| ObjectiveChronic atrophic gastritis is a common digestive refractory disease,belongs to a kind of chronic gastritis.It defined that mucosa glands were decreased,with or without intestinal metaplasia and dysplasia hyperplasia in histopathology.It always diagnosis by pathological biopsy under gastroscope,which was single,unacceptability,and there is no effective and accurate serological index that may greatly hinders the development of study of chronic atrophic gastritis.Chronic atrophic gastritis is a premalignant disease of the stomach.Its treatment is aimed at improving symptoms and nourishing gastric mucosa.Western medicine does not reverse atrophy,intestinal metaplasia and dysplasia.Interestingly,more and more studies have reported that Traditional Chinese medicine can reverse atrophy.Therefore,this study would focuces on exosomal miRNAs,looking for chronic atrophic gastritis serum biomarkers,and then to explore the mechanism of traditional Chinese medicine that how to reverse atrophy.That may be useful in clinical work.Methods1.Clinical study screening phase: collected the clinical pathological data of CAG and normal controls(CNAG)and samples of serum and gastric mucosa.Using Exo Quick to extract serum exosomes,nano-tracking technology(NTA)and flow cell technology were used to determine the serum.High throughput sequencing technology was utilized to analyze the expression of s RNAs in the serum exosomes of the two groups(s RNA-seq).Candidate miRNAs were preliminaries screened according to the expression of abundant and significant difference,and the diagnostic value was evaluated by the subject’s work characteristic curve analysis(ROC),and bioinformatics analysis was carried out.2.Clinical research validation phase: through validation of candidate miRNAs q RT-PCR technology,at the same time by ELISA kit to detect the CAG known serum marker stomach gastrin-releasing(GAS),pepsinogen(PG I,II),and so on,by ROC analysis to evaluate and compare the diagnostic value of them,with diagnostic value of biomarkers and key miRNAs;The expression of key miRNAs was analyzed according to the classification of gastric mucosa histopathological indexes.Finally,the key miRNAs were predicted by RNAhybrid,miRanda and Target Scan,and key miRNAs and possible target genes were detected in gastric mucosal tissues.3.Weiweiqing intervention and mechanism to explore: on the basis of studying existing Weiweiqing intervention,pathological histology as the main curative effect evaluation index,folic acid as the control group,evaluate its clinical curative effect in intervention model the CAG pixu(spleen deficient);Through q RT-PCR technology,the expression of key miRNAs and target genes in the serum exosomes and gastric mucosa tissues before and after the treatment of Weiweiqing were detected and the mechanism of action was explored.Results1.By comparing the sex and age of 30 subjects in the two groups of CAG and CNAG,there was no statistical difference and comparability.To extract serum secrete body outside,verification results showed that 6 samples of the particle size is about 70% in 20 nm-200 nm,particle distribution coefficient(PDI)between 0.08-0.7,surface markers CD81,CD63 positive ratio of over 80.0%,is the Exo-Quick kit preparation of precipitation secrete body rich in external bleeding body,but can’t rule out contain other extracellular vesicles,can be used for further research.Then,s RNA-seq results showed that each sample received an average of 37,000,000 reads,about 50.2% could be compared to the genome,with miRNAs accounting for 42.4%.More than 10 reads/ samples had 220 miRNAs,from 111 different pre-mirnas,including 7 new miRNAs.The top 10 miRNAs with the highest expression levels were hsa-miR-148a-3p,hsa-miR-122-3p,hsa-miR-486-3p,hsa-miR-451 a,hsa-miR-122-5p,hsa-miR-3591-3p,hsa-miR-486-5p,hsa-miR-151a-3p,hsa-miR-92a-3p,and hsa-miR-320 a.The expression of hsa-miR-3591-3p,hsa-miR-122-3p and hsa-miR-122-5p was significantly increased in the CAG group,with significant difference.Hsa-miR-451 a,hsa-miR-151a-3p,hsa-miR-92a-3p were significantly down-regulated.Therefore,they are defined as candidate miRNAs.ROC analysis indicated that the most promising potential was hsa-miR-3591-3p(AUC 1.00,95%CI:1-1,P =0.049),100% specificity and 100% sensitivity;Secondly,hsa-miR-122-5p(AUC 0.78,95%CI:0.36-1,P =0.28),hsa-miR-122-3p(AUC 0.56,95%CI:0.03-1,P =0.83).Because the sample size was too small,the ROC analysis of these six candidate miRNAs was not possible.6 candidate miRNAs with the target gene for GO significant enrichment analysis showed that the main concentration was in biological processes:cellular process,cell components:cell,cell part,molecular function:binding.Enrichment of target genes most pathway Signal transduction,working:the Overview,Global and the Overview maps,Endocrine system and Immune system.The largest concentration of target genes was Protein processing in endoplasmic reticulum,Neurotrophin signaling pathway,Fluid shear stress and atherosclerosis.Therefore,it can be found that these candidate miRNAs are mainly related to cellular components and immune responses.2.Using Elisa kits,the concentration of GAS,PG I,PG II,in CAG and CNAG patients were(16.77,6.25)pg/m L vs(14.28,2.51)pg/m L,(83.92,71.73)ng/m L vs(64.86,102.62)ng/m L,(4.33,5.30)ng/m L vs(3.07,3.31)ng/m L,(22.05,16.93)vs(12.83,10.9),respectively.GAS,PG I/II in the two groups are statistically significant.According to the results of s RNA-seq,30 samples of CNAG and CAG were selected,and 6 candidate miRNAs were verified.The results showed that in the comparison between the two groups,the most potential hsa-miR-3591-3p in the s RNA-seq results had a low expression level,and the number of cases that were included in the actual analysis was less than that and the expression trend was inconsistent.The expression tendency of hsa-miR-122-3p,hsa-miR-122-5p was consistent with the results of s RNA-seq,obviously up-regulated,but there were fewer cases of hsa-miR-122-3p with lower expression and larger Ct value.In addition,there was no significant difference in expression of hsa-miR-92a-3p,hsa-miR-151a-3p,and hsa-miR-451 a.ROC curve shows the GAS,PG I/II,hsa-miR-122-3p,hsa-miR-122-5p of the area under the curve(AUC)were 0.74,0.67,0.76,0.67,and the diagnostic value of other indicators are significantly higher than single,has a certain difference between the CAG patients and normal control group;The best critical values of GAS,PG I,PG II and PG I/II were 16.13,53.18,5.45 and 23.84 respectively.Hsa-miR-122-3p,hsa-miR-3591-3p were all included in the analysis,and the reliability was slightly worse.Logistic regression was used to model them,and the combination 8 was GAS,PG I/II,hsa-miR-122-5p AUC,0.84(95%CI: 0.74-0.94),and the sensitivity and specificity were 67.86% and89.66% respectively,and the diagnostic value was the best.According to the results of s RNA-seq and RT-q PCR,the expression of hsa-miR-122-3p and hsa-miR-122-5p in the CAG group was significantly up-regulated,and the correlation analysis indicated a positive correlation(r=0.92,P <0.05),and they were defined as key miRNAs.In gastric mucosa pathological histology index classification,analyze the expression of key miRNAs status,the results show that hsa-miR-122-5p in moderate and severe atrophy may express,it also may as the degree of severity,hsa-miR-122-5 p increases,and hyperplasia,chronic inflammation,no obvious correlation between activity;Hsa-miR-122-3p showed no differential expression in different pathological indexes.Therefore,the expression of hsa-miR-122-5p in serum exosomes may be related to atrophic and intestinal metaplasia,while the expression of hsa-miR-122-3p in serum exosomes is less stable,and its reliability is not hsa-miR-122-5p.Finally,hsa-miR-122-5p was significantly lower in the gastric mucosal tissues of the CAG group and was statistically significant,and the ROC analysis results were in agreement with the serum results.Prediction of target genes in six target genes may be associated with the onset of the CAG,further validation CCLN found the CAG group,OLCN,SLC7A1 does not change significantly expression,and NOD2 expression significantly lowered,P value is 0.09,tend to be statistically significant,and gastric mucosa tissue of miR-122-5p to maintain consistent trend,and serum secrete in the miR-122-5p keep the opposite trend;SLC1A5 showed a downward trend with a P value of 0.14.3.The database was retrieved,and a total of 64 patients completed the study,of which 38 patients were in Weiweiqing group and 26 in folic acid group.There was no statistical difference in the clinicopathological features of the pretreatment group,and the baseline data were consistent and comparable.Compared with before treatment,all the patients in the group decreased,chronic inflammation and pathological total score decreased significantly.After the treatment,atrophy and chronic inflammation decreased,suggesting that the treatment of Weiweiqing may be the main effect of reversing atrophy and reducing inflammation.After treatment with folic acid group,the atrophy decreased significantly,suggesting that folic acid may reverse atrophy.Weiweiqing and folic acid were equally effective in improving the pathologic score,the disappearance rate of atypical hyperplasia,and the improvement rate of histopathological indexes of patients.Through qRT-PCR,the expression level of hsa-miR-122-5p in serum exosome or gastric mucosa tissue,NOD2,SLC1A5 were analysed in some subjects,the results showm hsa-miR-122-5p showed a trend of slight downregulation after treatment,gastric mucosa tissue NOD2 and miR-122-5p showed a trend of increase;compared with the Wuxiao group,the Youxiao group has down-regulated hsa-miR-122-5p and up-regulated NOD2 and SLC1A5;compared with folic acid group,the decrease of hsa-mir-122-5p in the serum exosome,the increase of NOD2 were more obvious after the treatment of Weiweiqing group.All though,those results was no statistical difference,but the trend of change were meet to the hypothesis that hsa-mir-122-5p would downregulate and target gene NOD2 would upregulate with the improvement of gastric mucosal lesion after treatment.Therefore,the Weiweiqing may play a role in miR-122-5p/NOD2.ConclusionSerum exosomal hsa-miR-122-5p has the potential to be a diagnostic marker of non-invasive CAG,which can effectively distinguish between chronic atrophic gastritis and normal population or chronic non-atrophic gastritis.The variation tendency of the expression was not consistent with the gastric mucosa,and its source was not yet determined.It was linked to atrophy and intestinal metaplasia,and the target gene was NOD2.The new molecular labeling with GAS and PG I/II has superior diagnostic efficacy for CAG.The treatment of CAG may be through the regulation of hsa-miR-122-5p /NOD2 to reverse the atrophy of gastric mucosa or reduce the inflammatory response.Other candidate miRNAs: hsa-miR-92a-3p,hsa-miR-122-3p,hsa-miR-151a-3p,hsa-miR-451 a,and hsa-miR-3591-3p need more clinical sample size to test their expression differences and diagnostic value. |
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