MicroRNA-143 regulates adipocyte differentiation

MicroRNAs (miRNAs) are endogenously expressed 20-24 nucleotide RNAs thought to repress protein translation through binding to a target mRNA (1-3). Only a few of the more than 250 predicted human miRNAs have been assigned any biological function. In an effort to uncover miRNAs important during adipocyte differentiation, antisense oligonucleotides (ASOs) targeting 86 human miRNAs were transfected into cultured human pre-adipocytes, and their ability to modulate adipocyte differentiation was evaluated. Expression of 254 miRNAs in differentiating adipocytes was also examined on a miRNA microarray. Here we report that the combination of expression data and functional assay results identified a role for miR-143 in adipocyte differentiation. miR-143 levels increased in differentiating adipocytes, and inhibition of miR-143 effectively inhibited adipocyte differentiation. In addition, protein levels of the proposed miR-143 target ERK5 (4) were higher in ASO-treated adipocytes. These results demonstrate that miR-143 is involved in adipocyte differentiation and may act through target gene ERK5.     

The role of microRNA-26b in human adipocyte differentiation and proliferation

Recent findings indicate that microRNAs (miRNAs) are involved in the regulatory network of adipogenesis and obesity. Thus far, only a few human miRNAs are known to function as adipogenic regulators, fanning interest in studies on the functional role of miRNAs during adipogenesis in humans. In a previous study, we used a microarray to assess miRNA expression during human preadipocyte differentiation. We found that expression of the miR-26b was increased in mature adipocytes. MiR-26b is an intronic miRNA located in the intron of CTDSP1 (carboxy terminal domain, RNA polymerase II, polypeptide A, small phosphatase 1). Target prediction and Renilla luciferase analyses revealed the phosphatase and tensin homolog gene (PTEN) as a putative target gene. In this study, we found that miR-26b was gradually upregulated during adipocyte differentiation. To understand the roles of miR-26b in adipogenesis, we adopted a loss-of-function approach to silence miR-26b stably in human preadipocytes. We found that miR-26b inhibition effectively suppressed adipocyte differentiation, as evidenced by decreased lipid droplets and the ability of miR-26b to decrease mRNA levels of adipocyte-specific molecular markers and triglyceride accumulation. Furthermore, the cell growth assay revealed that miR-26b inhibition promoted proliferation. Nevertheless, it had no effect on apoptosis. Taken together, these data indicate that miR-26b may be involved in adipogenesis and could be targeted for therapeutic intervention in obesity.     

过表达miR-191抑制猪ADSCs向脂肪细胞分化

在脂肪组织发育过程中存在许多差异表达的microRNAs（miRNAs）,而 miR-191 是从实验室前期Solexa测序结果中筛选出的差异表达miRNAs之一.本研究对不同物种miR-191的成熟及前体序列进行同源性分析,并检测其在猪不同发育阶段各组织中的表达情况,发现miR-191的成熟及前体序列在不同物种间都非常保守,通过real-time qPCR检测发现,miR=191在猪脂肪组织不同发育阶段呈高丰度差异表达,其与Solexa测序结果一致.为了更好地研究miR-191在脂肪细胞分化过程中的功能,通过基因克隆的方法,利用Ad-Easy体系,构建过表达miR 191的腺病毒载体,转染 293A细胞,成功包装出重组 miR-191腺病毒并能高效侵染猪脂肪基质细胞（adipose derived stromal cells, ADSCs）.通过real time qPCR检测表明,感染重组 miR-191 腺病毒后的ADSCs能大量表达miR-191|油红O染色可以观察到,过表达miR=191的猪ADSCs在诱导分化后与对照组相比,脂滴明显减少. 进一步研究发现,miR-191过表达的猪ADSCs中,SREBP-1C（sterol regulatory element binding protein=1C）和LPL（lipoprotein lipase）的mRNA水平显著降低,脂解关键基因HSL（hormone sensitive lipase）和ATGL（adipose triglyceride lipase）mRNA水平显著升高. 同时,Western印迹结果显示,过表达miR-191的猪ADSCs在诱导分化过程中,PPARγ（peroxisome proliferator-activated receptor γ）、C/EBPα（CCAAT/enhancer binding protein α）和A-FABP（adipocyte fatty acid binding protein）的蛋白表达水平显著下调. 实验表明,过表达miR-191抑制了猪ADSCs的分化过程. 这为深入研究miR-191在猪ADSCs分化过程中的调控机制奠定基础.     

Pseudogene PTENP1 sponges miR-214 to regulate the expression of PTEN to modulate osteoclast differentiation and attenuate osteoporosis

Background aimsOsteoporosis (OP) is a common bone metabolic disease with a high incidence. Our study aimed to explore the pseudogene PTENP1/miR-214/PTEN axis to modulate the osteoclast differentiation in osteoporosis.MethodsPatients with osteoporosis were recruited in our study, and RANKL-induced osteoclast differentiation and ovariectomy-induced osteoporosis mouse model were established in vitro and in vivo, respectively.ResultsPseudogene PTENP1 and PTEN were significantly down-regulated and miR-214 was up-regulated in osteoporosis patients. In addition, overexpression of PTENP1 or silence of miR-214 inhibited the expression levels of osteoclast specific markers and osteoclast differentiation induced by RANKL. Overexpression of PTENP1 or silence of miR-214 also inhibited the levels of phosphorylation of PI3K and AKT, p65 nuclear translocation, IκBα degradation and the expression level of NFATc1. AlsoSilence of PTENP1 or overexpression of miR-214 induced the osteoclast differentiation under normal physiological condition. Pseudogene PTENP1 sponged miR-214 to regulate the expression of PTEN.ConclusionsIn an ovariectomy-induced osteoporosis mouse model, obvious pathological changes in bone tissues were found, and bone marrow mononuclear cells in this group were more likely to differentiate into osteoclasts. Therefore, pseudogene PTENP1 sponged miR-214 to regulate the expression of PTEN to inhibit osteoclast differentiation and attenuate osteoporosis by suppressing the PI3K/AKT/NF-κB signaling pathway.     

MicroRNAome Comparison between Intramuscular and Subcutaneous Vascular Stem Cell Adipogenesis

Background

As an important factor affecting meat quality, intramuscular fat (IMF) content is a topic of worldwide concern. Emerging evidences indicate that microRNAs play important roles in adipocyte differentiation. However, miRNAome has neither been studied during porcine intramuscular preadipocyte differentiation, nor compared with subcutaneous preadipocytes. The objectives of this study were to identify porcine miRNAs involved in adipogenesis in primary preadipocytes, and to determine whether intramuscular and subcutaneous adipocytes differ in the expression and regulation of miRNAs.

Results

miRNAomes in primary intramuscular and subcutaneous adipocytes during differentiation were first sequenced using the Solexa deep sequencing method. The sequences and relative expression levels of 224 known (98.2% in miRbase 18.0) and 280 potential porcine miRNAs were identified. Fifty-four of them changed in similar pattern between intramuscular vascular stem cells (IVSC) and subcutaneous vascular stem cells (SVSC) differentiation, such as miR-210, miR-10b and miR-99a. Expression levels of 10 miRNAs were reversely up-or down-regulated between IVSC and SVSC differentiation, 19 were up-or down-regulated only during IVSC differentiation and 55 only during SVSC differentiation. Additionally, 30 miRNAs showed fat-depot specific expression pattern (24 in cells of intramuscular origin and 6 in cells of subcutaneous origin). These adipogenesis-related miRNAs mainly functioned by targeting similar pathways in adipogenesis, obesity and syndrome.

Conclusion

Comparison of miRNAomes in IVSC and SVSC during differentiation revealed that many different miRNAs are involved in adipogenesis, and they regulate SVSC and IVSC differentiation through similar pathways. These miRNAs may serve as biomarkers or targets for enhancing IMF content, and uncovering their function in IMF development will be of great value in the near future.     

Biological role of MicroRNA-103 based on expression profile and target genes analysis in pigs

MicroRNAs (miRNAs) are endogenously expressed RNAs consisting of 20–24 nucleotides. These molecules are thought to repress protein translation by binding to target mRNAs. However, biological functions have not been assigned to most of the 175 porcine miRNAs registered in miRBase (release 15.0). In an effort to uncover miR-103 important in pigs, we examined the integrative tissue expression profile and gene ontology (GO) term enrichment of predicted target genes to determine the global biological functions of miR-103. Our results demonstrated that miR-103 is involved in various biological processes including brain development, lipid metabolism, adipocyte differentiation, hematopoiesis, and immunity. Moreover, we also experimentally verified effects of miR-103 in porcine preadipocytes. miR-103 levels increased in differentiating adipocytes, and inhibition of miR-103 effectively inhibited preadipocyte differentiation. In addition, mRNA levels of the putative miR-103 target RAI14 were higher in miR-103 inhibitor-treated adipocytes. These results demonstrate that miR-103 is involved in porcine preadipocyte differentiation and may act through the putative target gene RAI14. In a word, our data provide new insights into the global biological role of miR-103.     

miR-27a is a negative regulator of adipocyte differentiation via suppressing PPARγ expression

microRNAs (miRNAs) are non-coding small RNAs regulating gene expression, cell growth, and differentiation. Although several miRNAs have been implicated in cell growth and differentiation, it is barely understood their roles in adipocyte differentiation. In the present study, we reveal that miR-27a is involved in adipocyte differentiation by binding to the PPARγ 3′-UTR whose sequence motifs are highly conserved in mammals. During adipogenesis, the expression level of miR-27a was inversely correlated with that of adipogenic marker genes such as PPARγ and adiponectin. In white adipose tissue, miR-27a was more abundantly expressed in stromal vascular cell fraction than in mature adipocyte fraction. Ectopic expression of miR-27a in 3T3-L1 pre-adipocytes repressed adipocyte differentiation by reducing PPARγ expression. Interestingly, the level of miR-27a in mature adipocyte fraction of obese mice was down-regulated than that of lean mice. Together, these results suggest that miR-27a would suppress adipocyte differentiation through targeting PPARγ and thereby down-regulation of miR-27a might be associated with adipose tissue dysregulation in obesity.     

MicroRNAs Regulate Human Adipocyte Lipolysis: Effects of miR-145 Are Linked to TNF-α

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and have multiple effects in various tissues including adipose inflammation, a condition characterized by increased local release of the pro-lipolytic cytokine tumor necrosis factor-alpha (TNF-α). Whether miRNAs regulate adipocyte lipolysis is unknown. We set out to determine whether miRNAs affect adipocyte lipolysis in human fat cells. To this end, eleven miRNAs known to be present in human adipose tissue were over-expressed in human in vitro differentiated adipocytes followed by assessments of TNF-α and glycerol levels in conditioned media after 48 h. Three miRNAs (miR-145, -26a and let-7d) modulated both parameters in parallel. However, while miR-26a and let-7d decreased, miR-145 increased both glycerol release and TNF-α secretion. Further studies were focused therefore on miR-145 since this was the only stimulator of lipolysis and TNF-α secretion. Time-course analysis demonstrated that miR-145 over-expression up-regulated TNF-α expression/secretion followed by increased glycerol release. Increase in TNF-α production by miR-145 was mediated via activation of p65, a member of the NF-κB complex. In addition, miR-145 down-regulated the expression of the protease ADAM17, resulting in an increased fraction of membrane bound TNF-α, which is the more biologically active form of TNF-α. MiR-145 overexpression also increased the phosphorylation of activating serine residues in hormone sensitive lipase and decreased the mRNA expression of phosphodiesterase 3B, effects which are also observed upon TNF-α treatment in human adipocytes. We conclude that miR-145 regulates adipocyte lipolysis via multiple mechanisms involving increased production and processing of TNF-α in fat cells.     

miR-324-5p promotes adipocyte differentiation and lipid droplet accumulation by targeting Krueppel-like factor 3 (KLF3)

miRNAs, a kind of noncoding small RNA, play a significant role in adipose differentiation. In this study, we explored the effect of miR-324-5p in adipose differentiation, and found that miR-324-5p could promote adipocytes differentiation and increase body weight in mice. We overexpressed miR-324-5p during adipocytes differentiation, by oil red O and bodipy staining found that lipid accumulation was increased, and the expression level of adipogenic related genes were significantly increased. And the opposite experimental results were obtained after inhibiting miR-324-5p. In vivo, we injected miR-324-5p agomiR in obese mice and found that body weight, adipocyte area, and adipogenic-related gene expression level were significantly increased but lipolytic genes were decreased. To further explore the mechanism of miR-324-5p regulation in lipid accumulation, we constructed Krueppel-like factor 3 (KLF3) 3′-untranslated region luciferase reporter vector and KLF3 pcDNA 3.1 overexpression vector, and found that miR-324-5p was able to directly target KLF3. Overall, in this study we found that miR-324-5p could promote mice preadipoytes differentiation and increase mice fat accumulation by targeting KLF3.     

Pygopus2 ameliorates mesenteric adipocyte poor differentiation to alleviate Crohn's disease ‐like colitis via the Axin2/GSK3β pathway

ObjectivesCrohn''s disease (CD) mesenteric adipose tissue (MAT) inflammation affects enteritis through the interaction between the mesentery and intestine, and we previously found that poorly differentiated mesenteric adipocytes were related to its inflammatory features. Pygopus2 (Pygo2) is a key negative regulator of adipocyte differentiation. We aimed to determine whether Pygo2 participates in CD mesenteric lesions and whether Pygo2 knockdown would be beneficial in a CD model (Il‐10 ^−/− mice).MethodsPygo2 expression in MAT from control and CD patients and Il‐10 ^−/− mice was measured by immunohistochemistry. Lentiviral transfection was used to regulate Pygo2 expression in Il‐10 ^−/− mice, and the effects on mesenteric adipocyte differentiation, inflammation, and dysfunction during spontaneous colitis, as well as the possible mechanism, were investigated.ResultsPygo2 expression was increased in MAT from CD patients and Il‐10 ^−/− mice, and its expression correlated with poor adipocyte differentiation and inflammation. Pygo2 knockdown significantly ameliorated colitis in Il‐10 ^−/− mice. Moreover, the downregulation of Pygo2 gene expression could promote adipocyte differentiation and inhibit adipocyte inflammation in vivo and in vitro, and the effects were at least partly mediated by the Axis inhibition protein 2 (Axin2)/glycogen synthase kinase 3 beta (GSK3β) pathway.ConclusionsThe increase in Pygo2 may be related to mesenteric adipocyte poor differentiation and inflammatory features of CD, and Pygo2 inhibition could alleviate CD‐like colitis by improving mesenteric lesions by regulating the Axin2/GSK3β pathway.

The increase in Pygopus2 (Pygo2) may be related to mesenteric adipocyte poor differentiation and inflammatory features of CD, and Pygo2 inhibition could alleviate adipocyte differentiation and mesenteric lesions by regulating the Axin2/GSK3β pathway.     

Adipose Rheb deficiency promotes miR-182-5p expression via the cAMP/PPARγ signaling pathway

Dysregulation of microRNAs (miRNAs) in adipocytes plays a critical role in the pathogenesis of obesity. However, the signaling mechanisms regulating miRNAs production in adipose tissue remain largely unclear. Here, we show that adipose tissue-specific knockout of Ras homolog enriched in brain (Rheb), a direct upstream activator of mTOR, increases miR-182-5p level in mouse subcutaneous white adipose tissues. Interestingly, the inhibition of mTOR signaling by rapamycin has no effect on miR-182-5p level in primary subcutaneous white adipocytes, suggesting the presence of a mTOR-independent mechanism regulating Rheb-mediated miR-182-5p expression. Consistent with this view, Rheb-ablation activates the cAMP/PPARγ signaling pathway. In addition, treatment of white adipocytes with pioglitazone, a PPARγ agonist, dramatically upregulates miR-182-5p levels. Our study reveals a unique mechanism by which Rheb regulates miR-182-5p in adipocytes. Given that increasing miR-182-5p in adipose tissue promotes beige fat development, our study also suggests a unique mechanism by which Rheb promotes thermogenesis and energy expenditure.