Study On The Role And Molecular Mechanism Of RECK In Gastric Cancer Progression

Gastric cancer(GC)is one of the most common malignant tumors worldwide,with the incidence and mortality rate ranking high in China.Due to the inconspicuous early symptoms of GC and the absence of reliable early diagnostic methods,the majority of patients were already in advanced stages at diagnosis.This ultimately leads to a diminished five-year survival rate and an unfavorable prognosis.The incidence and mortality rates of gastric cancer have been decreasing in recent years as a result of the implementation of early detection methods and the increased awareness of risk factors identification.Nevertheless,as a consequence of the considerable genetic and phenotypic heterogeneity of GC,which leads to the differences in biological functions of tumor cells within the same tumor tissues,certain patients with GC were resistant to a variety of anticancer treatments.Hence,exploring and identifying the prospective pivotal regulators associated with GC,as well as elucidating their molecular mechanisms underlying the tumorigenesis and progression of GC,is crucial for the development of molecular-targeted therapies and early diagnostic methods of GC.With the advent of "big data" in life science research,an enormous amount of sequencing data has been generated.Analyzing this data can assist in the discovery and identification of molecular indicators related to early tumor diagnosis,prognosis,and anti-cancer treatments for cancers.Reversion-inducing cysteine-rich protein with Kazal motifs(RECK),a negative cell surface regulator of matrix metalloproteinases(MMPs),is strongly suppressed in the majority of malignant tumors and functions as a suppressor of malignant tumor behaviors,including cell proliferation,invasion,and metastasis.Previous research has demonstrated that the expression of RECK is noticeably reduced in GC.Nevertheless,there is limited knowledge of the potential molecular mechanisms for the antitumor effects of RECK in GC.In this study,utilizing differential expression analysis,prognosis analysis,and correlation analysis between gene expression and clinical variables in the TCGA STAD cohort,RECK was identified as a potential key regulator of GC.Subsequently,multiple datasets were employed to evaluate the expression pattern of RECK in GC.In addition,more investigations were conducted to examine the correlation between RECK and a range of factors,including pathological types,tumor microenvironment,and tumor infiltrating immune cells in GC.Moreover,the impact of RECK on the biological functions of AGS and HGC-27 cells was confirmed through RECK overexpression and silencing,and the MAPK/ERK pathway was further demonstrated to mediate the regulatory effects of RECK on AGS and HGC-27 cells.Additionally,based on gene coexpression analysis and western blot assay,CALD1 had the potential to function as a downstream molecule of RECK and ERK.Subsequent investigations demonstrated that RECK exerted a regulatory influence on AGS and HGC-27 cells by specifically targeting CALD1.The above findings demonstrated that gene RECK is an independent prognostic factor in gastric cancer and exerts its biological function by inhibiting the MAPK/ERK signaling pathway as well as targeting the CALD1.These results above ultimately elucidated the regulatory function of RECK in the progression of GC and its underlying mechanism and provide a certain theoretical foundation and scientific research basis for the prognosis and molecular diagnosis of gastric cancer.Part I Identification of key regulators in GCDifferential analysis was performed on the gene expression profiles of the TCGA STAD cohort to identify 4128 differentially expressed genes(DEGs).After utilizing Kaplan-Meier survival analysis,the univariate and multivariate Cox regression analyses,and ROC curve analysis based on DEGs,a final selection of 29 independent prognostic genes with high predictive accuracy was ultimately determined.Subsequently,correlation analysis of 29 independent prognostic genes with clinical features was performed.Three genes were obtained,with a filter based on the number of clinical features significantly correlated with gene expression,and the clinical features significantly correlated with gene expression included age,grade,stage,and T.Meanwhile,according to the relevant research,RECK was a suppressor in the majority of malignant tumors.However,there were fewer relevant reports regarding the detailed regulatory mechanisms of RECK in GC.Therefore,RECK was explored and studied in depth as a potential key regulator of GC.Subsequently,the downregulation of RECK gene expression in GC was confirmed via various cohorts and gastric cancer cell lines.In addition,the correlation analysis revealed a strong connection between RECK gene expression and tubular GC as well proliferative GC in terms of tumor histological types.Additionally,meta-survival analysis based on multiple datasets identified RECK as a prognostic factor in GC.In order to investigate the potential biological role of RECK in the progression of GC,various data platforms(TIMER,TIP,and EMTome)were utilized to analyze the relationship between RECK gene expression and factors such as immune score,tumor immune cell infiltration,gene expression of immune checkpoint in the tumor microenvironment,and tumor metastasis.The results showed a strong correlation between RECK gene expression and these factors.Furthermore,function enrichment analysis showed a significant correlation between RECK expression and critical cellular processes,including MAPK signaling,DNA replication,cell cycle,and cell apoptosis in GC.The comprehensive functional investigation of RECK indicated that RECK might serve as a crucial regulatory molecule in GC.Part II:Investigations for the biological functions of RECK in GC cellsTo elucidate the biological function of RECK in GC progression,we initially enhanced the expression of RECK in AGS and HGC-27 cell lines by transfecting the pcDNA3.1-RECK plasmid.Simultaneously,we employed molecular biology techniques,such as gene silencing,to decrease the expression of RECK in AGS and HGC-27 cells.Using CCK-8,colony formation,and EdU cell staining assay,we have demonstrated that RECK suppressed the growth of AGS and HGC-27 cells.Additionally,RECK hindered the progression of the cell cycle in the G1 phase and enhanced the apoptosis of AGS and HGC-27 cells.The results from wound healing,transwell cell migration,and invasion assays demonstrated that RECK played a crucial role in suppressing the migration and invasion of AGS and HGC-27 cells.Thus,we utilized Western blot assay to identify alterations in protein expression levels related to the functional regulation of RECK in cell proliferation,apoptosis,cell migration,and invasion of AGS and HGC-27 cells.The findings demonstrated that RECK effectively increased the protein levels of p21,Bax,E-cadherin,and ZO-1,while significantly reducing the protein levels of PCNA,cyclin D1,cyclin D3,CDK4,CDK6,Bcl-2,Ncadherin,Vimentin,and MMP-2.These results indicate that RECK potentially plays an essential part in modulating the biological characteristics of gastric cancer by regulating the expression of these molecules.To deeply investigate the molecular functions of RECK in AGS and HGC-27 cells,western blot assay was conducted to determine the impact of RECK overexpression or silencing on the expression of key molecules in the MAPK signaling pathway identified by functional enrichment analysis,specifically p38,p-p38,JNK,p-JNK,ERK,and p-ERK.The findings demonstrated that RECK reduced the expression levels of p-ERK and prevented the activation of the MAPK/ERK signaling pathway.However,RECK did not have any impact on the activity of the p38 and JNK signaling pathways.The aforementioned findings suggested that RECK played an essential role in inhibiting the GC progression.It was speculated that RECK performed its biological activity by influencing the MAPK/ERK signaling pathway.Part Ⅲ RECK exerts its biological functions through the MAPK/ERK signaling pathway.To thoroughly examine the molecular mechanisms of RECK in GC progression,based on the understanding that RECK regulated the activity of the MAPK/ERK signaling pathway,we utilized an ERK activator,TBHQ,and an ERK inhibitor,PD98059,to treat RECK-overexpressing and silenced AGS and HGC-27 cells,respectively.Subsequently,the colony formation,EdU cell staining,and cell cycle assays showed that TBHQ was effective in reducing the inhibitory impact of RECK overexpression on the proliferation of AGS and HGC-27 cells,whereas PD98059 was able to weaken the pro-proliferative effect of RECK knockdown on GC cells.Additionally,the flow apoptosis analysis demonstrated that TBHQ effectively counteracted the pro-apoptotic impact of RECK overexpression on AGS and HGC-27 cells.Conversely,PD98059 was successful in reversing the anti-apoptotic effects of RECK knockdown on AGS and HGC-27.Wound healing,transwell cell migration,and invasion assays similarly showed that TBHQ was able to alleviate the inhibitory effect of RECK overexpression on the migration and invasion of AGS and HGC-27 cells,while PD98059 successfully hindered the pro-migratory and invasive effects of RECK knockdown on GC cells.Furthermore,western blot assay revealed that TBHQ effectively mitigated the enhancing impact of RECK overexpression on the protein expression of p21,Bax,E-cadherin,and ZO-1,while also counteracting the inhibitory effect on the protein expression of PCNA,cyclin D1,cyclin D3,CDK4,CDK6,Bcl-2,N-cadherin,Vimentin,and MMP-2.On the other hand,PD98059 was able to reverse the suppressive effect of RECK knockdown on p21,Bax,E-cadherin,and ZO-1 protein expression,as well as the promotional effect on PCNA,cyclin D1,cyclin D3,CDK4,CDK6,Bcl-2,N-cadherin,Vimentin,and MMP-2 protein expression.The findings indicated that RECK molecules influenced the behavior of GC cells by modulating the MAPK/ERK signaling pathway.Part Ⅳ CALD1 mediates the biological functions of RECKTo further investigate the molecular mechanism of RECK in regulating the GC progression,a co-expression study was conducted to screen CALD1,a closely associated molecule with RECK,and it was speculated that CALD1 may play a crucial role in the biological function of RECK.Furthermore,we analyzed the immunohistochemical results of GC tissues in the HAP database and observed a significant decrease in the expression level of CALD1.The down-regulation of CALD1 expression level in AGS and HGC-27 cells was further confirmed through qPCR and Western blot assays.Subsequently,western blot assay was applied to show that both RECK and ERK had the capacity to influence the protein expression of CLAD1 in AGS and HGC-27 cells,suggesting that CALD1 might be a downstream molecule of RECK and ERK.Consequently,more investigations were conducted to explore whether CALD1 was involved in the regulation of biological processes by RECK in GC cells.To assess alterations in the proliferation and apoptosis of GC cells,co-transfection techniques were conducted by transfecting combinations of pcDNA3.1-RECK and siCALD1,as well as siRECK and pcDNA3.1-CALD1,into AGS and HGC-27 cells.Then colony formation,EdU cell staining,and cell flow assays were utilized to measure changes in proliferation and apoptosis of AGS and HGC-27.Additionally,wound healing,transwell cell migration,and invasion assays were employed to evaluate alterations in the migration and invasion of AGS and HGC-27 cells.The aforementioned findings indicated a strong co-expression between CALD1 and RECK.Furthermore,CALD1 had a function in mediating RECK’s regulation on cell proliferation,apoptosis,migration,and invasion in GC cells.