Identification of miRNA in Sheep Intramuscular Fat and the Role of miR-193a-5p in Proliferation and Differentiation of 3T3-L1

Abstract

Intramuscular fat (IMF) is one of the most critical parameters affecting meat quality and mainly affected by genetic factors. MicroRNA as an important regulatory factor, which is still a lack of research in the development of sheep IMF deposition. We used RNA sequencing (RNA-seq) and cell-level validation to explore the role of miRNA in IMF deposition. As for this purpose, longissimus thoracis et lumborum (LTL) samples of 2 month-old (Mth-2) and 12 months-old (Mth-12) Aohan fine-wool sheep (AFWS) were used to identified miRNAs expression. We found 59 differentially expressed miRNAs (DE-miRNA) between these age groups and predicted their 1,796 target genes. KEGG functional enrichment analysis revealed eight pathways involved in lipid metabolism-related processes, including fatty acid elongation and the AMPK signaling pathway. A highly expressed DE-miRNA, miR-193a-5p, was found to serve a function in 3T3-L1 preadipocyte differentiation. Luciferase assay demonstrated that miR-193a-5p directly binds to the 3′-UTR region of ACAA2. By constructing mimics and inhibitor vector transfecting into 3T3-L1 cells to explore the effect of miR-193a-5p on cell proliferation and differentiation, we demonstrated that overexpression of miR-193a-5p inhibited 3T3-L1 preadipocyte proliferation, as evidenced by decreased mRNA and protein expression of CDK4 and CyclinB. CCK-8 assay showed that miR-193a-5p significantly inhibited cell proliferation. Similarly, the overexpression of miR-193a-5p inhibited 3T3-L1 preadipocyte differentiation and adipocyte-specific molecular markers’ expression, leading to a decrease in PPARγ and C/EBPα and ACAA2. Inhibition of miR-193a-5p had the opposite effects. Our study lists the miRNAs associated with intramuscular lipid deposition in sheep and their potential targets, striving to improve sheep meat quality.

Comparative Transcriptome Analysis of Slow-Twitch and Fast-Twitch Muscles in Dezhou Donkeys

“…In this interaction network, some genes related to myofiber type pathways associated with the troponin complex (such as TNNI2 , TNNC2 and TNNT3 ), actin binding ( ACTN3 ) and glycolysis/gluconeogenesis (such as GRK6 , PKM , EPHA , ALDOA and PGM1 ), which are negatively regulated by eca-miR-193a-5p or/and eca-miR-1379, showed higher expression in BF (higher fast muscle fiber population). According to previous reports, miRNA-193a-5p mostly functions in the context of cell proliferation and differentiation, including tumor or cancer development and 3T3-L1 preadipocyte proliferation and differentiation [ 44 , 45 , 46 ]. For instance, the overexpression of miR-193a-5p can inhibit 3T3-L1 preadipocyte proliferation and differentiation by targeting ACAA2 [ 46 ].…”

“…According to previous reports, miRNA-193a-5p mostly functions in the context of cell proliferation and differentiation, including tumor or cancer development and 3T3-L1 preadipocyte proliferation and differentiation [ 44 , 45 , 46 ]. For instance, the overexpression of miR-193a-5p can inhibit 3T3-L1 preadipocyte proliferation and differentiation by targeting ACAA2 [ 46 ]. A study by Ju et al showed that miR-193-3p may contribute to the regulation of oxidative myofibers in chickens by targeting the PPARGC1A gene [ 47 ].…”

LncRNA RPL34-AS1 suppresses the proliferation, migration and invasion of esophageal squamous cell carcinoma via targeting miR-575/ACAA2 axis

“…In the present study, our results indicated that RPL34-AS1 interacted with miR-575 to promote the expression of ACAA2, and inferred a novel mechanistic role of RPL34-AS1/miR-575/ACAA2 axis in regulating the progression of ESCC. The upregulation of miR-193a-5p inhibited 3 T3-L1 preadipocyte differentiation, leading to a decrease in fatty acid related gene ACAA2 [ 44 ]. Furthermore, nonalcoholic steatohepatitis (NASH) related differential lncRNAs were associated with predicted protein-coding targets of ACAA2 [ 45 ].…”

Genome-Wide Analysis of microRNAs Identifies the Lipid Metabolism Pathway to Be a Defining Factor in Adipose Tissue From Different Sheep

“…The reads aligned to the reference genome were aligned with the miRNA precursor and mature sequences in the miRBase V.22.1 ( 33 ) to obtain known miRNAs. The Rfam ( 34 ) was used to filter ncRNAs and repeats sequences such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA), and at that time, the types and numbers of these sequences were counted. The sRNAs that cannot be aligned with Rfam and miRBase were aligned to the reference genome, and the surrounding sequences were intercepted using miRDeep2 ( 35 ) software for secondary structure prediction to identify new miRNAs.…”