Analysis Of MiRNA Expression And Functional Prediction Of Target Genes In Colon Tissues Of Milk Allergen Mice

β-Lactoglobulin（β-Lg）allergy is one of the food safety issues which seriously affects the healthy growth and development of infants and young children.There are mainly frequently gastroesophageal reflux,vomiting,chronic diarrhea,antifeeding etc.clinical symptoms.At present,the exact pathogenesis ofβ-Lg allergic inflammation is still unclear.Recent studies suggest that microRNA（miRNA）,as an important regulator of intestinal mucosal immunity,plays a key role in various diseases such as inflammatory bowel disease,pathogen infection,intestinal tumor,and has become a hot research spot of molecular regulation mechanisms of diseases.However,it has rarely been reported so far to analyze the role of miRNA imbalance and its target gene expression in the development of cow’s milk allergy.This study was the first to use Illumina/Solexa high-throughput sequencing technology to screen the expression profile of miRNA in colon tissues of allergic mice,the target differential expression gene,gene function annotation and KEGG signaling pathway analysis.To provide a new theoretical basis for elucidating the pathogenesis of β-Lg allergic inflammation,and a new intervention target for the pretreatment of allergic inflammation.（1）Establishment and verification of allergic animal modelsFemale KM mice 5-6 weeks old were randomly divided into normal group and allergy group（n=20）.The mice in the cow’s milk allergy group were randomly injected with 0.2 mL of β-Lg（1 mg/mL）once a week for 3 weeks.At the 4th week,the mice were sacrificed by cervical dislocation after oral stimulation of β-Lg allergen.At the end of the model construction,the indicators of allergic signs,body weight and immune organs,eosinophils,tissue antibody content and related inflammatory cytokines were measured.The results showed that compared with the normal group,the weight ofβ-Lg-sensitized mice was decreased,β-Lg-specific IgE,eosinophil count and pro-inflammatory cytokines IL-4 and IL-17 levels were significantly increased（P<0.05）,suggesting thatβ-Lg allergy animal model was successfully constructed.（2）Differential expression lineage of miRNAThe total RNA of colon tissue inβ-Lg allergy group and normal group was extracted and sequenced.17 differentially expressed miRNAs were screened,and 8 miRNAs were up-regulated（miR-5101-R+1,miR-1934₅p,miR-196b_R+2,miR-365-5p,miR-504-5p,miR-433-3p,miR-1193-3p,miR-224-5p）,and 9 miRNAs were down-regulated（miR-4454_L-2,miR-7977-p5,miR-106a-5p,miR-194-5p,miR-6691-5p,miR-30a-3p,miR-150,miR-669a-5p,miR-19a-5p）.The differentially expressed miRNAs was verified by qRT-PCR,indicating that the sequencing results were reliable.（3）Target gene prediction and functional verification of miRNATarget gene prediction was performed on the differentially expressed miRNA-enriched KEGG signaling pathway.The target genes which related to allergic inflammation and actived in the pathway were mainly Mapkl,Pdgfrβ,Jak2,Stat3,IKbkb,I16rα,Mapkl4,Tgfβr2.The validation results by qRT-PCR showed that the screening and predicting target gene was reasonable and relevant.After GO signal annotation and KEGG pathway analysis,549 GO entries related to allergic inflammation and differential gene expression were obtained,and 8 KEGG signaling pathways were significantly enriched.Among them,JAK-STAT and MAPKs were associated with allergic inflammation.The difference in signal pathway was significant and the number of differentially expressed genes was the highest（P<0.01）.All analysis indicates that the screened and verified miRNAs and their target genes may participate in the regulation of related allergic inflammatory pathways and exert their functions,which can be followed up.Based on the test results,the screened and verified the miRNA expression profile data ofβ-Lg allergic mice by small RNA sequencing,was highly reliable and can be used for subsequent analysis.At the same time,the three screened differentially signaling pathways（PI3K/Akt,JAK-STAT and MAPKs）and the predicted and verified function of miRNA and target genes can provide theoretical basis and research directions for the study of the pathogenesis of β-Lg allergic inflammation,the specific mode of regulation needs to be further analyzed in subsequent experiments.