Effects Of DDX56 On Proliferation,Apoptosis,Invasion And Migration Of Gastric Cancer And Its Molecular Mechanism

Objective DEAD-box helicase(DDX)family exerts a critical effect on cancer initiation and progression through alternative splicing,transcription and ribosome biogenesis.Increasing evidence has demonstrated that DEAD-box helicase 56(DDX56)is over-expressed in several cancers,which plays an oncogenic role.Till the present,the impact of DDX56 on gastric cancer(GC)remains unclear.Methods We conducted high-throughput sequencing(RNA-seq)to demonstrate aberrant DDX56 levels within 10 GC and matched non-carcinoma tissue samples.DDX56 levels were detected through qRT‐PCR,western blotting(WB)and immunochemical staining in GC patients.We conducted gain-and loss-of-function studies to examine DDX56’s biological role in GC development and the precise mechanisms.In vitro,we carried out 5?Ethynyl?2′?deoxyuridine(EdU),scratch,Transwell,and flow cytometry(FCM)assays for detecting GC cell growth,invasion,migration and apoptosis.Additionally,gene set enrichment analysis(GSEA),WB assay,and Encyclopedia of RNA Interactomes(ENCORI)were carried out for analyzing DDX56-regulated downstream genes and signaling pathways.In vivo,tumor xenograft experiment was performed for investigating how DDX56 affected GC development within BALB/c nude mice.Results Functionally,DDX56 knockdown inhibited the G1/S arrest of cell cycle,invasion and migration of AGS and MKN28 cells,and enhanced their apoptosis.Ectopic DDX56 expression enhanced the cell growth,migration and invasion,and inhibited apoptosis.Knockdown of DDX56 suppressed GC growth in the tumor models of BALB/c nude mice.Mechanistically,DDX56 post-transcriptionally suppressed FOXO1/p21 Cip1 protein expression,which could activate its downstream cyclin E1/CDK2/c-Myc signaling pathways.Conclusion The expression of DDX56 gene elevated in human gastric cancer tissues,and gastric cancer patients with high DDX56 expression have poor survival prognosis.DDX56 gene enhances the proliferation,invasion and migration of gastric cancer cells by regulating the FOXO1 / p21 Cip1 / c-Myc signaling pathway,and suppresses cell apoptosis.This sheds lights on the GC pathogenic mechanism and offers a potential anti-cancer therapeutic target.Part1 Bioinformatics method and RNA-Sequence high-throughput sequencing technology were used to screen the DDX56 gene and explore its correlation with the clinicopathological characteristics of gastric cancerObjective RNA-Sequence high-throughput sequencing technology were used to screen DDX56(DEAD-box helicase DDX56),detected the expression of DDX56 gene in gastric cancer tissues,and explored the relationship with the clinicopathological characteristics of gastric cancer.Methods 1.Clinical tissue sample mRNA was subjected to deep sequencing using Illumina Hi Seq TM 2500 high-throughput sequencing technology,and the target genes were screened after alignment and analysis.2.The RNA expression levels of DEAD-box helicase(DDX / DHX)family genes in gastric cancer and normal gastric mucosal tissues were compared by using the Gastric adenocarcinoma(STAD)dataset of TCGA database.The expression of DDX56 gene was detected in the pan-cancer data with the help of Fire Browse software platform.The expression of DDX56 in gastric cancer was analyzed through the GEPIA online software platform and the star Base public database.Using the Kaplan-Meier Plotter public data platform,correlations between DDX56 expression levels and the prognosis of gastric cancer patients were analyzed.Collected information about gastric cancer patients and tumor tissue samples,in order to analyzed the expression level of DDX56 in gastric cancer tissues by conducting tissue RNA,tissue protein and immunohistochemistry,respectively.The measurement data were expressed by mean ± standard deviation((?) ± s).Measurement data between two groups were compared by t-test,and measurement data between multiple groups were described by one-way analysis of variance(One-Way ANOVA)test.Chi-square test was used for counting data.P<0.05 indicates that the difference is statistically significant.Results 1.High-throughput sequencing results suggested that DDX56 gene expression levels were significantly higher in gastric cancer tissues than in adjacent normal gastric mucosa tissues(P<0.01).2.The bioinformatics analysis showed that the expression level of DEAD-box helicase family genes increased in gastric cancer tissues,and the expression difference of DDX56 in gastric cancer was significantly representative(P<0.001).Comparison of pan-cancer data suggested that DDX56 was increased in gastric cancer,urothelial cancer,esophageal cancer and lung adenocarcinoma(P<0.05),and patients with high DDX56 expression have a poor prognosis than GC patients with low DDX56 expression(P<0.0001).3.The data of this part of the study suggested that the expression level of DDX56 in gastric cancer was higher than that in adjacent normal tissues in mRNA and tissue protein detection in clinical tissue samples(P < 0.05).Conclusion The RNA helicase DDX56 expression level elevated in human gastric cancer tissues,and gastric cancer patients with high DDX56 expression have a poor survival prognosis.Part2 Effect of DDX56 gene on the biological function of gastric cancer cells in in vivo and in vitro experimentsObjective To investigate the effect of DDX56 gene on gastric cancer cells in vitro and in the subcutaneous neoplasia model of nude mice.Methods 1.Gastric cancer cell lines with silenced and overexpressed DDX56 gene were constructed by plasmid transfection and lentiviral infection.2.Explore the effect of DDX56 gene on gastric cancer cell proliferation,cell invasion and migration,and apoptosis through Edu assay,CCK8,flow cell cycle detection assay,Transwell assay,scratches assay,flow cell apoptosis detection and Western blotting.3.Gastric cancer cells stably transfected with DDX56 gene were planted subcutaneous in nude mice,and the proliferation ability of gastric cancer cells after silencing DDX56 gene was explored by subcutaneous graft tumor model.The measurement data were expressed by mean ± standard deviation((?)±s).Measurement data between two groups were compared by t-test,and measurement data between multiple groups were described by one-way analysis of variance(One-Way ANOVA)test.Chi-square test was used for counting data.P<0.05 indicates that the difference is statistically significant.Results 1.According to results of qRT-PCR and Western blotting,the sh-DDX56-2 silencing DDX56 gene was selected to construct the AGS cell line and M K N 28 cell line.In addition,the overexpressing DDX56 gene was successfully constructed in AGS cells and MKN28 cells(P<0.05).2.After the above series of cell function experiments,it is suggested that silencing DDX56 gene can inhibit the cell cycle progression of G1/S of gastric cancer cells,reduce cell invasion and migration ability and promote apoptosis.On the other hand,overexpression of DDX56 gene can promote G1/S phase transformation of gastric cancer cells,enhance cell invasion and migration ability and inhibit apoptosis(P<0.05).3.In the subcutaneous xenograft model of nude mice,AGS cells silencing DDX56 gene resulted in lower tumor volume and weight in nude mice compared with control group.Similarly,immunohistochemistry confirmed that the proliferation factor Ki-67 protein,as well as the target gene DDX56 protein,was decreased in the transplanted tumors silencing the DDX56 gene(P<0.05).Conclusion The DDX56 gene can enhance the proliferation,invasion and migration of gastric cancer AGS cells and MKN28 cells,and inhibit apoptosis.Parts3 Regulation mechanism and potential molecular interactions of DDX56 gene on GC cell proliferation,apoptosis,invasion and migrationObjective To explore the influence of DDX56 gene in the regulation of proliferation,apoptosis,invasion and migration of gastric cancer cells.Methods 1.Further gene set enrichment analysis(GSEA)and RNA Interactome Encyclopedia(ENCORI)were used to provide theoretical support for finding the downstream target genes and signaling pathways of the DDX56 gene.2.Using successfully constructed silent DDX56 and overexpressing DDX56 gene cell lines,DDX56 with FOXO1(forkbox O1),p21 Cip1(cyclin-dependent kinase inhibitor 1A),CCNE1(cyclin E1),CDK2(cyclin-dependent kinase 2),MYC(c-Myc proto-oncogene),and GAPDH(glyceraldehyde-3-phosphate dehydrogenase)proteins were determined by Western blotting.The measurement data were expressed by mean ± standard deviation((?)±s).Measurement data between two groups were compared by t-test,and measurement data between multiple groups were described by one-way analysis of variance(One-Way ANOVA)test.Chi-square test was used for counting data.P <0.05 indicates that the difference is statistically significant.Results 1.GSEA database analysis suggested that DDX56 gene is mainly enriched in cell cycle pathway,apoptosis pathway,shear body pathway and RNA degradation pathway.ENCORI database suggested that DDX56 gene is negatively associated with FOXO1 at mRNA level and positively associated with CCNE1,CDK2 and MYC(P<0.05).2.In both AGS cells and MKN28 cells,the Western blotting results prompt that silencing of the DDX56 gene increased the protein expression level of FOXO1 and p21 Cip1 protein,and the protein expression levels of CCNE1,CDK2,and MYC were decreased(P <0.05),compared with the control group.On the other hand,overexpression of the DDX56 gene in AGS cells versus MKN28 cells,reducing the expression levels of FOXO1 protein and p21 Cip1 protein,and improve the expression of CCNE1,CDK2 and MYC protein(P <0.05).3.These results suggested that the DDX56 gene could inhibit the transformation of G1 phase to S phase of gastric cancer cell cycle by relieving FOXO1 / p21 Cip1 protein,enhancing the cell cycle promotion effect of CCNE1 / CDK2 / MYC protein.Conclusion The DDX56 gene suppresses the expression of FOXO1 / p21 Cip1 protein and activates the downstream cyclin E1 / CDK2 / c-Myc signaling pathway,thus affecting gastric cancer cell proliferation.