The Mechanism Of CXXC4 Promoting GDF15 Transcription In Gastric Cancer Development And Its Relative Clinical Analysis

Background:Gastric cancer(GC)is one of the most common malignant gastrointestinal cancers which seriously threat to the human health and the advancement of society.In spite of the extraordinary progress has been made in clinical diagnosis and treatment recently,gastric cancer remains a major cause of death worldwide with the morbidity and mortality ranked fourth and second among all malignant tumors respectively.Epidemiological studies have shown that in China,most gastric cancer patients are in middle-advanced stage when diagnosed initially,with the characteristics of high recurrence rate and poor prognosis.Therefore,it is urgent to find new effective treatment strategies for gastric cancer patients.As we all know,the occurrence and development of gastric cancer is a complex multi-stage process with various factors involved in,such as inheritance,environment and so on.Among these factors,genetic and epigenetic aberrations play a quite critical role.Abnormal activation of oncogenes and inactivation of tumor suppressor genes have led to changes in many important signaling pathways affecting proliferation,differentiation,cell cycle and cell fate decisions,which ultimately led to the development of gastric cancer.CXXC4(CXXC finger protein 4)was first discovered in renal cell carcinoma as a negative regulator of the canonical Wnt signaling pathway.CXXC4 contains a specific CXXC zinc finger domain,and a large number of studies have confirmed that this domain can bind to DNA and regulate the transcriptional expression of genes.In our previous study,we found that CXXC4 was a novel gastric cancer-associated tumor suppressor.CXXC4 was down-regulated in gastric cancer and its low expression was closely related to the poor prognosis of gastric cancer patients.CXXC4 was a direct downstream target gene of EZH2(enhancer of zeste homolog 2),which played a negative role in the regulation of Wnt/β-catenin and Ras/MAPK signaling,thereby inhibited the growth of gastric cancer cells.GDF15(Growth differentiation factor 15)is a member of the transforming growth factor-β(TGF-β)superfamily.It is widely involved in many human pathophysiological responses,such as stress response,bone formation,ischemia-reperfusion injury and cancer as well.Overwhelming evidence has shown that GDF15 not only promotes tumorigenesis,but also inhibits tumorigenesis in different cellular.This double-edged sword function of GDF15 is closely related to the tumor type,stage,cell condition,tumor microenvironment and so on.At the same time,GDF15 has also been found to be a useful predictor for clinical treatment and prognosis in oncology.Objective:Our previous findings reveal that CXXC4 can effectively inhibit the proliferation of gastric cancer cells and play a role in tumor suppression.CXXC4 exhibits low expression in gastric cancer patients and its low expression is associated with poor prognosis in gastric cancer patients.The purpose of this article is to further explore the specific molecular regulatory mechanism of CXXC4 in inhibiting gastric cancer cell proliferation.And try to find out whether CXXC4 exerts this inhibition through its CXXC zinc finger domain.Besides,identify and explore the underlying molecular regulatory mechanism between CXXC4 and its downstream target gene.At the same time,analyze the clinicopathological correlation of the target gene expression in gastric cancer by using the clinical tissue samples and public database.The present study hopes to provide an effective new target for clinical diagnosis and treatment of CXXC4-deficient gastric cancer patients.Method:First,constructed a gastric cancer cell line SGC7901-WT with doxcycline to stably inducing CXXC4 expression.The effect of high expression of CXXC4 on gastric cancer cells was then examined by MTS cell survival assay,flow cytometry and western blot.Nucleocytoplasmic separation and immunofluorescence experiment were performed to determine the cellular localization of CXXC4.The yeast two-hybrid system was used to assess the DNA binding capacity of both wild type(WT)and mutant type(MT)of CXXC domain.At the same time,the effect of CXXC MT on gastric cancer cells was explored through MTS,flow cytometry and western blot.After that,by comparative analyzing the gene expression profile data of cells before and after doxycycline exposure,combined with apoptosis-related gene expression data and quantitative real-time PCR(qRT-PCR)results,we identified the potential downstream target gene of CXXC4--GDF15.Following siRNA-mediated knockdown of CXXC4,the expression changes of GDF15 were further verified on both mRNA and protein levels through qRT-PCR and western blot experiment respectively.Plasmid DNA transfection,lentiviral transfection and siRNA technology were used to simulate different status of GDF15 expression,upon which flow cytometry,western blot,cell plate formation assay and MTS assay were carried out to detect the influences of GDF15 expression on cell apoptosis.Afterwards,immunohistochemical staining was performed to compare the expression of GDF15 in gastric cancer tissues and adjacent tissues.TCGA and Oncomine databases were further analyzed to explore the clinicopathological correlation of GDF15 in gastric cancer.Finally,the molecular mechanism for the regulation of GDF15 by CXXC4 was accessed via chromatin immunoprecipitation(ChIP)assay,qRT-PCR and western blot.Results:The novel gastric cancer suppressive gene CXXC4 was found primarily expressed in the nucleus.By comparing the gene expression profile data of cells before and after doxycycline exposure combing with apoptosis-related gene expression gene set,we screened and further confirmed the direct downstream target gene of CXXC4--GDF15.CXXC4 promotes apoptosis of gastric cancer cells and inhibits the development of gastric cancer by up-regulating GDF15 expression at the transcriptional level.The CXXC domain mutated CXXC4(CXXC4-MT)lost the ability to transcriptionally regulate GDF15 expression and cannot induce gastric cancer cell apoptosis.Specifically,we demonstrated that CXXC4 achieved such transcriptional regulation by enhancing the binding ability of the transcription factor Sp1 to the GDF15 promoter region.Results of immunohistochemical staining and public database analysis indicated that GDF15 was up-expressed in adjacent tissues compared to tumor tisssues.And high expression of GDF15 was significantly associated with longer overall survival(OS)and disease-free survival(DFS)in gastric cancer patients.Conclusion:For the first time that we find out GDF15 is a direct downstream target gene of CXXC4.CXXC4 can promote the transcriptional expression of GDF15 by enhancing the binding ability of transcription factor Sp1 to the GDF15 promoter region,causing apoptosis of gastric cancer cells,thereby inhibiting the development of gastric cancer.While CXXC MT loses this regulatory function.GDF 15 is relatively low expression in gastric cancer patients and the low expression of GDF 15 is associated with poor prognosis in gastric cancer patients.This study basically clarified the anti-cancer effects and inter-regulatory molecular mechanisms of CXXC4 and GDF 15.The current study also provides the theoretical foundation and promising targets for the prevention and treatment of CXXC4-deficient gastric cancer patients.