To Investigate The Core Gene Expression Transforming Intestinal Metaplasia To Gastric Adenocarcinoma And The Intervention Mechanism Of Berberine Based On DSP Technology

Gastric carcinoma(GC)is a global and multiple digestive tract tumor disease,and the high incidence and mortality of GC make it the top five most common tumors worldwide.The increase of intestinal metaplasia(IM)leads to the increase of the incidence and mortality of Gastric carcinoma,but the progression of intestinal metaplasia to Gastric carcinoma requires a certain time cycle and provides an adequate window period for clinical observation and intervention,so intestinal metaplasia is an important gateway for precancerous lesions of Gastric carcinoma.At present,the pathogenesis of intestinal metaplasia is not clear,so there is a lack of targeted treatment and methods.Therefore,it is significant to delay or block the progression of intestinal metaplasia-Gastric carcinoma by dissecting the pathological mechanism of intestinal metaplasia.Digital Space Analysis Technology-DSP(GeoMx Digital Spatial Profiler,DSP)is a high-throughput,multi-omics,self-selected region of interest detection method developed by integrating multi-omics based on NanoString,which can not only be used to analyze proteins and RNAs in biobanks,but also be used to analyze immune markers in patient samples and has potential prognostic and predictive potential for clinical decision-making.Clinical sequential samples of intestinal metaplasiagastric carcinoma were collected,and protein and gene information of intestinal metaplasia were analyzed in situ using the DSP platform.In this study,we analyzed the intestinal metaplasia-gastric cancer differential genes obtained based on DSP technology by a variety of bioinformatics methods,verified their expression trends using IHC experiments in clinical cohorts,and focused on the core genes of intestinal metaplasia-gastric carcinoma.Compounds and herbs that may have an intervention effect on IM based on network pharmacology prediction,and the verification of the effect of berberine,a traditional Chinese medicine monomer,on intestinal metaplasia intervention and block was carried out in cell experiments,and at the same time,the TCM syndrome types of IM were predicted according to the distribution rule of herbs that may intervene IM obtained by network pharmacology.To provide a basis for further analysis of the dynamic deduction process of IM occurrence and development,and provide a new basis for clinical intervention of integrated traditional Chinese and western medicine and blocking IM.Object:The clinical sequential cases of intestinal metaplasia and Gastric carcinoma were collected and analyzed in situ by DSP technique to obtain differentially expressed genes(DEGs)of intestinal metaplasia and Gastric carcinoma.A variety of bioinformatics methods were used to analyze and demonstrate the core gene of intestinal metaplasia and Gastric carcinoma,which was verified by clinical IHC,and the core gene of intestinal metaplasia and Gastric carcinoma was refocused.To explore the regularity of TCM intervention IM based on network pharmacology and identify berberine as a TCM monomer for intervention IM.MC cell models were constructed and evaluated to explore the mechanism of intestinal metaplasia-Gastric carcinoma at the cellular level from different levels such as "intestinal metaplasia marker-cell morphology-protein expression-gene expression",and cellular experiments of berberine intervention IM were also completed to clarify the effect and mechanism of berberine intervention and blocking intestinal metaplasia.Method:Part Ⅰ Analysis of Core Genes of Intestinal Metaplasia-Gastric adenocarcinoma Based on DSP Technology MiningCollect real-world clinical IM sequential cases,combine clinical symptoms,endoscopic diagnosis,pathological diagnosis to complete the inclusion and exclusion of IM sequential cases,of which the histopathological changes of IM are the gold standard for its diagnosis.By designing the clinical specimen screening process,DSP experimental protocol,sample quality control,tissue section criteria and references,it is determined that the application of DSP technology in IM meets the research requirements.Based on DSP technology for morphological marker staining,ROIs were selected,oligo was collected,NGS libraries were established and quality controlled,and ILLumina sequencing platform was used for data normalization to obtain highly accurate genome sequence information,providing assurance for the biological significance of gene expression changes in subsequent validation experiments.After obtaining the expression level of ROIs gene,T-test was used for statistical test and the screening criteria for differential genes were set as pval<0.05&|log2FC|>1.2 to obtain differential genes between intestinal metaplasia-Gastric carcinoma groups,and the distribution and clustering characteristics of differential genes were visualized using volcano plots and heat maps.Multiple bioinformatics analysis methods such as KEGG,GO,and GSEA were used to enrich intestinal metaplasia-Gastric carcinoma differential genes.In order to obtain the signal pathways and gene functions that may be involved in the regulation of differential genes in different backgrounds of CD45 and PanCK,KEGG and GO analysis were used to complete.GSEA enriched gene global expression profile analysis was used to confirm the intestinal metaplasia-Gastric carcinoma core obtained by DSP screening,and the core genes were screened by the genes with high | ES | score in The Leading-Edge Subset,and comprehensive analysis was performed to predict the biological regulatory mechanisms that may be involved in it in combination with literature studies.Part Ⅱ IHC validation and ROC curve analysis of core genes in intestinal metaplasia-gastric adenocarcinomaNine core genes of intestinal metaplasia-Gastric carcinoma were obtained by DSP technology combined with a variety of bioinformatics analysis methods.In order to further focus on the core genes of intestinal metaplasia-Gastric carcinoma,30 patients with clinical cross-sectional intestinal metaplasia and 30 patients with gastric adenocarcinoma were collected for IHC validation in clinical trials.After cross-confirmation analysis by psychologists with professional titles of two chief physicians,core genes were further focused,IM target genes were screened based on the database,and genes obtained from the two pathways were analyzed using KEGG,GO and other methods to try to discover the mechanism of intestinal metaplasiaGastric carcinoma through the coincident genes of the two or signaling pathways involved in regulation,laying the foundation for subsequent cell experiments.IHC showed that the expression trends of JUN,ETS2 and RPL8 were consistent in intestinal metaplasia-Gastric carcinoma tissues,and the expression showed low expression in the intestinal metaplasia group and high expression in the Gastric carcinoma group.Combined with the clinical significance,we performed ROC curve analysis of the above 3 genes to evaluate the diagnostic efficacy of the 3 genes.According to the results of immunohistochemical semiquantitative analysis,ROC curve was drawn,and the diagnostic efficacy and correlation of JUN,ETS2,and RPL8 for intestinal metaplasia-Gastric carcinoma were evaluated from the AUC,Youden index,sensitivity,and specificity of ROC curve.Part Ⅲ To investigate the regularity of traditional Chinese medicine intervention for intestinal metaplasia based on network pharmacology and prediction of TCM syndrome typesBased on DisGenet,GeneCards,OMIM and other databases to mine the disease targets of IM,IM targets with DL≥0.18 were screened in the Pharmacology Database and Analysis Platform of Traditional Chinese Medicine System(TCMSP),target components and their corresponding compounds were collected one by one,and compound-related traditional Chinese medicines were continued to be collected.After screening and collecting the targets,compounds and herbs,Cytoscape3.8.0 was used to display and analyze the target-compound-herbs relationship in a visual complex network.Statistical analysis of the frequency of taste meridians and efficacy of traditional Chinese medicine matched with IM targets was used to analyze the potential medication regularity of IM and predict the distribution of TCM clinical syndromes of IM.In order to verify the accuracy and scientificity of the network pharmacological prediction results,the obtained core compounds were molecularly docked with IM targets to provide further scientific evidence for the potential compound-targets that had been mined.Part Ⅳ MC cell modeling,model evaluation and berberine intervention validationGES-1 cells are currently cryopreserved and passaged human gastric epithelial cell lines after immortalization treatment,and are commonly studied in cell models associated with precancerous gastric lesions.In this study,N-methyl-N’-nitroN-nitrosoguanidine(MNNG),a nitrosamine compound,was used to induce GES-1 cells to transform into IM cell model named MC(Metaplastic cells,MC)cells.The MC cell model was systematically evaluated in terms of cell morphology and protein expression of IM markers before and after induction.Based on network pharmacology,berberine,a traditional Chinese medicine monomer,was selected to intervene intestinal metaplasia,and the optimal concentration of berberine to intervene intestinal metaplasia was screened by MTT assay.After administration intervention,WB detected the expression of IM-related markers CDX2,MUC2,PGC and intestinal metaplasia-gastric carcinoma core genes JUN,ETS2,and RPL8 in each group to explore the intervention effect of berberine on intestinal metaplasia and its mechanism.Results:Part Ⅰ1.Develop the inclusion and exclusion criteria of clinical sequential specimens of intestinal metaplasia-Gastric carcinoma,design the screening process of enrolled specimens,complete the collection of clinical sequential specimens of intestinal metaplasia-Gastric carcinoma,and confirm the histological sections by pathological diagnosis for future use.2.Nine intestinal metaplasia-Gastric carcinoma DEGs common on the basis of CD45 and PanCK background obtained based on DSP technology:PABPC1,RPS4X,JUN,RPL8,ETS2,RPL30,OLFM4,IFITM3,and ZNF703.Combined GSEA enrichment was performed to complete the analysis demonstration of the above 9 genes.3.KEGG and GO analysis of DEGs from multiple perspectives using a variety of bioinformatics methods shows that ribosomes,B-cell receptors,and cancer pathways are the main signaling pathways that may be involved in the regulation on the basis of CD45 and PanCK backgrounds;cytoplasmic ribosomes,translation initiation,and cell matrix junctions are the main biological gene functions that may be involved in biological processes.Part Ⅱ1.Thirty patients with intestinal metaplasia and 30 patients with gastric adenocarcinoma were collected from the two groups,and the trend of 9 gene expression was verified by IHC.Once again,the core genes:JUN,ETS2,RPL8 were focused and combined with comprehensive analysis of database IM target genes to validate each other at the cellular level.2.To evaluate the clinical diagnostic efficacy of the three DEGs focused again,ROC curve parameters showed that the immunohistochemical semi-quantitative results of JUN,ETS2,and RPL8 and ROC curve analysis showed that one or more of the three DEGs had good sensitivity and specificity for the diagnosis of intestinal metaplasia-Gastric carcinoma,and also further explained the association between intestinal metaplasia and Gastric carcinoma.Part Ⅲ1.A total of 109 targets that may act on IM were obtained based on network pharmacology and data mining,and finally 33 targets completed the matching of effective compounds,396 target-matched compounds,and 309 herbs were matched based on compounds.It is predicted that quercetin,kaempferol,berberberine and other compounds are core compounds acting on intestinal metaplasia,and Coptis chinensis,Ephedra sinica,Ligustrum lucidum and other traditional Chinese medicines are core herbs intervening intestinal metaplasia.Molecular docking validation was also performed.2.Frequency statistics showed that the taste of traditional Chinese medicine for intervening intestinal metaplasia of gastric mucosa was mainly bitter,pungent and sweet,and the medicinal properties were mainly cold,warm and flat,which mainly belonged to the three meridians of liver,stomach and lung,and the efficacy was mainly clearing away heat,detoxifying,dampness and removing blood stasis.It is predicted that dampness-heat syndrome,blood stasis and toxin syndrome and phlegm-dampness syndrome are TCM clinical syndromes of intestinal metaplasia.Part Ⅳ1.Model establishment and model evaluation,comparing the cell morphology and growth characteristics before and after model establishment,all showed characteristic changes,and the passage and continuation process could observe that the cells showed typical "island-like" clonal growth.WB showed that the expression of intestinal metaplasia marker-related proteins CDX-2 and MUC2 was increased,and the expression of gastric type marker PGC was decreased.At the same time,AB-PAS/HID-AB special staining experiments for cells were carried out,that is,the modeling was successful.2.MTT assay was used to screen the optimal concentration of berberine for intervening intestinal metaplasia at 100μmol/L.The results of WB showed that the expression trends of CDX2 and MUC2 in GES-1 cell group,MC cell group,MC cell berberine low dose group,medium dose group and high dose group were basically the same,all of which were negatively expressed in GES-1 cell group,strongly positively expressed in MC cell group,and gradually decreased in berberine low,medium and high dose groups.The expression trend of PGC in each group was the opposite.The core genes obtained by DSP and IM targets obtained by the database were enriched in the ribosomal signaling pathway,and the protein expression of JUN,ETS2,and RPL8 in MC cells after berberine intervention showed a decreasing trend.Conclusion:Based on DSP(Digital Spatial Profiler)spatial multi-omics technology combined with a variety of bioinformatics analysis methods to mine and analyze the core genes of intestinal metaplasia-Gastric carcinoma,nine core genes that may play a role in the process of intestinal metaplasia-Gastric carcinoma,including PABPC1,RPS4X,JUN,RPL8,ETS2,RPL30,OLFM4,IFITM3,and ZNF703,which are co-expressed in the context of CD45 and PanCK,were preliminarily obtained.After IHC validation of clinical sample cohorts,the core genes focusing on the central role of co-expression in the context of CD45 and PanCK were JUN,ETS2,and RPL8,and the diagnostic efficacy of ROC curves for JUN,ETS2,and RPL8 was evaluated,which confirmed the sensitivity,specificity,and accuracy of JUN,ETS2,and RPL8 in the diagnosis of intestinal metaplasia-Gastric carcinoma and is expected to be a biomarker for the diagnosis of intestinal metaplasia-Gastric carcinoma,which can be explored and validated in clinical practice based on this.To investigate the regularity of traditional Chinese medicine(TCM)in the intervention of intestinal metaplasia and the prediction of TCM syndrome types based on network pharmacology and data mining.Thirty-three compounds potentially acting on intestinal metaplasia and 309 herbs were screened.The top five candidate core targets of TCM intervention in intestinal metaplasia were TNF,CDKN1A,VEGFA,IL6,and TP53;the predicted core compounds were quercetin,kaempferol,berine,apigenin,and genistein;and the predicted core herbs were Coptis chinensis,Ephedra sinica,Ligustrum lucidum,Phyllanthus emblica,and Carthamus tinctorius.After counting the frequency of four properties,five flavors,meridians and functional distribution of 309 herbs,the regularity of traditional Chinese medicine medication that may act on intestinal metaplasia was sorted out and summarized,and the distribution of TCM clinical syndromes of intestinal metaplasia lesions was predicted by measuring the prescription of medicine and measuring the syndrome with the prescription.The intestinal metaplasia cell model of MC was established,and the intestinal metaplasia cell model of MC cells was evaluated and identified from the level of "cell morphology-intestinal metaplasia marker-protein expression-special staining".The intervention effect of berberine on intestinal metaplasia was verified.The expression of CDX2 and MUC2 in MC cells showed a gradually decreasing trend in berberine low,medium and high dose groups,and the expression of PGC in MC cells showed a gradually increasing trend in berberine low,medium and high dose groups,which was mutually confirmed with the results obtained from network pharmacology.At the same time,WB was used to detect the protein expression of JUN,ETS2 and RPL8 in MC cells after berberine intervention.It was confirmed at the cellular level that berberine could not only effectively intervene IM,but also block the progression of IM.