| ObjectivesThe aims to search for key molecules related to "inflammation and cancer" of chronic atrophic gastritis and explore the intervention mechanism of traditional Chinese medicine.To screening the key mRNAs related to "inflammation-cancer" of chronic atrophic gastritis and verify its crucial role in "inflammation transforming to cancer" in the background of chronic atrophic gastritis purpose to preliminarily reveal the molecular mechanism of "inflammation shifting to cancer" of chronic atrophic gastritis.Based on the randomized clinical study of evaluating the clinical effectiveness of fortifying the spleen,clearing heat and activating blood method for chronic atrophic gastrtitis,the key mRNAs are detected to expoler the possible mechanism of TCM blocking the process of "inflammation transforming to cancer"in chronic atrophic gastritis.Method1.Screening and validation of key mRNAs related to "inflammation and cancer" in chronic atrophic gastritisThis part was aiming at mRNA molecule.Homo specimens of gastric mucosa was collected from four pathological phase of "inflammation transforming to cancer"(chronic non-atrophic gastritis(n=5),chronic atrophic gastritis(n=5),precancerous lesions of gastric cancer(n=5),gastric adenocarcinoma(n=5).A total of 20 samples were put in high throughput second-generation sequencing to screening mRNAs associated with "inflammation and cancer" in chronic atrophic gastritis.Functional enrichment analysis and protein interaction were performed with FunrichV3.0.On this basis,the homo gastric mucaso microarray dataset related to normal,chronic atrophic gastritis and gastric adenocarcinoma from TGCA and GEO database was selected for bioinfomatic analysis to screen the mRNAs associated with "inflammation and cancer".A total of 172 samples’s microarray data was anlysised by by methods of GEO2R(GEO),CRN(TGCA)and GEPIA(TGCA)to search the differential significantly expressed genes.Functional enrichment analysis were performed with FunrichV3.0.Protein interaction was anlysised by string database.The candidate key mRNAs was affirmed by integrating microarray data and high-throughput second-generation sequencing data.The identified mRNA molecules were verified by small samples,and the sample size was further expanded to verify at the gene level and protein level respectively.Real-time fluorescence quantitative PCR was performed on 176 human gastric mucosa samples(chronic non-atrophic gastritis(n=60),chronic atrophic gastritis(n=59),precancerous gastric lesions(n=30),and gastric adenocarcinoma(n=27)).Immunohistochemical tests were performed on 121 human gastric mucosa samples(chronic non-atrophic gastritis(n=33),chronic atrophic gastritis(n=34),precancerous gastric lesions(n=25),and gastric adenocarcinoma(n=29)).To clarify the important role of key mRNA in the "inflammation-cancer" transformation of chronic atrophic gastritis.Based on the expression level of key mRNA in the pathological stage of "gastritis-cancer"in the stomach,chronic non-atrophic gastritis was used as the control group to draw the ROC curve of single mRNA and combined mRNAs for the prediction of pre-cancer diseases and gastric cancer,and analyze the specificity and sensitivity of the ROC curve for the prediction of pre-cancer diseases and gastric cancer.At the same time,the correlation with OLGA and OLGIM staging was analyzed to clarify the important clinical significance of key mRNA in the transformation of "phlogistic cancer" in chronic atrophic gastritis.2.Regulating the action mechanism of the key mRNA related to"inflammation and cancer"in the treatment of chronic atrophic gastritis by invigorating the spleen,clearing heat and activating bloodIn this part,the effectiveness of invigorating spleen,clearing heat and promoting blood circulation in the treatment of chronic atrophic gastritis was first evaluated through a multicenter randomized controlled trial.The subjects were selected from 11 grade a hospitals of traditional Chinese medicine in guangdong province according to inclusion and exclusion criteria.The experimental drug was jianpi qingrehuoxue fang(weiqing granules),and the control drug was folic acid tablets.The course of treatment was 24 weeks.The study period was from June 2015 to December 2018.On the basis of completing the effectiveness of invigorating spleen,clearing heat and activating blood for the treatment of chronic atrophic gastritis,the subjects with effective treatment and frozen gastric mucosa specimens before and after treatment were selected for the study on the therapeutic mechanism.Key mRNA related to "inflammation and cancer" of chronic atrophic gastritis was targeted,and the expression levels of key mRNA before and after treatment in the two groups were detected by real-time fluorescence quantitative PCR technology to evaluate the difference in expression levels before and after treatment.To explore the possible mechanism of jianpi qingrehuoxue method and folic acid tablets in regulating the expression of key mRNA in the treatment of chronic atrophic gastritis and blocking the transformation of "inflammation and cancer".Result1.Screening and validation of key mRNA related to "inflammation and cancer" in chronic atrophic gastritisA total of 1,781 mrnas related to "inflammation and cancer" of chronic atrophic gastritis were screened out from 20 human gastric mucosa samples at different pathological stages by highthroughput second-generation sequencing technology.There were 802 inflammatory-cancer related pathways,128 molecular functions,241 cell components and 68 biological processes.Among them,there were 90 pathways with P<0.05,30 molecular functions,38 cell components,and 16 biological processes.Protein interaction relationship 885.The gene chip data mining of TGCA and GEO screened out 13 "inflammatory-cancer" related mrnas in chronic atrophic gastritis.There were 114 pathways related to "inflammation and cancer",8 molecular functions,18 cell components and 7 biological processes.There were 25 pathways with P<0.05,6 molecular functions,5 cell components and 0 biological processes.There were 23 adjacent interacting proteins and 27 interacting relationships.By integrating and analyzing gene chip data and high-throughput second-generation sequencing data,5 candidate mrnas related to "phlogistic cancer" in chronic atrophic gastritis were selected.All the 5 candidate mRNA molecules were detected by real-time fluorescence quantitative PCR in 28 human gastric mucosa samples of different pathological stages of "gastritiscarcinoma",and the results showed that the expression levels of 5 mRNA were statistically different in the four pathological stages(P<0.05).In terms of small sample verification results,LYZmRNA and SSTmRNA with consistent expression trends in the four stages of"inflammation and cancer" were selected for expanded sample size verification.LYZmRNA and SSTmRNA in 176 cases of gastric inflammation-cancer of different pathological stages of specimens of gastric mucosa in real-time fluorescent quantitative PCR detection,the results showed that LYZmRNA in chronic atrophic gastritis and chronic atrophic gastritis,the stomach before lesions,gastric adenocarcinoma four pathologic stage expression level in non-normal distribution(P>0.05),2-ΔΔCt median 1.18>0.79>0.52>0.35,respectively,the statistical differences between groups(P<0.05),and the overall expression levels showed a trend of diversity decline.SSTmRNA in chronic atrophic gastritis and chronic atrophic gastritis,the stomach before lesions,gastric adenocarcinoma four pathologic stage expression level in non-normal distribution(P>0.05),2-ΔΔ Ct median 0.941>0.53>0.32>0.12,respectively,the statistical differences between groups(P<0.05),and the overall expression levels showed a trend of diversity decline.The results were verified at the protein level,and the immunohistochemical results showed that the positive expression intensity of LYZ in the four groups presented a gradually declining trend(2=7.47,p=0.00)。SST was differentially expressed in the four groups,and the positive rate showed a gradually decreasing trend.(2=19.64,p=0.00)。Therefore,it is clear that LYZmRNA and SSTmRNA play an important role in the "inflammation-cancer" transformation of chronic atrophic gastritis.The ROC curve of LYZmRNA showed that AUC=0.636 and P=0.01 in the chronic nonatrophic gastritis group as the control group.AUC=0.711,P=0.00 in the precancerous group.In the gastric adenocarcinoma group,AUC=0.867,P=0.00.The ROC curve of SSTmRNA showed that AUC=0.618 and P=0.02 in the chronic non-atrophic gastritis group as the control group.AUC=0.728,P=0.00 in the precancerous group.In the gastric adenocarcinoma group,AUC=0.831,P=0.00.The ROC curve of LYZmRNA+SSTmRNA showed that,taking chronic non-atrophic gastritis as the control,AUC=0.718,P=0.00;AUC=0.762,P=0.00 in the precancerous group.In the gastric adenocarcinoma group,AUC= 0.894,P=0.00.AUC>0.5,P<0.05 indicated that SSTmRNA and LYZmRNA had good specificity and sensitivity in the prediction of gastric pre-cancer diseases and gastric adenocarcinoma,and the combined application had better diagnostic effect.The expression level of LYZmRNA was related to the stages of OLGA and OLGIM,and the expression level of stage 0 was different from that of stage Ⅰ and stage Ⅱ,respectively(P<0.05).The expression level of SSTmRNA was related to the stages of OLGA and OLGIM,and the expression level of stage 0 was different from that of stage I,II and III,respectively(P<0.05).2.Regulating the action mechanism of the key mRNA related to "inflammation and cancer"in the treatment of chronic atrophic gastritis by invigorating the spleen,clearing heat and activating bloodIn the clinical study to evaluate the effectiveness of invigorating the spleen and clearing away heat in the treatment of chronic atrophic gastritis,a total of 8 centers participated,3 centers withdrew,and a total of 177 subjects were included.Among them,131 subjects who completed the study were included in the main outcome evaluation.Among them,65 cases were in the jianpi qingrehuoxue prescription group and 66 cases were in the folic acid tablet group,and there was no difference in gender and age distribution between groups or within groups(P>0.05).The pathological score changes before and after treatment were evaluated by comparing the subjects’ own before and after treatment,and the results showed that Z=3.55 and P=0.00 showed statistically significant differences.In order to further clarify the difference between the two treatment effects,the pathological degree of CAG in the two groups of subjects before treatment was at the same level,with comparability(P>0.05).There was no significant difference in CAG pathological score between the two groups after treatment(Z=-0.201,P=0.84).The changes of OLGA and OLGIM stages in the spleenstrengthening,heat-clearing and blood-activating prescription group(before and after treatment)were statistically different(P=0.00).The changes of OLGA and OLGIM stages in the folic acid tablets group(before and after treatment)were statistically significant(P=0.00).There was no significant difference between the two groups in OLGA and OLGIM(Z=-0.13,P=0.89).Z=-1.33,P=0.18).The pathological efficacy of jianpi qingrehuoxue recipe in the treatment of CAG was 80.00%,and that of folic acid tablets was 83.33%,with no statistical significance(Z=-0.21,P=0.83).In complete curative effect mechanism study,spleen heat huoxue fang group,n=21,folic acid group,n=21,before and after treatment in the gastric mucosa LYZmRNA and SSTmRNA expression level,the results show that the spleen heat huoxue fang and folic acid can increase LYZmRNA and expression level of SSTmRNA spleen heat huoxue fang(before-after treatment),P=0.029,folic acid tablets group(before-after treatment),P=0.00,the difference is statistically significant.Conclusion1.Five mRNA may play an important role in the transformation of "inflammation-cancer"in chronic atrophic gastritis.Among them,LYZmRNA and SSTmRNA have been preliminarily verified and confirmed to play an important role in "inflammatory-cancer"transformation.2.Fortifying the spleen and stomach and promoting blood circulation and detoxification method and folic acid tablets are effective in the clinical treatment of chronic atrophic gastritis,which can effectively improve gastric mucosal atrophy,intestinal metaplasia and other gastric mucosal lesions.It may play a therapeutic role in blocking the "inflammation-cancer" transformation by up-regulating the expression levels of LYZmRNA and SSTmRNA. |
