Effect Of MiR-183 And MiR-135a In Hircine Preadipocyte Differentiation And Adipogenesis

MicroRNAs (miRNAs) are a family of short,non-coding,about 22 nt RNAs that actively participate in many biological processes including embryo development, cell proliferation and differentiation, lipid metabolism and tumorigenesis,through post-transcriptional regulation of target genes. Many groups have reported that miRNAs play important roles in adipocytes proliferation and differentiation, lipid metabolism. This study is aimed to establish the correlation between miRNAs and intramuscular fat content and lay a foundation for regulating the fat acid synthesis by miRNAin goat bydetecting of thefunctions of miR-183 and miR-135a in the adipogenesis of hircine preadipocytes.Here, we cloned the sequences of chi-miR-183 and chi-miR-135a. qPCR was used to quantify miR-183 and miR-135a expression patterns in ten tissues of adult Tibetan goat including heart, liver, spleen, lung, kidney, brain, longissimus dorsi muscle, subcutaneous adipose,perirenal adipose and mesenteric adipose.Then, we obtained hircine preadipocytes from the subcutaneous adipose of neck and back of three-day-old Nanjiang Yellow goat with collagenase digestion. The mature adipocytes were induced by adipogenic differentiationfrom preadipocytes and cells were stained with Oil Red O to examine the formation of lipids. qPCR was employed to determine the relative expression of miR-183, miR-135a and PPARy during the differentiation of preadipocytes.To explore therole of miR-183 and miR-135a in hircine preadipocytes differentiation,miRNA mimics and inhibitors were used to perform miR-183 and miR-135a overexpression or knockdown, respectively.Then, qPCR was used to quantify the relative expression of ACC, FAS, C/EBPa, PPARy, SREBP-1. Results were shown as follows:1.We obtained the sequences of chi-miR-183 and chi-miR-135a, the sequence of chi-miR-183 was 5’-UAUGGCACUGGUAGAAUUCACUG-3’and the sequence of chi-miR-135a was 5’-UAUGGCUUUUUAUUCCUAUGUGA-3’.2. MiR-183 and miR-135a expressed in all tissues and the relative expression of miR-183 and miR-135a had significant differencesamong organizations. MiR-183 and miR-135a had different expression patterns among tissues, while miR-183 and miR-135a had a similar expression profile among subcutaneous adipose, perirenal adipose and mesenteric adipose.3. After 5-day culturing, cell growth arrested resulted fromcontact inhibition. Small lipid droplets began to appear in a few cells, meanwhile the cell came into differentiation phase. On the 8th day of adipogenesis, the intracellular lipid droplets fused to large fat droplets and cells exhibited the characteristics of mature adipocytes. The mRNA expression of PPARywas up-regulated during the differentiation of hircine preadipocytes and the highest expression level was observed on the 6th day of differentiation.4. MiR-183 and miR-135a had different expression profiles during preadipocytes differentiation. The expression of miR-183 increased whilethat of miR-135a decreased.5. Overexpression ofmiR-183 in hircine preadipocytes significantlyincreased the expression of adipocytespecific transcription factors including PPARy, C/EBPa, SREBP-1, FAS and ACCalong with promoted lipid deposition in hircine adipocytes.Suppress expression of miR-183 coulddramatically reduce the mRNA expression of adipogenicmarkers PPARγ, C/EBPα, SREBP-1, FAS and ACC,and attenuate the lipid deposition in hircine adipocytes.These results suggested that miR-183promotes the differentiation of hircine preadipocytes.6. Overexpression of miR135adownregulated theexpression of PPARγ, C/EBPα, SREBP-1, FAS and ACC,and impaired lipid accumulation in the differentiation of hircine preadipocytes. In addition, the inhibited miR-135a level in hircine adipocytes upregulated theexpression of PPARγ, C/EBPα, SREBP-1, FAS andACC with the promotion of lipid accumulation.Inconclusion, we demonstrated that miR-135a inhibits hircine preadipocytes differentiation.