【作者】 李培武；

【导师】 刘凤斌；

【作者基本信息】 广州中医药大学 ， 中医内科学（专业学位）， 2019， 博士

【摘要】 目的:本论文以探究胃“炎-癌”相关差异表达环状RNAs（Circular RNAs,circRNAs）-微小核糖核酸RNAs（MicroRNA,miRNAs）、探讨健脾清热活血方治疗慢性萎缩性胃炎的疗效与机制为主要研究目标。以circRNAs-miRNAs为目标分子,通过对慢性非萎缩性胃炎（Chronic Non-Atrophic Gastritis,CNAG）-慢性萎缩性胃炎（Chronic Atrophic Gastritis,CAG）-胃癌前病变（Precancerous Lesions of Gastric Cancer,PLGC）-胃腺癌（Gastric Adenocarcinoma,GAC）等4个不同病理阶段患者临床胃黏膜标本,进行高通量二代测序、同时进行筛选并确定相关差异表达的circRNAs,确定差异表达circRNAs调控的miRNAs并预测其下游mRNAs,验证其在胃“炎-癌”转化中的关键作用及对胃“炎-癌”转化早期发现、早期诊断的临床应用价值,初步揭示胃“炎-癌”转化的关键分子机制。基于关键circRNAs,在评价健脾清热活血方干预CAG、PLGC有效的基础上,探讨中医药调控circRNAs-miRNAs等关键分子,阻断胃“炎-癌”转化的可能机制。方法:1.胃“炎-癌”转化相关差异表达circRNAs的测序分析1.1胃“炎-癌”转化相关差异表达circRNAs的高通量二代测序本研究运用高通量测序技术方法（二代）筛选20例胃“炎-癌”转化4个不同病理阶段（CNAG-CAG-PLGC-GC）差异表达的circRNAs,探索胃“炎-癌”转化相关差异表达分子（circRNAs、miRNAs、mRNA）,为寻求中医药干预靶点提供参考。1.2胃“炎-癌”转化相关差异表达circRNAs的筛选本部分研究首先对前期测序得到的2749个circRNAs进行筛查,缩小差异表达circRNAs的范围以便于下一步研究。主要按照3个步骤进行:（1）建立与CAG发病、发展相关的 circRNAs 组合,即 CAG group,将CNAG-vs-CAG,CNAG-vs-PLGC,CAG-vs-PLGC的DEGs组合,取并集,得到73个与CAG发病与进展相关的circRNAs组合。（2）建立与 GAC 发病、发展相关的 circRNAs 组合,即 GC group,将 CNAG-vs-GAC,CAG-vs-GAC,PLGC-vs-GAC的DEGs组合,取并集,得到127个与GAC发病相关的circRNAs组合。（3）CAG group 和 GAC group 取交集,得到 12 个与胃“炎-癌”CNAG-CAG-PLGC-GAC转化相关差异表达的circRNAs。最后通过miRanda计算、提供了注释上circBase的circRNA结合miRBase中miRNA的结果,确定了具有miRNA的结合位点的4个circRNAs。1.3胃“炎-癌”转化相关差异表达circRNAs的验证与circRNAs-miRNAs-mRNAs的构建本研究整合前期胃“炎-癌”相关差异表达circRNA高通量二代测序和筛选数据,通过miRanda计算、提供了注释上circBase的circRNA结合miRBase中miRNA的结果,确定了具有miRNA的结合位点的4个circRNAs。以hsa-circ-0000798为例,通过生物信息学方法预测circRNAs与miRNAs的结合位点,miRNAs与mRNAs的结合位点。经 miRanda 软件预测,hsa-circ-0000798 可能与 19 个 miRNAs。通过 RT-qPCR 验证,明确has-miR-4478在胃“炎-癌”转化不同阶段的表达趋势与has-circ-0000798相反,符合ceRNA机制。经Targetscan软件联合miRanda软件预测分析,has-miR-4478与10749个mRNAs结合。课题组前期研究明确1781个mRNA与胃“炎-癌”转化相关,经取交集,得出与hsa-miR-4478结合的mRNAs中参与胃“炎-癌”转化的共231个。以变化趋势一致,表达量相对高,差异变化倍数大为筛选条件,候选与hsa-miR-4478结合的mRNAs中参与胃“炎-癌”转化的共28个。经FunrichV3.0富集28个mRNAs的功能,细胞组分23个,分子功能15个,生物学过程11个。最终,初步构建circRNAs-miRNAs-mRNAs的调控网络,分析其相关的功能及通路。通过初步功能分析,本研究初步确定 SLC34A2、SOX10、HAP1 作为 hsa-circ-0000798-hsa-miR-4478 的关键靶基因,其中与SLC34A2、SOX10与经典“炎-癌”转化信号通路——Wnt/β-cantenin信号传导密切相关。依据关键circRNAs RT-qPCR结果作图绘制受试者工作特征曲线（Receiver Operating Characteristic Curve,ROC）,计算曲线下面积（AUC）,评估其用于预测PLGC、GAC的特异性及敏感性。同时分析其与胃炎评估系统（Operative Link for Gastritis Assessement,OLGA）、胃炎肠上皮化生评估系统（Operative Link for Gastritis Intestinal Metaplasia Assessment,OLGIM）的相关性,阐释其在胃“炎-癌”转化中诊断、病情与预后评估的重要作用。2.基于胃“炎-癌”转化差异表达circRNAs测序结果探讨健脾清热活血方的干预研究临床研究采用多中心、随机对照试验评价健脾清热活血方——胃萎清颗粒治疗慢性萎缩性胃炎的有效性。参加临床试验的患者来源于广东省内的11家三甲中医院,按照规定的纳入标准、排除标准筛选受试者,试验组口服健脾清热活血方（胃萎清颗粒）,对照组口服叶酸片,治疗时间24周,以基于胃黏膜病理学改变的OLGA、OLGIM两个分级分期评估系统为主要结局指标。按照OLAG、OLGIM分级分期系统评价要求和circRNAs检测质量要求,38例胃黏膜标本冻存于-80℃冰箱里的受试者纳入疗效机制研究。结果:1.胃“炎-癌”转化相关差异表达circRNAs的测序分析1.1胃“炎-癌”转化相关差异表达circRNAs的高通量二代测序结果得到4个不同病理阶段6个组间差异表达的circRNAs多达数千个,其中有2749个circRNAs与胃“炎-癌”转化存在一定相关性,信息量丰富,但同时也给后续研究带来挑战。这一研究结果说明了胃“炎-癌”转化的复杂性,与目前该领域相关研究报道相符合。如何从2749个circRNAs中进一步筛查胃“炎-癌”转化差异表达的关键circRNAs,以进行下一步功能、诊断、预测研究,是本研究的重点和难点。1.2胃“炎-癌”转化相关差异表达circRNAs的筛选确定具有 miRNAs 结合位点的 4 个 circRNAs（hsa-circ-0000798、hsa-circ-0005286、hsa-circ-0004928、hsa_circ₀062649）,进入下一步验证及机制研究,具有重要的研究意义和临床应用价值。1.3胃“炎-癌”转化相关差异表达circRNAs的验证与circRNAs-miRNAs-mRNsA的构建以hsa-circ-0000798为例,通过生物信息学方法预测circRNAs与miRNAs的结合位点,miRNAs与mRNAs的结合位点。经miRanda软件预测,hsa-circ-0000798可能与19个miRNAs,如下表所示,通过RT-qPCR验证,明确has-miR-4478在胃“炎-癌”转化不同阶段的表达趋势与has-circ-0000798相反,符合ceRNA机制。经Targetscan软件联合miRanda软件预测分析,has-miR-4478与10749个mRNAs结合。课题组前期研究明确1781个mRNA与胃“炎-癌”转化相关,经取交集,得出与hsa-miR-4478结合的mRNAs中参与胃“炎-癌”转化的共231个。以变化趋势一致,表达量相对高,差异变化倍数大为筛选条件,候选与hsa-miR-4478结合的mRNAs中参与胃“炎-癌”转化的共28个。经FunrichV3.0富集28个mRNAs的功能,细胞组分23个,分子功能15个,生物学过程11个。最终,初步构建circRNAs-miRNAs-mRNAs的调控网络,分析其相关的功能及通路。通过初步功能分析,本研究初步确定SLC34A2、SOX10、HAP1 作为 hsa-circ-0000798-hsa-miR-4478 的关键靶基因,其中与 SLC34A2、SOX10与经典“炎-癌”转化信号通路——Wnt/β-cantenin信号传导密切相关,为后续研究提供基础。与OLGA Ⅰ/Ⅱ期相比较,hsa-circ-0000798在OLGA Ⅲ期明显呈低表达,差异有统计学意义,这与前期测序、筛选、验证得到的hsa-circ-0000798在CNAG-CAG-PLGC-GC 中表达呈下降相符合（即 hsa-circ-0000798、hsa-circ-0005286、hsa-circ-0004928与GAC的发生、发展呈负相关）,说明hsa-circ-0000798能在胃“炎-癌”转化过程中发挥早期发现、早期诊断的作用。与OLGA Ⅰ/Ⅱ期相比较,hsa-circ-0005286、hsa-circ-0004928在OLGA Ⅲ期明显呈低表达,但差异没有统计学意义,可能与样本量有关,有待下一步进一步研究验证。依据hsa-circ-0000798 RT-qPCR、hsa-circ-0005286 RT-qPCR、hsa-circ-0004928 RT-qPCR 结果分别绘制 ROC曲线,计算AUC区间,结果显示3个circRNAs在诊断CAG、PLGC、GAC中具有不同价值。3 个 circRNAs RT-qPCR 诊断 CAG 准确性较低（0.5≤AUC 区间<0.7）,3 个 circRNAs RT-qPCR 诊断 PLGC 准确性较高（0.7≤AUC 区间<0.9,P<0.01）,hsa-circ-0004928 RT-qPCR 诊断 GAC 准确性较高（0.7≤AUC 区间<0.9,P<0.01）,hsa-circ-0000798 RT-qPCR、hsa-circ-0005286 RT-qPCR 诊断 GAC 具有最高准确性（AUC 区间>0.9,P<0.01）。结合前面3个circRNAs RT-qPCR与OLGA、OLGIM分级分期评估系统的相关性,我们可以认为,经测序、筛选、验证确定的胃“炎-癌”转化相关差异表达的3个circRNAs在胃“炎-癌”转化过程中发挥重要作用,可应用于临床中PLGC、GAC的早期发现、早期诊断,值得进一步探索其功能。2.基于胃“炎-癌”转化差异表达circRNAs测序结果探讨健脾清热活血方的干预研究本研究采用OLGA、OLGIM对胃萎清组、叶酸组治疗前后评分进行对比、分析,结果显示治疗后OLGA、OLGIM明显改善、差异有统计学意义（P<0.05）,两组改善差异没有统计学意义（P>0.05）,肯定了胃萎清颗粒治疗CAG的有效性。在肯定胃萎清颗粒治疗CAG有效的基础上,进一步分析治疗有效患者干预前后胃黏膜hsa-circ-0000798 RT-qPCR、hsa-circ-0005286 RT-qPCR、hsa-circ-0004928 RT-qPCR的表达量,结果显示胃萎清颗粒、叶酸片均能上调胃黏膜hsa-circ-0000798 RT-qPCR、hsa-circ-0005286 RT-qPCR、hsa-circ-0004928 RT-qPCR 的表达水平,胃萎清组和叶酸组两组合并（n=30）hsa-circ-0005286 RT-qPCR治疗前后差异具有统计学意义（P<0.05）,但单独胃萎清组（n=15）、单独叶酸组（n=15）差异没有统计学意义（P>0.05）,这可能与单独胃萎清组或单独叶酸组样本量少有关。hsa-circ-0000798 RT-qPCR、hsa-circ-0004928 RT-qPCR表达量有上调,但治疗前后差异没有统计学意义。提示 hsa-circ-0000798、hsa-circ-0005286、hsa-circ-0004928 可能是药物干预的靶点,并更进一步验证 hsa-circ-0000798 RT-qPCR、hsa-circ-0005286 RT-qPCR、hsa-circ-0004928 RT-qPCR 的临床应用价值。结论:以“CNAG-CAG-PLGC-GAC”等4个不同病理阶段临床胃黏膜标本为研究对象,经高通量二代测序、筛选、临床验证确定了胃“炎-癌”转化相关差异表达的3个circRNAs（hsa-circ-0000798、hsa-circ-0005286、hsa-circ-0004928）,其在胃“炎-癌”转化过程中发挥重要作用,可应用于临床中PLGC、GAC的早期发现、早期诊断。健脾清热活血方治疗CAG有效,可能通过调节hsa-circ-0000798、hsa-circ-0005286、hsa-circ-0004928发挥阻断胃黏膜萎缩的治疗作用。更多还原

【Abstract】 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/β-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/β-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 Ⅰ/Ⅱ,hsa-circ-0005286 and hsa-circ-0004928 were significantly lower in OLGA stage Ⅲ,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.更多还原