| Beef cattle industry is an important component of modern husbandry production.The proportion of high-grade beef production has been an important indicator of beef cattle industry development level for one country.In our country,cattle breed resources are abundant,but due to lack of effective systematic meat quality breeding for a long time,so the beef quality is uneven,and high quality beef production is low,mainly for few of beef intramuscular fat and maldistribution of fatty deposition.Therefore it is necessary to conduct the thorough research to the rule of cattle fatty deposition.miRNAs is a kind of important small non-coding RNA molecules,mainly to regulate the transcription by matching the target gene mRNA,widely participating in mammals growth and development,cell differentiation and apoptosis,etc.miR-143 and miR-378 have significant control effect on the fat deposition based on the foregone scientific researches.This study mainly included the following several parts:(1)Identifying of suitable endogenous control genes for miRNA quantitative real time PCR assays in cattle solid tissues;(2)Constructing beef cattle preadipocytes cell lines successfully;(3)Screening monoclonal cell strains by transfected lentiviral vector for up/down-regulated miR-143 and miR-378;(4)Bioinformatic predicting and verifying the effect of both targeting specific genes;(5)Detecting the action on preadipocytes differentiation,proliferation and migration ability,etc..Currently,the main conclusions were as follows:(1)U6,miR-191 and let-7f were statistically superior to the other endogenous controls in cattle solid tissues;The combined utilization of miR-191 and U6 would be more suitable to as normalizers of miRNA expression levels than individual U6 snRNA or certain a miRNA for miRNA qPCR assays.(2)miR-143 and miR-378 express were extensive and different obviously in cattle tissues or organs by fluorescence quantitative RT-PCR detection;the adipogenesis of preadipocytes cultured in vitro rooted in intramuscular fat isolated from longissimus dorsi of beef cattle,was confirmed by oil red O staining;miR-143 and miR-378 expression levels increased with adipocyte differentiation.(3)The monoclonal cell strains presented higher expression of adipogenesis landmark genes such as PPARγ,C/EBPβ,ADI,SREBP1 c and KLF5,and lower Pref-1 expression for miR-143 and miR-378 stable overexpression by lentiviral vectors transfection,and otherwise cell doubling time and intracellular fat content had varying degrees of increment compared with empty vector transfection or no treatment control group.And that would be inversed when miR-143 and miR-378 stable expression inhibition by lentiviral vectors transfection.Thus these proved that miR-143 and miR-378 could promote preadipocytes differentiation.(4)Following bioinformatic prediction the miR-143 and miR-378 target genes by TargetScan and PicTar methods,GO and KEGG functional analysis showed that miR-143 participates in the regulation of cell growth and proliferation,involving the MAPK and TGFβ signaling;miR-378 works in biological processes of apoptosis,death,and transcription regulation,involving fat cytokine signaling pathways;Through base pairs analysis of miRNAs and candidate target genes,it was said that TGFβ2 and MAP2K5 would be candidate target genes of miR-143 and CaMKK2 would be one of candidate target genes of miR-378.(5)Dual luciferase report system and Western Blot analyses confirmed that the TGFβ2 and MAP2K5 were target genes of miR-143,and CaMKK2 was one target gene of miR-378.The former suggested that miR-143 contributes to preadipocytes differentiation via TGFβ and MAPK signaling pathways possibly and the later suggested that miR-378 played a role in preadipocytes differentiation via AMPK signaling pathway possibly.It was verified that miR-143 and miR-378 may promote preadipocytes differentiation by a series of trials such as constructing preadipocytes cell lines,inducing adipogenesis,and so on,based on the existing research.New traget genes and possible signaling passway involved of miR-143 and miR-378 were proved to a certain degree.The further clarification on functional mechanisms of miR-143 and miR-378 during intramuscular fat deposition would be helpful for new varieties breeding of beef cattle. |
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