| Backgrand:Gastric cancer（GC）is a commonly occurring tumor in the digestive system.The pathogenesis of gastric cancer is widely accepted to be the Correa cascade reaction[1].This process involves various molecular regulations,signal transductions,and pathway activations or inhibitions.The Correa cascade explains the histopathological evolution process of gastric cancer,which includes Chronic non-atrophic gastritis（CNAG）,Chronic atrophic gastritis（CAG）,Intestinal metaplasia（IM）,and ultimately,gastric cancer（GC）.IM is an intermediate stage in the Correa cascade and is considered a Precancerous lesion（PLGC）of GC.It is closely associated with the development of intestinal GC.lnc RNAs exhibit high tissue specificity and some have been used as novel markers in clinical settings.Additionally,lnc RNAs have diverse regulatory functions and play a significant role in various disease processes,particularly in relation to the occurrence and progression of tumors.However,there are limited reports on the involvement of lnc RNAs in IM regulation.Therefore,our objective is to identify lnc RNAs that can serve as early warning markers for predicting GC risk and investigate their potential mechanisms and modes of action in IM.Objective:The aim to this study is to use whole transcriptome sequencing to screen biomarkers that can predict the risk of gastric cancer（GC）.We aim to detect the expression levels of these biomarkers in clinical tissues and investigate the molecular mechanism underlying their role in intestinal metaplasia（IM）.Method:（1）Gastric mucosal tissue specimens,including CNAG,CAG,IM,and GC tissues,were collected from 18 patients at our hospital.Full transcriptome sequencing was performed on these specimens.The counts of each gene in the samples were standardized using DESeq2software,and differential gene analysis was conducted to identify DElnc RNA,DEmi RNA,and DEm RNA.Through interactive analysis,differentially expressed lnc RNAs were identified.The functions and pathways of neighboring genes of different lnc RNA genes in each comparison group were analyzed using GO/KEGG.（2）The modules related to the malignant transformation process of the Correa cascade reaction were analyzed using the WGCNA analysis method.The Hub gene was screened and the PPI network was constructed.Additionally,by constructing the m RNA-lnc RNA co-expression network,lnc RNAs that may be related to inhibiting IM were identified.These lnc RNAs were then verified in CNAG by q RT-PCR to determine their association with promoting IM.Among the differentially expressed molecules,lnc-BRF2-7 showed the most significant expression difference in gastric mucosal tissues between the IM and GC groups.（3）To investigate the function of lnc-BRF2-7,we constructed an overexpressed/silenced cell model of lnc-BRF2-7.Additionally,we induced an enteric cell model of normal gastric epithelial cells（GES-1）using chenodeoxycholic acid（CDCA）.We analyzed the effects of lnc-BRF2-7 on IM marker Kruppel like factor 4（KLF4）and Caudal type homeobox gene transcription 2（CDX2）expression through q RT-PCR and Western blot（WB）analysis.We also verified the anti-apoptotic effect of lnc-BRF2-7 using flow cytometry and WB.Furthermore,we confirmed the expression of IM markers through immunohistochemistry（IHC）and WB,and detected the expression of lnc-BRF2-7 using q RT-PCR.（4）The molecular mechanism of lnc-BRF2-7 in IM was investigated in this study.Subcellular mapping of lnc-BRF2-7 was performed using nuclear plasmic separation.The ce RNA regulatory network was constructed using RNA-seq data,and the intersection with the RNA22database revealed a potential ce RNA regulatory mechanism involving mi R-135a-5p and lnc-BRF2-7.The effects of mi R-135a-5p mimics and inhibitors on the expression of KLF4 and CDX2 were validated using q RT-PCR and WB.Dual luciferase reporter gene experiments and AGO2-RIP assays confirmed that lnc-BRF2-7 binds to mi R-135a-5p through the ce RNA regulatory mechanism,leading to the regulation of KLF4 and CDX2 expression and promoting the development of IM.Result:（1）Compared to the expression of differentially expressed lnc RNAs in the CAG vs CNAG group（59 up-regulated and 51 down-regulated）,the number of differentially expressed lnc RNAs in the IM vs CNAG group was higher（141 up-regulated and 91 down-regulated）.The CNAG group had the highest number of differentially varied lnc RNAs（244 up-regulated and 185 down-regulated）,indicating that more changes in lnc RNA expression gradually occurred during the transformation process from CNAG to CAG to IM to GC,and these lnc RNAs may be involved in the regulation of the inflammatory cancer transformation process.Through interaction analysis,18 lnc RNAs were identified as co-down-regulated during the inflammatory cancer transformation process.GO/KEGG analysis of the neighboring genes of these 18 lnc RNAs revealed that their functions were mainly associated with glandular development,pancreatic development,and cardiomyocyte differentiation.On the other hand,35 lnc RNAs neighboring genes that were co-up-regulated were primarily involved in regulating glandular development,dendritic cell chemotactic migration,cell adhesion,and fibroblast apoptosis.（2）WGCNA analysis revealed a negative correlation between the Cyan module and the Correa cascade reaction process.Key lnc RNAs were identified and validated using q RT-PCR.In the IM groups,GATA6-AS1exhibited a down-regulation,indicating its potential negative regulatory role in the process of inflammatory cancer transformation.On the other hand,the Orange module showed a positive correlation with the Correa cascade reaction.Key lnc RNAs associated with this module were identified and confirmed through q RT-PCR.Specifically,lnc-BRF2-7demonstrated a gradual up-regulation in the CNAG,IM,and GC groups.Notably,lnc-BRF2-7 displayed significant differences in both sequencing data and q RT-PCR validation,suggesting its potential role in promoting IM.Therefore,we focused our study on exploring the mechanism of lnc-BRF2-7.（3）Overexpression or silence of lnc-BRF2-7 in normal gastric epithelial cells（GES-1）was investigated using q RT-PCR and WB.The findings demonstrated that overexpression of lnc-BRF2-7 promoted the expression of intestinal metaplasia（IM）markers KLF4 and CDX2,while silence of lnc-BRF2-7 inhibited their expression.In CDCA-induced enteric cell membrane,the expression of lnc-BRF2-7 was upregulated compared to the control group.Silencing lnc-BRF2-7 restored the induction effect of CDCA on KLF4 and CDX2,suggesting that lnc-BRF2-7 promotes IM by enhancing the expression of these IM markers.Flow analysis and WB revealed that CDCA treatment in GES-1 cells resulted in anti-apoptotic activity,characterized by increased expression of anti-apoptotic protein Bcl-2 and decreased expression of pro-apoptotic protein caspase3.Partial reversal of the CDCA-induced anti-apoptotic activity was observed after si-lnc-BRF2-7 treatment,indicating that CDCA may induce the increase of anti-apoptotic activity in GES-1 cells through lnc-BRF2-7,potentially contributing to the transformation of gastric epithelium into intestinal epithelium.IM mouse models were constructed using CDCA,and immunohistochemistry and WB analysis showed elevated expression of IM markers.Furthermore,the CDCA-induced IM mouse model exhibited upregulation of lnc-BRF2-7 expression in gastric tissue,suggesting that lnc-BRF2-7 plays a role in promoting IM.（4）The molecular mechanism of lnc-BRF2-7 in promoting the occurrence and development of IM was further investigated.Initially,a ce RNA network was constructed using RNA-seq data,and the ce RNA regulatory mechanism involving mi R-135a-5p and lnc-BRF2-7 was identified through RNA22 database prediction.The expression of mi R-135a-5p in human gastric mucosa IM tissues was quantified using q RT-PCR,revealing a decrease in its expression which correlated negatively with the expression of lnc-BRF2-7.Cell experiments confirmed this correlation,as q RT-PCR analysis demonstrated that overexpression/silencing of lnc-BRF2-7 led to the inhibition/promotion of mi R-135a-5p expression.The specificity of the interaction between the two was confirmed through dual luciferase reporter gene testing,while RIP testing validated the ce RNA mechanism.Subsequently,bioinformatics software was employed to predict binding sites between mi R-135a-5p and CDX2 as well as KLF4.Cell experiments revealed that mi R-135a-5p negatively regulated the expression of CDX2 and KLF4,which was further confirmed by dual luciferase reporter gene assays.In the IM cell model,the inhibitory effect of mi R-135a-5p inhibitors on CDX2 and KLF4 was partially reversed by si-lnc-BRF2-7.These findings suggest that lnc-BRF2-7promotes IM by competitively binding mi R-135a-5p to target KLF4/CDX2.Conclusions:（1）The Correa cascade process led to the gradual emergence of changes in lnc RNA expression,suggesting their potential involvement in the regulation of inflammatory cancer transformation.（2）Key long non-coding RNAs（lnc RNAs）associated with the transformation of inflammatory cancer were identified through the use of WGCNA)and validated through quantitative Polymerase Chain Reaction（q PCR）analysis of clinical samples.It was found that GATA6-AS1 may inhibit inflammatory mediators（IM）and gastric cancer（GC）,while lnc-BRF2-7may promote IM and GC.（3）The expression of lnc-BRF2-7 was found to be upregulated in human IM specimens,IM cell models,and IM mouse models.In vitro experiments further confirmed its role in promoting IM.（4）lnc-BRF2-7 regulates the expression of the IM marker KLF4 CDX2 by competitively binding to mi R-135a-5p,thereby promoting the development of IM. |
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