A Study On LncRNA Signature As Biomarker Of Gastric Cancer And The Mechanism Of PHF10 Involving Gastric Carcinogenesis Through Inhibition Of Gastric Epithelial Cell Differentiation

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LncRNA signature as biomarker of gastric cancerBackground and Objectives: Gastric cancer?GC?is one of the most aggressive malignancies and has a poor prognosis.Identifying novel diagnostic and prognostic markers for GC is of great importance.Long non-coding RNAs?lncRNAs?,which are involved in multiple processes during the development and progression of cancer,may act as potential biomarkers of GC.The aim of this study was to screen a lncRNA signature for diagnosis and prognosis of GC.Methods: We performed data mining using 4 microarray data sets of GC downloaded from the Gene Expression Omnibus?GEO?database with different classifiers and risk score analyses to screen a lncRNA signature that has diagnostic and prognostic values for GC.Then expression of the screened lncRNAs was validated by quantitative reverse transcription polymerase chain reaction?qRT-PCR?in 30 pairs of gastric cancer tissues.Results: We identified a 5-lncRNA signature?AK001094,AK024171,AK093735,BC003519 and NR₀03573?displaying both diagnostic and prognostic values for GC.Kaplan-Meier survival analysis and Log-rank test showed that the risk score based on this 5-lncRNA signature was closely associated with overall survival time of the patients?P=0.0001?.Further analysis revealed that the risk score is an independent predictor of patient prognosis.Quantitative RT-PCR performed on 30 pairs of gastric cancer tissues confirmed that the 5 lncRNAs were dysregulated in GC,and ROC curves showed the high diagnostic ability of combining the 5 lncRNAs,with an AUC of 0.95±0.025.Gene set enrichment analysis?GSEA?also suggested that these 5 lncRNAs involed in several cancer-related pathways.Conclusion: This 5-lncRNA signature maybe a good biomarker for diagnosis and prognosis of GC.Part ? The mechanism of PHF10 involving gastric carcinogenesis through inhibition of gastric epithelial cell differentiationBackground and Objectives: Dysdifferentiation is one of the hallmarks of malignant tumors.Gastric cancer?GC?is a typical disease with dysdifferentiation.The underlying mechanism of dysdifferentiation in GC is not clear at present.Our previous study found that the transcription factor PHF10 was negatively correlated with the degree of histological differentiation in GC,suggesting that it may be involved in the process of dysdifferentiation in GC development.Therefore,the purpose of this study is to clarify the expression pattern of PHF10 in gastric carcinogenesis and progression,its clinical significance,the role of PHF10 in regulating GC cell differentiation and its underlying molecular mechanisms.Methods: Quantitative RT-PCR?qRT-PCR?and IHC were used to detect the expression levels of PHF10 at the different stages of the transformation from normal gastric mucosa to GC.The relationship between PHF10 expression and clinical pathological parameters was analyzed.After overexpressing or silencing PHF10 stable GC cell lines were constructed,the biological functions of PHF10 were investigated,qRT-PCR for gastric differentiation markers expression,transmission electron microscopy?TEM?for subcellular structures,sphere formation assay for GC cells stemness,and tumor xenograft experiment in nude mice for adenous duct formation ability of GC cells.Dual-luciferase reporter gene assay and chromatin immunoprecipitation?ChIP?assay were performed to detect the interaction between transcriptional factors and gene promoter regions.The signaling pathways which PHF10 involved were analyzed by qRT-PCR and Western blotting?WB?and Co-IP.Results: This study showed a trend of increasing levels of PHF10 during the transformation from normal gastric mucosa to GC.PHF10 was significantly upregulated in GC,and PHF10 expression level was negatively correlated with the degree of cancer differentiation,and positively related with gastric cancer progression and poor patient prognosis.Cytological experiments showed that knockdown of PHF10 could promote gastric cancer cell to express gastric epithelial cell maturation markers,and induce inhibition of expression of gastric epithelial cell or gastric cancer cell stemness markers,and induce GC cells to appear differentiation-like subcellular structure in TEM,and inhibit sphere formation of GC cells and GES-1 cells,and induce adenous duct formation of GC cells in tumor xenograft experiment in nude mice.While overexpression of PHF10 could inhibit expression of gastric epithelial cell maturation markers and enhance expression of gastric epithelial cell or GC cell stemness markers in both GC cells and GES-1 cells,and promote sphere formation of GC cells and GES-1 cells.When overexpressing E2F1 in GC cells,both PHF10 m RNA and protein were upregulated.When knockdowning E2F1 in GC cells,both PHF10 m RNA and protein were down-regulated.Interestingly,E2F1 protein but not m RNA could also be upregulated by PHF10,suggesting existence of a positive feedback regulation loop between E2F1 and PHF10.Dual-luciferase reporter gene assay and ChIP-q PCR further validated that PHF10 is a direct target of E2F1.Co-IP confirmed that PHF10 was a member of the chromatin remodeling complex SWI/SNF in GC cells,and was important for the stability of the complex.PHF10 could directly inhibit transcription of DUSP5 via this complex,thus increasing levels of p ERK1/2,which was confirmed by qRT-PCR,WB,dual-luciferase reporter gene assay and ChIP-q PCR.In addition,we observered positive relationship between E2F1 and PHF10 and negative relationship between E2F1 and DUSP5,or between PHF10 and DUSP5 in GC tissues.Finally,in reverse experiments,by overexpression of E2F1 or knockdown of DUSP5 in PHF10 silenced GC cells,or by knockdown of E2F1 or overexpression of DUSP5 in PHF10 overexpressed GC cells,we observed the differentiation phenotype changes mediated by PHF10 were partially or completely reversed.Conclusion: E2F1 dependent dysregulation of PHF10 can mediate dysdifferentiation of GC cells through the DUSP5-p ERK1/2 pathway,thus promoting the tumorigenesis and progression of GC.