Research On Mirna Expression Profiles In Lactation And Non-Lactation And The Target Genes Of Mir-103 And Novel-mir-57 In Buffalo

Water buffalo are important milk-producing animals in the tropical and subtropical regions and they contribute to more than 5%of the world's milk supply.The fat and protein content of Buffalo milk were 2.22 times and 1.72 times of Holstein cow milk.For this reason,it is important to understand the gene regulatory networks involved in the lactation physiology of the water buffalo to recover the molecular mechanism of its high nutrient value.MicroRNAs(miRs)are a group of small(approximately 19-25 nucleotides(nt)),non-coding and endogenous RNAs that regulate expression of protein by binding to target mRNAs so as to repress translation or to promote degradation of the target mRNA.It is estimated that the expression of 30%of the protein-coding genes is regulated by miRs.It has currently been established that miRs play important roles in cell differentiation,proliferation,and apotheosis in animals.The mammary gland of adult animals could undergo a cycle of cell proliferation,differentiation,de-differentiation,and death,making it an ideal model organ for study of the molecular mechanisms underlying mammary gland physiology and lactation.Work on the miRNome in human and mouse tissues has shown that,in humans,the expression of 23 miRs is specific to the breast tissue,but,in mice,only 9 miRs are specific to mammary tissues.This suggests that miRs play an important role in mammary gland physiology.Several studies have focused on the role of miRs in lactation physiology of mammary glands of other milk-producing livestock.The miR profile in the lactating and non-lactating mammary gland tissue of the water buffalo,however,has yet to be published.The miRs profiles in lactating and non-lactating mammary gland tissues of the water buffalo were constructed.The different expression patterns of 18 miRs were verified and analyzed.Bbu-miR-103 and novel-miR-57 were selected as the significant different expression miRs,and the target genes of which were further studied.The research was to establish the molecule base for illustrating the molecular mechanism of lactation matter and energy metabolic pathway in B.bubalis.(Bubalus bubalis).1.Construction and analysis of different miRs expression profiles in the lactation and non-lactation of buffalo mammary glandThe samples of buffalo mammary gland in lactation(milking peak)and non-lactation(dry period)were collected.Two miRs expression profiles in lactation and non-lactation of buffalo were constructed by Solexa high-through sequencing technology.12,569,467 and 12,768,110 high-quality reads between 18nt and 31nt were obtained in non-lactation and lactation separately.The statistic analysis results showed that the width patterns of sRNA in lactation and non-lactation of China Swamp buffalo were between 18 and 31n,which reached a peak in 22nt.sRNA of 22nt occupied 33.4%of total sRNA in lactation of mammary gland.There were 676 mature miRs and 662 pre-microRNA of Bos taurus in miRbasel7.0,which belong to 500 miRs families.While 359 mature miRs and 363 pre-microRNA were verified in buffalo mammary gland by Solexa sequencing,which belong to 259 miRs families.230 novel miRs from 262 candidate miR with novel hairpin were discovered in the buffalo mammary gland.Five novel miRs were considered as buffalo special miRs.Analyses of the first nucleotide bias of the 17-29 nt miRs candidates showed uridine(U)to be the most common(94.15 and 97.90%)at the 5' end of the 19 nt and 25 nt novel miRs.The chromosome locations for all miRs were investigated.Approximately 68.77%(240/349)of the known pre-miRs that were successfully located in chromosomes in the mammary gland tissues were also found in the intergenic regions.Known miRs located in the auto-chromosome and X chromosome(11.65%and 5.83%),which were 74 miRs and 37 miRs(total 635).The numbers of novel miRs in 21 and X autosome were 38 and 32 separately.Known miRs located in the auto-chromosome and X chromosome,the density of miRs in one autosome ranged from 0.09 to 1.05 miRs per 1 Mbp.21 chromosome was the maximum distribution chromosome,in which the density of total miRs,miRs in lactation and miRs in non-lactation were 1.05/Mbp,1.34 and 1.35 separately.The density of miRs in the same chromosome between lactation and non-lactation were nearly the same.2.Differential expression of miRs in lactating and non-lactating mammary tissueIn non-lactating tissues,bbu-miR-148a,bbu-let-7b,bbu-let-7a,bbu-miR-21,bbu-miR-143,bbu-miR-200c,bbu-miR-26a,bbu-miR-200a,and bbu-let-7f were the dominant expressed miRs,with more than 20,000 reads.They constituted 53.8%of the total known miR sequencing reads,suggesting that they have abundant expression in non-lactating mammary tissue.However,in lactating tissue,7 miRs(bbu-let-7b,bbu-let-7a,bbu-miR-26a,bbu-miR-125b,bbu-miR-21,bbu-miR-29a,and bbu-let-7c),each had more than 20,000 reads,were the most abundant.Comparing the two libraries showed the expression of these miRs such as bbu-miR-148a,bbu-miR-143,bbu-miR-200a,bbu-miR-141,bbu-miR-30a-5p decrease to less than half their non-lactating expression levels.Some miRs,such as bbu-miR-26a,bbu-miR-29a,bbu-miR-125b,bbu-let-7c,and bbu-miR-99a,displayed no less than twice the sequencing frequencies of the non-lactating mammary library.The predicted target genes of 20 significantly different expression miRs were classified according to KEGG functional annotations.Interestingly,most of the 109 miR targets were part of the MAPK signaling pathway.The other important pathways were Jak-STAT,PRL signal transduction and insulin pathways.3.Validation of 18 significantly different expression miRs in lactation and non-lactation with QRT-PCRComparative analysis of the different miR expression profiles in the two tissues,18 miRs were selected and the expression levels of them were determined by Q-PCR assay in both tissues.They were divided into high expression,low expression and novel miRs:(a)9 conserved miRs with high expression,bbu-miR-30a-5p,bbu-miR-141,bbu-miR-103,bbu-miR-101,bbu-miR-30a-5p,bbu-miR-148a,bbu-miR-29a,bbu-miR-125b,bbu-miR-497,and bbu-miR-125a;(b)4 miRs with low-level expression in lactating and non-lactating:bbu-miR-490,bbu-miR-217,bbu-miR-592,and bbu-miR-2370*;(c)5 novel miRs with high levels of expression:novel-miR-57,novel-39,novel-148,novel-76,and novel-123b.The expression of bbu-miR-103,bbu-miR-125a,bbu-miR-30a-5p,and bbu-miR-148a were 5.29,4.06,3.43,and 2.43 fold higher(P<0.05)in lactating than non-lactating tissues.Interestingly,the expression of bbu-miR-29a was 0.3 fold and significantly(P<0.05)lower in lactating than in non-lactating tissue.However,there no significant difference(P>0.05)in levels of expression of bbu-miR-141,bbu-miR-125b,bbu-miR-497,and bbu-miR-101 was recorded between these tissues.Among miRs with the low-level expression,miR-490 and miR-592 was 4.96 and 3.82 fold higher in lactating than non-lactating tissue(P<0.05).Among the 5 novel miRs,there were no significant differences(P>0.05)between the novel-39,novel-148,novel-76,and novel-123b in lactating and non-lactating tissues.Interestingly,the expression of novel-miR-57 was 29.79 fold higher(P<0.01)in lactating tissue than in non-lactating tissue.Based on these results,miR-103 and novel-miR-57 were selected for further analysis in lactating and non-lactating tissues.4.Analysis of the functional role of Buu-miR-103 by targeting to PANK3 in milk fat synthesisBased on the vector backbone of Lenti-Pac HIV Expression Packaging system,the vector containing the precursor of bta-mir-103-1 cloned was constructed and named LpEZX-pre-MR103(HIV),the plasmid was 8219 bp in size.It was packaged in 293T cell lines with the packaging plasmid(?NRF)and the envelop plasmid encoding the vesicular stomatitis virus-G glycoprotein(VSV-G);the infection titer was determined as 3.42 × 106 PFU/ml and 3.47 × 106 PFU/ml of NC and miR-103 on 293T cell lines.Then the buffalo mammary epithelial cells(BMECs)were cultured by tissue blocks,which were infected using Lentiviral vector with miR-103.They all displayed a green light from green fluorescence protein under microscope after 48h,which showed that miR-103 and NC viral particles had successfully entered the buffalo mammary epithelial cells and produced excessive miR-103.The inhibitor of miR-103 was synthesized and transfected at the same time.The over expression and suppression of miR-103 was negatively correlated with Pantothenate kinase 3(PANK3).Hence,the target of miR-103 was speculated as PANK3.Over expression of miR-103 down-regulated the expression of PANK3,and significantly enhanced the expression of acetyl CoA carboxylase alpha(ACACA)and steroid regulatory element binding protein(SREBP1c).The effects of over expression of miR-103 on the 8 key fat synthesis metabolism pathways were determined.The results indicated that bbu-miR-103 enhances fatty acid synthesis through de novo synthesis and also plays a role in triglyceride synthesis,fat lipid synthesis and secretion,and fatty acid absorption but does not have a significant effect on fatty acid transfer metabolism.5.Hunt for the target gene of Novel-miR-57 oriented to DOK4 in Bcap-37 cells and Buffalo mammary gland epithelial cell(BMECs)Novel-miR-57 was the most significantly differentially expressed miR of the novel miRs in lactating and non-lactating tissues,but no homology miR was foun in miRBase.The secondary structure of novel-miR-57 was predicted by MiRscan,and results showed there to be 7 stem-loops with a binding free energy of-28.0 kcal/mol.The mature sequence of novel-miR-57 was located on the first stem-loop.The 3' UTRs of annotated buffalo mRNA were intercepted by the self-programming software-Ensembl(v80)as database for predicting the target gene of bbu-miR-57.The mRNA was considered as target gene if its binding free energy to 3' UTR of 2-8 seed sequence of novel-miR-57 were below-20 kcal/mol.Finally,34 buffalo mRNAs were identified as the possible target of novel-miR-57 such as CYP7B1,CACNG3,DOK4,COL17A1,ESN1,and others.The GO assignment and KEGG pathway analysis of the 34 target mRNAs were predicted.The nematode miR-39 served as the external control,the significantly different target genes of novel-miR-57 were searched for by consulting literature,designing specific primers,and QRT-PCR analysis in lactating and non-lactating mammary gland tissues.Six genes were not detected by QRT-PCR.The other genes were divided into two groups if the change fold of gene relative expression was two or three digital number.It turned out that the change fold of gene relative expression of 10 genes,including RCL1,UBE3C,and NFRKB,in non-lactating tissues were 0.98 to 10.59 to that in lactating tissue,while the expression of 11 genes,including BRMS1L,ACTL,ADORO,RCL1,UBE3C,and NFRKB in lactating tissue was higher than or equal to that in non-lactating tissues.The most significant differentially expressed genes were DLX3(128.03 fold),CANCNG3(144.23 fold),DOK4(146.24 fold),NFKBID(160.96 fold),C17orf53(160.38 fold),RTN1(274.4 fold),and FBXO10(326.24 fold).The relative expression of these genes was far higher in non-lactating tissues than in lactating tissue(P<0.01),which would the possible target genes of novel-57.The mimics and inhibitor of novel-miR-57 were chemically synthesized and transfected into Bcap-37 cell lines,which indicated that only DOK4 of the seven genes was related to novel-miR-57.The expression of DOK4 were extremely significantly increased or decreased by novel-miR-57 inhibitor and mimics(P<0.01).When BMECs were transfected by the same 100 nM mimics and 200 nM of inhibitor of novel-miR-57,beyond all expectations,the gene expression of DOK4 was promoted and reduced by the novel mimics and inhibitor,respectively,and the changes were significant(P<0.01).This was the opposite of what was observed in the Bcap-37 cell line.The conclusion was that DOK4 was the target gene of Bcap-37 and BMECs,and the novel-miR-57 could selectively up-regulate or down-regulate the expression of DOK4 according to different physiological conditions and ultimately function in the lactation metabolism pathway.Conclusion:The target gene of significantly differentially expressed Bbu-miR-103 was PANK3 in buffalo mammary gland epithelial cells(BMECs).Bbu-miR-103 enhances fatty acid synthesis through de novo synthesis and also plays a role in triglyceride synthesis,fat lipid synthesis and secretion,and fatty acid absorption.The target gene of novel-miR-57 were hunted as DOK4 in both BMECs and Bcap-37 cell line,which appeared to correlate with epithelial cell differentiation.