel targets for the early diagnosis,prognostic evaluation,and treatment of gastric cancer.Metastasis and recurrence are the two major causes of cancer-related death worldwide,and are also important contributors to the poor prognosis of gastric cancer patients.Epithelial–mesenchymal transition(EMT)and the existence of cancer stem cells(CSC)are important factors affecting tumor metastasis and recurrence.Epithelial cells lose cell polarity and adhesion during the EMT process and transform into mesenchymal cells with an invasive or metastatic phenotype.These resulting characteristics often render surgical resection impossible,leading to treatment resistance and ultimately to tumor recurrence.CSCs are a subpopulation of intratumoral cells with stem cell-like properties that are thought to be the origin of various human cancers.Despite their relatively low abundance,CSCs have been shown to play critical roles in cancer cell self-renewal,drug resistance,and metastasis,and to be associated with recurrence and treatment failure.SOX7 belongs to the SOXF subfamily of transcription factors and has been shown to regulate a variety of biological processes,including hematopoiesis,angiogenesis,cardiogenesis,and endoderm differentiation during embryonic development.Previous studies showed that SOX7 exerted a tumor suppressor role in human cancers andparticipated in the EMT process by regulating the Wnt/β-catenin pathway.However,further studies are needed to identify other factors regulating the EMT.Other members of the SOX family have demonstrated roles in regulating cancer stem-like characteristics,but it is currently unclear if SOX7 plays a similar role.The current study investigated SOX7 expression levels in gastric cancer cell lines and tissues,as well as the methylation status of Cp G islands in the SOX7 gene promoter region and the specific regulatory mechanism responsible for catalyzing its methylation.We also analyzed the relationships between SOX7 expression levels and clinicopathological factors,and examined the role of SOX7 in gastric cancer cell proliferation,invasion,and migration,the EMT,and CSCs,and further explored the downstream regulatory mechanisms of SOX7 in gastric cancer.Methods: 1.SOX7 m RNA and protein levels were detected in the gastric mucosal cell line GES-1,five gastric cancer cell lines(HGC-27,MKN74,AGS,SNU-1,and MKN-45),and in 54 paired gastric cancer/paracancerous tissues using real-time quantitative polymerase chain reaction(q RT-PCR)and western blotting.The correlations between SOX7 expression and clinicopathological indicators were analyzed.2.Two gastric cancer cell lines with relatively low SOX7 expression and two cell lines with high SOX7 expression were selected for overexpression and knockdown,respectively.A control group was also selected.3.The proliferation,migration,invasion,and self-renewal capacities of the experimental and control cells were examined using CCK8,scratch-healing,Transwell,and tumor stem cell spheroidization assays,respectively.4.Stemness markers were detected by q RT-PCR and the regulatory effect of SOX7 on stemness was assessed.Expression levels of EMT-related genes and proteins were also detected by q RT-PCR and western blot,respectively,and the regulatory effect of SOX7 on the expression of EMT-related genes was examined.5.The methylation status of Cp G islands in the SOX7 promoter region in gastric cancer cell lines and gastric cancer tissues was determined by methylation-specific PCR(MSP).The two cell lines with higher degrees of methylation were treated with graded concentrations of a demethylating agent.The methylation status of the Cp G islands in the SOX7 promoter region were detected after drug treatment by MSP,and the expression levels of SOX7 m RNA and protein were detected by q RT-PCR and western blot,respectively.6.Two cell lines with higher degrees of methylation were transfected with small interfering RNAs for DNAmethyltransferases(DNMT1,DNMT3 A,DNMT3B)using the lipid method,and SOX7 expression levels were detected by q RT-PCR and western blot.7.NANOG expression levels in gastric cancer cell lines and gastric cancer tissues were detected by q RT-PCR.The role of NANOG expression level in the prognosis of gastric cancer patients was analyzed by bioinformatics,and the correlation between SOX7 and NANOG expression was analyzed statistically.8.The transcriptional binding site of SOX7 and the NANOG promoter region was predicted using the bioinformatics website JASPAR and the predicted binding sites were verified by luciferase reporter,electrophoretic mobility(EMSA),and chromosomal immunoprecipitation(Ch IP)assays.9.Cell-function experiments were carried out to examine the role of the SOX7/NANOG axis in the occurrence and development of gastric cancer,and to detect its effect on the expression of EMT-related genes at the transcriptional and translational levels.10.We examined the correlation between NANOG and DNMT1 expression by bioinformatics analysis,and detected changes in DNMT1 expression by q RT-PCR and western blot following overexpression and knockdown of NANOG.We also searched the literature for information on the transcriptional binding site of NANOG and the DNMT1 promoter region,and verified this by luciferase reporter gene assay.Result: 1.SOX7 m RNA and protein levels were relatively low in gastric cancer cell lines,as shown by q RT-PCR and western blot,and SOX7 m RNA levels were decreased in 54 pairs of gastric cancer tissues,according to q RT-PCR.We analyzed the relationship between SOX7 expression and clinicopathological parameters in the54 paired gastric cancer tissues using χ2 tests.Low expression of SOX7 was associated with high TNM stage(P = 0.028),low tumor differentiation(P = 0.027),depth of invasion(P = 0.027)0.003),and lymph node metastasis(P = 0.002).2.Overexpression of SOX7 attenuated the proliferation,migration,invasion,and spheroidization abilities of gastric cancer cells,increased the m RNA and protein levels of the epithelial marker E-cadherin,and decreased the interstitial EMT markers N-cadherin and m RNA and protein levels of vimentin and Slug,while knockdown of SOX7 had the opposite effects.3.The Cp G island in the promoter region of SOX7 was abnormally hypermethylated and the degree of methylation was negatively correlated with the level of SOX7 expression.Demethylating agents reduced the degree of methylation in the promoter region and restored SOX7 expression in a concentration-dependent manner.Knockdown of DNMT1,but not knockdown ofDNMT3A or DNMT3 B,restored the expression of SOX7.4.Expression levels of SOX7 and NANOG in gastric cancer tissues were negatively correlated,as shown by Pearson’s correlation analysis.Overexpression of SOX7 significantly reduced the expression of NANOG,while SOX7 knockdown significantly increased the expression of NANOG.JASPAR bioinformatics analysis predicted that the transcription factor SOX7 could bind to the NANOG promoter region,and the binding sites were confirmed by luciferase reporter gene,Ch IP,and EMSA assays.Cell-function experiments showed that overexpression of NANOG could partially reverse the tumor suppressor effect caused by overexpression of SOX7.5.NANOG expression was positively related to DNMT1 expression levels.Overexpression of NANOG resulted in increased expression of DNMT1,and DNMT1 expression levels were decreased after knockdown of NANOG.The binding site between NANOG and DNMT1 was confirmed by luciferase reporter gene assay.Conclusion: SOX7 expression levels were decreased in gastric cancer cell lines and gastric cancer tissues.DNMT1-mediated promoter hypermethylation resulted in down-regulation of SOX7 expression.In addition,SOX7 expression significantly inhibited the proliferation,migration,invasion and inhibited the progression of EMT and stemness maintenance in gastric cancer.SOX7 may thus inhibit gastric cancer progression by directly inhibiting the expression of NANOG.The DNMT1/SOX7/NANOG positive-feedback pathway plays an important role in the occurrence and development of gastric cancer.