Detection Of Maize-derived MicroRNAs And Prediction And Validation Of Their Potential Target Genes In Pigs

MicroRNAs(miRNAs)are a class of small noncoding RNAs that act as high-efficiency post-transcriptional regulators of gene expression and thus play many important roles in living organisms.In 2012,the first cross-kingdom miRNA-based interaction had been reported by Zhang Chenyu,Nanjing University professor,demonstrating that diet-derived plant miRNAs could regulate mammalian gene expression as mammalian functional miRNAs did.Since then,a growing number of studies have provided evidence that exogenous miRNAs may be transferred from one species to another and regulate gene expression in recipient cells,and these studies have broadened our view of cross-kingdom communication.As a vital biomedical research model and agricultural economic animal,swine(Sus scrofa)is an ideal model to study cross-kingdom communication between plant-derived miRNAs and mammals for which staple food is corn(Zea mays L.)and soybeans.In the present study,we performed bioinformatics analysis,real-time fluorescence quantification PCR(qRT-PCR),TA clone,Sanger sequencing,TargetScan,NCBI Blast,RNAhybrid,Mir-Trap system and dual-luciferase reporter assay system to provide evidence that maize-derived miRNAs could presented in porcine serum(or tissues)and mediated cross-kingdom regulation of porcine gene expression.The results are shown as follows:1 MIR168a and MIR166a were the most abundant plant miRNA sequences filtered from 25 porcine small RNA datasets downloaded from NCBI database,accounting for 58.51%and 37.24%in total plant miRNAs(mean value from 25 samples),respectively.2 Maize-derived miRNAs presented in porcine serum and tissues were detected by qRT-PCR.The level of five maize miRNAs in porcine serum were increased and reached peak values at 6 h or 12 h after fresh corn feeding.3 Approximately 58.2%of the total maize miRNAs in porcine serum detected by qRT-PCR were presented in serum exosomes extracted by ultracentrifuge.4 Compared to fasting samples,the level of zma-miR164a-5p in cafeteria feeding samples presented a significant increase in different tissues(heart,longissimus dorsi,spleen and kidney)detected by qRT-PCR.5 We predicted 50 potential target porcine mRNAs which had low minimum free energy values and highly matched seed regions for zma-miR164a-5p by TargetScan,NCBI Blast and RNAhybrid.Mir-Trap system and qRT-PCR were performed to confirm that the porcine mRNAs whose level showed a significant increase(THBS4,BCL-9 and PLAGL2,et.al)compared with the controls were the targets of zma-miR164a-5p.6 A dual-luciferase reporter assay was employed to demonstrate that zma-miR164a-5p could reduce luciferase activity by directly binding to the putative binding sites of 3 porcine genes(CSPG4,OTX1 and PLAGL2).Our results indicated that maize-derived miRNAs could be detected in porcine serum and tissues,and these exogenous plant miRNAs in food had the potential to regulate the expression of target genes in pigs.