Screening And Function Of Differentially Expressed MiRNAs Between Ovulatory And Subordinate Follicles In Goats(Capra Hircus)

Ovulation is an important process which is controlled by many factors.MicroRNA（miRNA）is an endogenous non-encoding small molecular RNA.Reports show miRNAs play an important role in ovarian development including follicular growth and development,atresia,luteal formation and disappearance,but the study of miRNAs on ovulation is relatively limited.In this study,we use the ovulatory and subordinate follicles as experimental materials to screen the miRNAs associated with follicular development and ovulation by Illumina sequencing,bioinformatic analysising and qPCR expressional validation.The main results are as follows:1、An average of 12,069,414 raw reads and 11,876,770 clean reads were found in six folliclars RNA libaries（GF1 GF6）by high-throughput sequencing,respectively.Averagely,the 22 nt sequence was 45% in clean reads from six libaries.There were 418 known miRNAs with typical structure from six groups by matching to the reference genome and comparing with the miRBase（21 version）.At the same time,110 sequences were predicted as novel miRNAs by using miREvo,mirdeep and other miRNA prediction software.2、In this study,6 different size of follicles were divided into two groups,GF_d and GF_s,and 9 miRNAs were differentially expressed in GF_d vs GF_s.Four of them（chi-miR-582-5p、novel₁30、chi-miR-214-3p、chi-miR-500-5p）were up-regulated,and five of them（chi-miR-383、chi-miR-130b-5p、chi-miR-92a-3p、chi-miR-125b-5p、novel-9）were down-regulated.Additionally,four miRNAs（novel-344 、chi-miR-10a-5p、chi-miR-10a-3p and chi-miR-196b）were up-regulated in GF1 when GF1 was compared with other groups.3、Target genes of miRNAs were predicted by MiRanda software,and all of them were enriched into 274 pathways by KEGG.Significant differentially expressed chi-miR-130b-5p was predicted to target to BMP15 and GDF9,chi-miR-214-3p targeted to CYP19A1,LHR,GDF9,novel-344 targeted to FSHR and IGF1 R,and chi-miR-10a-5p targeted to 17β-HSD.At the same time,BMP15,CYP1A1,17β-HSD,IGF1 R and LHR were up-regulated in GF_d,and GDF9 and FSHR were down-regulated in GF_d when the same follicular samples were performed to trancriptional mRNA sequencing.BMP15 and chi-miR-130b-5p,FSHR and novel-344,GDF9 and chi-miR-10a-5p had opposite expressional patterns,which conformed to the regulatory characteristics of miRNAs.4、Differentially expressed miRNAs（chi-miR-200 a,chi-miR-34c-5p,chi-miR-10a-3p,chi-miR-10a-5p,novel-344 and chi-miR-196b）and mRNA（GDF9,BMP15 and BMP6）were selected for the expressional identification by qPCR,and the results were consistent with the expressional analysis of Illumina sequencing.In this study,a number of different expressed miRNAs were found in ovulatory and subordinate follicles,and they were predicted to involve in the ovarian hormone synthesis.The results may provide reference datas for the research of follicular development and ovulation.