| ObjectivesThis paper aims to explore the expression of circRNAs-miRNAs related to gastric "inflammation-cancer" and to explore the efficacy and mechanism of jianpi qingre huoxue decoction in the treatment of chronic atrophic gastritis.With circRNAs-miRNAs as the target molecules,and through the chronic non-atrophic gastritis,chronic atrophic gastritis,precancerous lesions of gastric cancer,and gastric adenocarcinoma patients with four different pathological stages of clinical specimens of gastric mucosa,high-throughput second-generation sequencing,at the same time to screen and identify the differentially expressed the circRNAs,identify differentially expressed the circRNAs,identify the differentially expressed the circRNAs and predict its downstream mRNAs,to verify its key role in the "inflammatory-cancer"transformation of the stomach and its clinical application value for the early detection and early diagnosis of gastric "inflammatory-cancer" transformation,initially revealed the key molecular mechanism of gastric"inflammatory-cancer" transformation.Based on the key circRNAs,on the basis of evaluating the effective effects of Jianpi Qingre Huoxue Decoction on CAG and PLGC,this paper explores the possible mechanisms by which Chinese medicine regulates key molecules such as circRNAs-miRNAs and blocks the transformation of gastric“inflammatory-cancer”.Method1.Sequencing analysis of differentially expressed circRNAs related to gastric"inflammatory-cancer" transformation1.1 High-throughput second-generation sequencing of differentially expressed circRNAs associated with gastric"inflammatory-cancer" transformationThis study used high-throughput sequencing technology(second generation)to screen 20 cases of gastric "inflammatory-cancer" transformation of four different pathological stages(CNAG-CAG-PLGC-GC)differentially expressed circRNAs,to explore gastric "inflammatory-cancer" transformation Related differentially expressed molecules(circRNAs,miRNAs,mRNA)provide a reference for the search for Chinese medicine intervention targets.1.2 Screening of differentially expressed circRNAs related to gastric"inflammatory-cancer" transformationIn this part of the study,2749 circRNAs obtained from previous sequencing were first screened to narrow the range of differentially expressed circRNAs for further study.Mainly in three steps:(1)establish a combination of circRNAs related to the onset and development of CAG,namely CAG group,combine the DEGs of CNAG-vs-CAG,CNAG-vs-PLGC,CAG-vs-PLGC,and combine them.,73 combinations of circRNAs associated with the onset and progression of CAG were obtained.(2)Establish a combination of circRNAs related to the pathogenesis and development of GAC,namely GC group,combine the DEGs of CNAG-vs-GAC,CAG-vs-GAC and PLGC-vs-GAC,and combine them to obtain 127 cases with GAC.Related circRNAs combinations.(3)The intersection of CAG group and GAC group was obtained,and 12 circRNAs differentially expressed with gastric"inflammatory-cancer" CNAG-CAG-PLGC-GAC transformation were obtained.Finally,four circRNAs with miRNA binding sites were identified by miRanda calculations,providing the results of annotation of circBase circRNA binding to miRBase miRNAs.1.3 Validation of differentially expressed circRNAs related to gastric"inflammatory-cancer" transformation and construction of circRNAs-miRNAs-mRNAsThis study integrates high-throughput second-generation sequencing and screening data for differential expression of circulatory expression in pre-phase gastric "inflammatory-cancer",and provides a binding site for miRNAs by miRanda calculations,providing the results of circBase circRNA binding to miRBase miRNAs.4 circRNAs.Taking hsa-circ-0000798 as an example,the binding sites of circRNAs and miRNAs,and the binding sites of miRNAs and mRNAs were predicted by bioinformatics methods.According to miRanda software,hsa-circ-0000798 may be associated with 19 miRNAs.By RT-qPCR,it was confirmed that the expression trend of has-miR-4478 in different stages of gastric"inflammatory-cancer" transformation was opposite to that of has-circ-0000798,which was consistent with the ceRNA mechanism.Has-miR-4478 was combined with 10749 mRNAs by Targetscan software in combination with miRanda software.The previous study of the research group clarified that 1781 mRNAs were associated with gastric“inflammatory-cancer”transformation,and a total of 231 mRNAs involved in gastric“inflammatory-cancer”transformation were obtained from the mRNAs that bind to hsa-miR-4478.Consistent with the trend of change,the expression level was relatively high,and the fold change multiple was a large screening condition.A total of 28 mRNAs participating in the stomach-inflammation-cancer transformation were involved in the mRNAs that bind to hsa-miR-4478.The function of enriching 28 mRNAs by FunrichV3.0 has 23 cell components,15 molecular functions and 11 biological processes.Finally,a regulatory network of circRNAs-miRNAs-mRNAs was initially constructed to analyze its related functions and pathways.Through preliminary functional analysis,this study preliminarily identified SLC34A2,SOX10,and HAP1 as key target genes of hsa-circ-0000798-hsa-miR-4478,including SLC34A2,SOX10 and classical"inflammatory-cancer" transformation signaling pathways.-Wnt/?-cantenin signaling is closely related.The ROC curve was plotted based on the results of key circRNAs RT-qPCR,and the area under the curve(AUC)was calculated to evaluate the specificity and sensitivity of PLGC and GAC.At the same time,it analyzes its correlation with OLGA and OLGIM grading evaluation system,and explains its important role in the diagnosis,disease and prognosis evaluation of gastric "inflammatory-cancer" transformation.2.Interventional study on the results of Jianpi Qingre Huoxue decoction based on the differential expression of circRNAs in gastric"inflammatory-cancer"transformationClinical studies used a multicenter,randomized controlled trial to evaluate the effectiveness of Jianpi Qingre Huoxue Decoction,Weiweiqing Granule,in the treatment of chronic atrophic gastritis.The patients who participated in the clinical trial were from 11 top three Chinese medicine hospitals in Guangdong Province.The subjects were screened according to the prescribed inclusion criteria and exclusion criteria.The experimental group received oral spleen Qingre Huoxue Decoction(weiweiqing granule),and the control group received oral folic acid tablets.The treatment time was 24 weeks,and the two grading evaluation systems of OLGA and OLGIM based on pathological changes of gastric mucosa were the main outcome indicators.Through OLAG and OLGIM grading and staging system evaluation,38 subjects who were treated effectively and frozen-80? gastric mucosa specimens were included in the study of therapeutic mechanism.Result1.Sequencing analysis of differentially expressed circRNAs related to gastric"inflammatory-cancer" transformation1.1 High-throughput second-generation sequencing of differentially expressed circRNAs associated with gastric"inflammatory-cancer" transformationThe results were up to thousands of circRNAs differentially expressed in 6 different pathological stages.Among them,2749 circRNAs were associated with gastric“inflammatory-cancer”transformation,and the information was abundant,but also for subsequent research.Come to challenge.The results of this study illustrate the complexity of the "inflammatory-cancer"transformation of the stomach,consistent with current research reports in the field.How to further screen the key circRNAs differentially expressed in gastric "inflammatory-cancer" transformation from 2749 circRNAs for further functional,diagnostic and predictive studies is the focus and difficulty of this study.1.2 Screening of differentially expressed circRNAs related to gastric"inflammatory-cancer" transformationFour circRNAs with miRNAs binding sites(hsa-circ-0000798,hsa-circ-0005286,hsa-circ-0004928,hsa-circ-0062649)were identified,which entered the next step of verification and mechanism research,which have important research significance and clinical application value.1.3 Validation of differentially expressed circRNAs related to gastric"inflammatory-cancer" transformation and construction of circRNAs-miRNAs-mRNAsTaking hsa-circ-0000798 as an example,the binding sites of circRNAs and miRNAs,and the binding sites of miRNAs and mRNAs were predicted by bioinformatics methods.According to miRanda software,hsa-circ-0000798 may be associated with 19 miRNAs,as shown in the following table,by RT-qPCR,to confirm the expression trend of has-miR-4478 in different stages of gastric"inflammatory-cancer" transformation and has-circ-0000798,in contrast,conforms to the ceRNA mechanism.Has-miR-4478 was combined with 10749 mRNAs by Targetscan software in combination with miRanda software.The previous study of the research group clarified that 1781 mRNAs were associated with gastric "inflammatory-cancer" transformation,and a total of 231 mRNAs involved in gastric“inflammatory-cancer”transformation were obtained from the mRNAs that bind to hsa-miR-4478.Consistent with the trend of change,the expression level was relatively high,and the fold change multiple was a large screening condition.A total of 28 mRNAs participating in the stomach-inflammation-cancer transformation were involved in the mRNAs that bind to hsa-miR-4478.The function of enriching 28 mRNAs by FunrichV3.0 has 23 cell components,15 molecular functions and 11 biological processes.Finally,a regulatory network of circRNAs-miRNAs-mRNAs was initially constructed to analyze its related functions and pathways.Through preliminary functional analysis,this study preliminarily identified SLC34A2,SOX10,and HAP1 as key target genes of hsa-circ-0000798-hsa-miR-4478,including SLC34A2,SOX10 and classical"inflammatory-cancer"transformation signaling pathways-Wnt/?-cantenin signaling is closely related,which provide the foundation for the follow-up research.Compared with OLGA stage I/II,hsa-circ-0000798 was significantly lower in OLGA stage III,and the difference was statistically significant.This was compared with the previous sequencing,screening and verification of hsa-circ-0000798 in CNAG-CAG.The expression of-PLGC-GC showed a decreasing phase(ie,hsa-circ-0000798,hsa-circ-0005286,hsa-circ-0004928 were negatively correlated with the occurrence and development of GAC),indicating that hsa-circ-0000798 can be in the stomach.Early detection and early diagnosis play a role in the transformation of inflammation-cancer.Compared with OLGA stage ?/?,hsa-circ-0005286 and hsa-circ-0004928 were significantly lower in OLGA stage ?,but the difference was not statistically significant,which may be related to the sample size,which needs.further research and verification.According to the hsa-circ-0000798 RT-qPCR,hsa-circ-0005286 RT-qPCR,hsa-circ-0004928 RT-qPCR results,the ROC curve was drawn and the AUC was calculated.The results showed that the three circRNAs have the diagnostic CAG,PLGC and GAC.Different values.The accuracy of CAG in 3 circRNAs RT-qPCR diagnosis was low(0.5?AUC<0.7,P<0.01),hsa-circ-0000798 RT-qPCR,hsa-circ-0005286 RT-qPCR,hsa-circ-0004928 RT-qPCR were more accurate in the diagnosis of PLGC(0.7?AUC<0.9,P<0.01),hsa-circ-0004928 RT-qPCR were more accurate in the diagnosis of GAC(0.7?AUC<0.9,P<0.01),hsa-circ-0000798 RT-qPCR and hsa-circ-0005286 RT-qPCR diagnosed GAC with the highest accuracy(AUC=0.94,P<0.01).Combining the correlation of the previous three circRNAs RT-qPCR with the OLGA,OLGIM grading and staging system,we can conclude that the three circRNAs differentially expressed in the gastric“inflammatory-cancer”transformation identified by sequencing,screening and verification are in the stomach.-Cancer plays an important role in the transformation process,and can be applied to the early detection and early diagnosis of PLGC and GAC in clinical practice,and it is worth further exploring its function.2.Interventional study on the results of Jianpi Qingre Huoxue decoction based on the differential expression of circRNAs in gastric "inflammatory-cancer"transformationIn this study,OLGA and OLGIM were used to compare and analyze the scores of Weiweiqing group and folic acid group before and after treatment.The results showed that OLGA and OLGIM were significantly improved after treatment(P<0.05).There was no statistical difference between the two groups.Significance(P>0.05)confirmed the effectiveness of Weiweiqing Granules in the treatment of CAG.On the basis of affirming the efficacy of Weiweiqing Granule in the treatment of CAG,the expression of hsa-circ-0000798 RT-qPCR,hsa-circ-0005286 RT-qPCR and hsa-circ-0004928 RT-qPCR in gastric mucosa before and after treatment was further analyzed.The results showed that Weiweiqing granules and folic acid tablets could up-regulate the expression levels of gastric mucosa hsa-circ-0000798 RT-qPCR,hsa-circ-0005286 RT-qPCR,hsa-circ-0004928 RT-qPCR,and Weiweiqing group.The difference between the two groups(n=30)hsa-circ-0005286 and RT-qPCR was statistically significant(P<0.05),but the gastric wilting group(n=15)and the folic acid group alone(n=15)The difference was not statistically significant(P>0.05),which may be related to the small amount of sample in the stomach-sweet group or the folic acid alone group.Hsa-circ-0000798 RT-qPCR,hsa-circ-0004928 RT-qPCR expression was up-regulated,but the difference before and after treatment was not statistically significant.Tips hsa-circ-0000798,hsa-circ-0005286,hsa-circ-0004928 may be the target of drug intervention,and further verify hsa-circ-0000798 RT-qPCR,hsa-circ-0005286 RT-qPCR,hsa-Circ-0004928 Clinical application value of RT-qPCR.ConclusionWith"CNAG-CAG-PLGC-GAC" and so on four different pathological stages of clinical specimens of gastric mucosa as the research object,by the high flux second generation sequencing,screening,clinical validation determines three differentially expressed circRNAs(hsa-circ-0000798,hsa-circ-0005286,hsa-circ-0004928)which is related with the stomach "inflammation-Cancer"Transformation,It play an important role in the process of conversion of the stomach "inflammation-Cancer",and can be used in the clinical PLGC,GAC early detection,early diagnosis.Jianpi Qingre Huoxue dprescription is effective in the treatment of CAG,which may play a therapeutic role in blocking gastric mucosa atrophy by regulating hsa-circ-0000798,hsa-circ-0005286 and hsa-circ-0004928. |
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