MiRNA-based Biomarkers And Action Mechanism Of β₂-agonists Abuse In Goats

The abuse ofβ₂-agonists in husbandry have attracted attention around the world.To date,many detection methods have been developed to determine drug residues in different samples,such as instrumental analysis,immunoassay,and sensor methods,all these tools based on knownβ₂-agonists.Since the large amount drugs inβ₂-agonists family,including new and escaped drugs,advance methods need to be established to traceβ₂-agonists,especially the unknown and escapedβ₂-agonists.Due to the same mechanism of β₂-agonists,investigating the molecular level change caused byβ₂-agonists in animals may give a new aspect of theβ₂-agonists detection.In our previous study,six mRNA biomarkers to detectβ₂-agonists in goats were determined.As mi RNA can regulate the expression of mRNA,which is also more stable than mRNA,we chose miRNA biomarkers as further studies to improveβ₂-agonists detection method in this thesis.Mi RNA is a kind of RNA with length of 21-23nt widely existing in eukaryotes.Compared with mRNA,miRNA is more stable,can regulate the expression of mRNA.Therefore,this project took miRNA as the point,through in vivo and in vitro experiments,found a group of miRNAs as biomarkers to monitor the miuse ofβ₂-agonists in goats,and discussed the regulatory role of miRNA in the pathway ofβ₂-adrenergic receptor（ADRB2）gene.1.Firstly,we screened and verified the miRNA biomarkers ofβ₂-agonists in vitro.miRNA sequencing technology was applied in this study to search differentially expressed mi RNAs in experimental and control groups of skeletal muscle cells treated withβ₂-agonists.qRT-PCR results indicated that selected miRNAs（let-7a-5p,let-7c-5p,let-7e-5p,let-7f-5p,miR-15a,miR-30a-5p,mi R-98-5p,miR-195-5p,miR-99b-5p and miR-1271-5p）can distinguish control cell group fromβ₂-agonists-treated cell group.With an association with previous mRNA biomarkers（FOXO1,mTOR,ATP2A3,PDE4C,ADRB2,IGF-1,MYLK,PTH,GPR,ADCY3,PRKACB,and IL1B）and these miRNA biomarkres,a regulatory networks related toβ₂-agonists in goat was established.2.Then,in vivo experiments were carried out to verify the miRNA biomarkers ofβ₂-agonists selected in vitro.Animal experiments were further applied to verify the in vivo of miRNAs biomarkers.The results showed that the differential-regulation of miRNAs in goat muscle were consistent with the in-vitro experiment.DD-SIMCA and heat map analysis showed that 10 miRNAs could distinguish samples between muscle tissue experimental group and control group.The results of in-vivo experiments were consistent with in vitro experiments,indicating that this group of miRNA biomarkers could be used as biomarkers for monitoring the illegal use ofβ₂-agonists in goats.Based on this group of miRNAs biomarkers,the effectiveness of these miRNA biomarkers in urine of goats（experimental group and control group）treated withβ₂-agonists was further tested.The results showed that the mi RNAs biomarkers could distinguish goat blank urine fromβ₂-agonists-treated goat urine.The above results indicate that ten candidate miRNAs biomarkers could be used to monitor the abuse ofβ₂-agonists in goats based on muscle and urine,and it is expected to provide reference for animal noninvasive detection.3.Regulation mechanisms betweenβ₂-agonists-related mi RNAs biomarker and ADRB2 were clear,and miRNA biomarkers were further verifird through transcriptional regulation.Based on mRNAs and mi RNAs biomarkers ofβ₂-agonists,results in Chapters 1 and 2 predicted that the ADRB2 gene was regulated by let-7a-5p,let-7c-5p,let-7e-5p,let-7f-5p,miR-15a-5p,miR-30a-5p and miR-98-5p.This prediction were also confirmed by cell transfection,dual-luciferase assay and VIP analysis,after mimic transfection,the expression of ADRB2 was significantly down regulated,ranging from 48.3%to 70.6%,and the expression level of ADRB2 was significantly increased in inhibitor transfection group,ranging from 173.8%to 192.1%;compared with the control group,the luciferase activity of miRNA-mut（NC）group was significantly lower,among which let-7f-5p（74.3%）,miR-30a-5p（76.5%）and let-7a-5p（72.5%）were the highest.Above results showed that ADRB2 gene was regulated by let-7a-5p,let-7c-5p,let-7e-5p,let-7f-5p,miR-15a-5p,miR-30a-5p and mi R-98-5p,and the 3’UTR region of the ADRB2 gene is the target site of the above miRNAs.In conclusion,the correctness of the ADRB2 gene and the mi RNAs（let-7a-5p,let-7c-5p,let-7e-5p,let-7f-5p,miR-15a-5p,miR-30a-5p and miR-98-5p）associated withβ₂-agonists in goat were verified.4.Finally,the downstream metabolites ofβ₂-agonists-related genes were studied with metabonomics technology,and the regulatory relationship between differential metabolites and m RNA level markers was established,so as to clarify the regulatory relationship between miRNA and mRNA metabolites related toβ₂-agonists with mRNA level biomarkers as mediators.Based on non-targeted metabolomics technology,the metabolics map ofβ₂-agonists in goat muscle cells was revealed,and a non-targeted metabolome based on UPLC-TOF/MS for monitoringβ₂-agonists in muscle cells was established.Peakview software was used to extract differential information among treatment and control groups,stoichiometric analysis such as PCA and OPLS-DA showed that these compounds information could be used to show the existence ofβ₂-agonists in muscle cells.The qualitative and software quantitative results of HMDB and XCMS showed that compared with the control group,L-leucine,glycerophosphate,stearamide,phytosphingosine,neursphingosine,L-lysine,betaine,L-phenylalanine,etc.in the combination treatment group were up-regulated,and the up-regulation times were between2.985（argininosuccinate）and 10.723（lysine）;creatine,oleamide,choline,L-tyrosine,cholesterol,and sphingosine 1-phosphate etc.showed down-regulation,among which L-tyrosine had the highest down-regulation multiple（-7.892）.They were the common metabolites of fat and protein metabolism,which may provide a new way to explain the mechanism of energy redistribution caused byβ₂-agonists.Combining the transcriptional level markers and KEGG metabolic pathways,the regulatory network and mechanism of β₂-agonists in muscle cells were further clarified,which verified miRNA biomarkers through metabolic regulation.