Deciphering the miRNA transcriptome of Rongchang pig longissimus dorsi at weaning and slaughter time points.

MicroRNA (miRNA) is essential for the process of gene posttranscriptional regulation in skeletal muscle of many species, such as mice, cattle and so on. However, a little number of miRNAs have been reported in the muscle development of Chinese native pig breeds. In this study, the longissimus dorsi transcripts of Chinese native Rongchang pig at weaning and slaughter time points were analysed for miRNA-seq. The results showed that 19 novel and 186 known miRNAs involved in the Rongchang pig skeletal muscle development were identified. Based on these findings, we further confirmed that porcine miR-127, miR-299 and miR-432-5p were obviously down-expressed in adult pig (287 days of age), while miR-7134-3p and 664-5p were significantly up-expressed in weaning pig (35 days of age). In other words, these miRNAs could be the potential molecular markers and play vital roles in the muscle development process. Moreover, we found miR-127 could inhibit the proliferation and myogenesis of porcine satellite cells in longissimus dorsi muscle. Our findings will provide deep insight into miRNA function for pork quality research with Chinese indigenous pig breeds.

MicroRNA-664-5p promotes myoblast proliferation and inhibits myoblast differentiation by targeting serum response factor and Wnt1.

MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression at the post-transcriptional level and are involved in the regulation of the formation, maintenance, and function of skeletal muscle. Using miRNA sequencing and bioinformatics analysis, we previously found that the miRNA miR-664-5p is significantly differentially expressed in longissimus dorsi muscles of Rongchang pigs. However, the molecular mechanism by which miR-664-5p regulates myogenesis remains unclear. In this study, using flow cytometry, 5-ethynyl-2'-deoxyuridine staining, and cell count and immunofluorescent assays, we found that cell-transfected miR-664-5p mimics greatly promoted proliferation of C2C12 mouse myoblasts by increasing the proportion of cells in the S- and G2-phases and up-regulating the expression of cell cycle genes. Moreover, miR-664-5p inhibited myoblast differentiation by down-regulating myogenic gene expression. In contrast, miR-664-5p inhibitor repressed myoblast proliferation and promoted myoblast differentiation. Mechanistically, using dual-luciferase reporter gene experiments, we demonstrated that miR-664-5p directly targets the 3'-UTR of serum response factor (SRF) and Wnt1 mRNAs. We also observed that miR-664-5p inhibits both mRNA and protein levels of SRF and Wnt1 during myoblast proliferation and myogenic differentiation, respectively. Furthermore, the activating effect of miR-664-5p on myoblast proliferation was attenuated by SRF overexpression, and miR-664-5p repressed myogenic differentiation by diminishing the accumulation of nuclear ß-catenin. Of note, miR-664-5p's inhibitory effect on myogenic differentiation was abrogated by treatment with Wnt1 protein, the key activator of the Wnt/ß-catenin signaling pathway. Collectively, our findings suggest that miR-664-5p controls SRF and canonical Wnt/ß-catenin signaling pathways in myogenesis.

MicroRNA-323-3p promotes myogenesis by targeting Smad2.

Skeletal muscle is an important and complex organ with multiple biological functions in humans and animals. Proliferation and differentiation of myoblasts are the key steps during the development of skeletal muscle. MicroRNA (miRNA) is a class of 21-nucleotide noncoding RNAs regulating gene expression by combining with the 3'-untranslated region of target messenger RNA. Many studies in recent years have suggested that miRNAs play a critical role in myogenesis. Through high-throughput sequencing, we found that miR-323-3p showed significant changes in the longissimus dorsi muscle of Rongchang pigs in different age groups. In this study, we discovered that overexpression of miR-323-3p repressed myoblast proliferation and promoted differentiation, whereas the inhibitor of miR-323-3p displayed the opposite results. Furthermore, we predicted Smad2 as the target gene of miR-323-3p and found that miR-323-3p directly modulated the expression level of Smad2. Then luciferase reporter assays verified that Smad2 was a target gene of miR-323-3p during the differentiation of myoblasts. These findings reveal that miR-323-3p is a positive regulator of myogenesis by targeting Smad2. This provides a novel mechanism of miRNAs in myogenesis.

Lentivirus-mediated CTRP6 silencing ameliorates diet-induced obesity in mice.

The C1q/TNF-related protein 6 (CTRP6) is an adipokine involved in diverse biological processes. Formerly, we identified that CTRP6 regulates adipocyte differentiation, fatty acid oxidation and triglyceride accumulation in vitro. However, the effects of CTRP6 on adiposity in vivo have not yet been defined. This study aimed to confirm the involvement of CTRP6 in adipose accumulation and brown adipogenesis by intraperitoneal injection of the CTRP6-shRNA lentivirus into mice (CL mice). CL mice were significantly thinner than the control mice after feeding with a high fat diet (HFD), independent of food intake quantity. These HFD-fed CL mice displayed lower white and brown adipocyte sizes, and serum leptin levels, but an increase in serum adiponectin and insulin sensitivity relative to control mice. Additionally, the brown fat markers, such as UCP1, PRDM16, PGC1α and Cidea were found to be upregulated in the white and brown adipose tissue of the CL mice. These markers were also upregulated in a primary culture of mouse white and brown adipocytes treated with the CTRP6-shRNA lentivirus. Mechanistically, the knockdown of CTRP6 increased p38MAPK phosphorylation, but decreased expression of proteins involved in the Hedgehog signaling pathway (Sufu, Gli2 and Gli3). CTRP6 knockdown also upregulated expression of mitochondrial metabolic factors NRF-1, TFAM, CPT1 and Cyt C. Data from the current study show that CTRP6 knockdown protects against diet-induced obesity and promotes brown adipogenesis by the p38MAPK/Hh signaling pathway in conjunction with the upregulation of brown fat markers and mitochondrial metabolic factors.

Adiponectin AS lncRNA inhibits adipogenesis by transferring from nucleus to cytoplasm and attenuating Adiponectin mRNA translation.

Adiponectin (AdipoQ) is an adipocyte-derived hormone with positive function on systemic glucose and lipid metabolism. Long noncoding RNA (lncRNA) is emerging as a vital regulator of adipogenesis. However, AdipoQ-related lncRNAs in lipid metabolism have not been explored. Here, AdipoQ antisense (AS) lncRNA was first identified, and we further found that it inhibited adipogenesis. The half-life of AdipoQ AS lncRNA was 10 h, whereas that of AdipoQ mRNA was 4 h. During adipogenic differentiation, AdipoQ AS lncRNA translocated from nucleus to cytoplasm. AdipoQ AS lncRNA and AdipoQ mRNA formed an RNA duplex. Moreover, AdipoQ AS lncRNA delivered via injection of adenovirus expressing AdipoQ AS lncRNA decreases white adipose tissue (WAT), brown adipose tissue (BAT) and liver triglycerides (TG) in mice consuming a high fat diet (HFD). Interestingly, the non-overlapping region of AdipoQ AS lncRNA improved serum glucose tolerance and insulin sensitivity in HFD mice, but not AdipoQ AS lncRNA. In conclusion, AdipoQ AS lncRNA transfer from nucleus to cytoplasm inhibits adipogenesis through formation of an AdipoQ AS lncRNA/AdipoQ mRNA duplex to suppress the translation of AdipoQ mRNA. Taken together, we suggest that AdipoQ AS lncRNA is a novel therapeutic target for obesity-related metabolic diseases.

PDGFRα Regulated by miR-34a and FoxO1 Promotes Adipogenesis in Porcine Intramuscular Preadipocytes through Erk Signaling Pathway.

Suitable intramuscular fat (IMF) content improves porcine meat quality. The vital genes regulating IMF deposition are necessary for the selection and breeding of an IMF trait. However, the effect and mechanism of PDGFRα on IMF deposition are still unclear. Here, PDGFRα is moderately expressed in porcine longissimus dorsi muscle (LD), whereas it highly expressed in white adipose tissue (WAT). Moreover, PDGFRα-positive cells were located in the gaps of LD fibers which there were IMF adipocytes. Compared with 180-day-old and lean-type pigs, the levels of PDGFRα were much higher in one-day-old and fat-type pigs. Meanwhile the levels of PDGFRα gradually decreased during IMF preadipocyte differentiation. Furthermore, PDGFRα promoted adipogenic differentiation through activating Erk signaling pathway. Based on PDGFRα upstream regulation analysis, we found that the knockdown of FoxO1 repressed lipogenesis by downregulating PDGFRα, and miR-34a inhibited adipogenesis through targeting PDGFRα. Collectively, PDGFRα is a positive regulator of IMF deposition. Therefore, we suggest that PDGFRα is a possible target to improve meat quality.

Lipogenesis in myoblasts and its regulation of CTRP6 by AdipoR1/Erk/PPARγ signaling pathway.

The induced lipogenesis and its regulation in C2C12 myoblasts remain largely unclear. Here, we found that the cocktail method could significantly induce lipogenesis through regulating lipid metabolic genes and Erk1/2 phosphorylation in myoblasts. Meanwhile, the expression and secretion of CTRP6 were increased during ectopic lipogenesis. Moreover, CTRP6 knockdown down-regulated the levels of lipogenic genes and phosphorylated Erk1/2 (p-Erk1/2) in the early lipogenic stage, whereas up-regulated p-Erk1/2 in the terminal differentiation. Interestingly, the effect of CTRP6 siRNA was attenuated by U0126 (a special p-Erk1/2 inhibitor) in myoblasts. Furthermore, AdipoR1, not AdipoR2, was first identified as a receptor of CTRP6 during the process of mitotic clonal expansion. Collectively, we suggest that CTRP6 mediates the ectopic lipogenesis through AdipoR1/Erk/PPARγ signaling pathway in myoblasts. Our findings will shed light on the novel biological function of CTRP6 during myoblast lipogenesis and provide a hopeful direction of improving meat quality of domestic animal by lipogenic regulation in skeletal muscle myoblasts.

Effects of Adjunct Questions on L2 Reading Comprehension with Texts of Different Types.

Answering text-related questions while reading is a questioning strategy which is called adjunct questions or embedded questions, the benefits of which have been established in first-language reading as to enhance comprehension. The present study aims to study the effects different adjunct questions exert on second-language (L2) readers' comprehension of texts of various types. One hundred and forty-four intermediate-level Chinese EFL learners participated in this study and were divided randomly into six groups. Each group was given either a narrative or an expository text with 'what or why' questions or no questions. A brief topic familiarity questionnaire was attached to the end of each text paper. The results showed that inserted adjunct questions improved the readers' reading comprehension both in expository and narrative texts, but only narrative texts inserted with why questions had significant effects on the L2 reading comprehension. The findings suggested that text types and question types modulate the effects of inserted adjunct questions on the English reading of intermediate learners. Pedagogical implications and suggestions for future studies are provided.

Discovering novel biomarkers for diagnosis and treatment monitoring of active pulmonary tuberculosis by ion metabolism analysis.

Tuberculosis (TB) is a highly lethal infectious disease that poses a global threat. Timely and accurate biomarker for TB diagnosis and treatment monitoring remains a pressing need. Ions, the crucial trace element for humans, may be potential targets for TB diagnosis and the forecasting of TB development. To explore the potential of ions as biomarkers, we measured and compared the levels of various ions in whole blood and plasma samples from healthy control (HC), pulmonary TB patients (TB), cured pulmonary TB patients (RxTB), and other non-TB pneumonia patients (PN) by using ultra-high performance liquid chromatography-tandem mass spectrometry. Our study demonstrated that Cu (AUC = 0.670), Pb (AUC = 0.660), and Zn (AUC = 0.701) in whole blood exhibited promising diagnostic performance for TB. Then we used a neural network (NNET) for TB prediction, the AUC values used to differentiate definite TB from HC or PN in plasma were 0.867 and 0.864, respectively. The AUC values used to differentiate definite TB from HC or PN in whole blood were 0.818 and 0.660, respectively. Our correlation analysis showed that Zn (r= 0.356, p=0.001) and Cu (r= 0.361, p=0.0004) in plasma are most closely related to disease severity. Additionally, six ions (Cu, Sb, V, Mn, Fe, Sr) in plasma and whole blood were altered following anti-TB therapy. These results showed that ions could be diagnostic biomarkers for TB. Furthermore, the level of particular ions can forecast the degree of lung damage and the success of the TB treatment. In conclusion, this study highlights the possibility of using ions from blood samples to enable rapid tuberculosis diagnosis.

The Relationships among Chinese University EFL Learners' Feedback-Seeking Behavior, Achievement Goals, and Mindsets.

This study investigated the characteristics of feedback-seeking behavior and the underlying motivational antecedents including the mindsets and achievement goals of Chinese EFL learners. Questionnaire data were collected from 677 learners taking English classes at different levels in China for (1) their beliefs about English learning (a fixed or growth mindset), (2) goal orientation in achievement-related situations (development or demonstration goals), and (3) FSB (whether to seek feedback, by what strategies, and from whom). Results indicated that Chinese EFL learners with a growth mindset or demonstration-approach goals proactively seek feedback through variant strategies (i.e., feedback direct inquiry, indirect inquiry, and monitoring) while those with development-approach goals or a fixed mindset seek feedback by monitoring only due to learners' different perceptions of the cost and value attached to different strategies. Furthermore, a demonstration approach partially mediated the predictive role of a growth mindset on three FSBs, while the relationships between feedback monitoring and the two mindsets were partially or fully mediated by a development approach.

Amino acid profiling as a screening and prognostic biomarker in active tuberculosis patients.

BACKGROUND: Tuberculosis (TB) is one of the world's most deadly chronic infectious diseases; early diagnosis contributes to reducing disease transmission among populations. However, discovering novel diagnostic and prognostic biomarkers is still an important topic in the field of TB. Amino acid is the basic unit of protein composition, and its structure and physicochemical characteristics are more stable. Therefore, it is a potential target for TB diagnosis and the prediction of TB development. METHODS: In this study, the blood of healthy people (HC), TB patients (TB), cured TB (RxTB), and other non-TB pneumonia patients (PN) were collected to detect the levels of amino acids in whole blood and plasma using ultra-high performance liquid chromatography coupled with mass spectrometry. RESULTS: We detected that the amino acid levels correlated with participants status (TB, HC, RxTB, or PN) and the degree of lung damage. The results showed that phenylalanine had a good effect on the screening of TB (AUC = 0.924). We then built a TB prediction model. The model, which was based on the ratio of plasma amino acid content to whole blood amino acid content, showed good performance for the screening of TB, with 84% (95% CI = 60-97) sensitivity and 97% (95% CI = 83-100) specificity. The result of correlation between the HRCT score and amino acid level indicated that the glutamine content of plasma was significantly inversely associated with disease severity. Additionally, ornithine levels in the plasma of RxTB group reduced and four amino acids of which the ratio in plasma to whole blood showed significantly changed. CONCLUSIONS: Taken together, amino acid profiling can be used for TB screening, and a multiparameter profiling model is better. The profiling can also reflect the severity of lung damage. Moreover, the amino acid profile is useful for reflecting the efficacy of TB treatment.

Linguistic features and psychological states: A machine-learning based approach.

Previous research mostly used simplistic measures and limited linguistic features (e.g., personal pronouns, absolutist words, and sentiment words) in a text to identify its author's psychological states. In this study, we proposed using additional linguistic features, that is, sentiments polarities and emotions, to classify texts of various psychological states. A large dataset of forum posts including texts of anxiety, depression, suicide ideation, and normal states were experimented with machine-learning algorithms. The results showed that the proposed linguistic features with machine-learning algorithms, namely Support Vector Machine and Deep Learning achieved a high level of performance in the detection of psychological state. The study represents one of the first attempts that uses sentiment polarities and emotions to detect texts of psychological states, and the findings may contribute to our understanding of how accuracy may be enhanced in the detection of various psychological states. Significance and suggestions of the study are also offered.

Isoleucine increases muscle mass through promoting myogenesis and intramyocellular fat deposition.

Isoleucine (Ile), as a branched-chain amino acid (BCAA), has a vital role in regulating body weight and muscle protein synthesis. However, the regulatory effect of Ile on muscle mass under high-fat diet (HFD) conditions and intramyocellular lipid deposition remains largely unclear. In this study, a feeding experiment with HFD with or without 25 g L-1 Ile was performed using 32 wild male C57BL/6J mice randomly divided into two groups. The results showed that Ile significantly increased both muscle and fat mass, as well as causing insulin resistance and meanwhile upregulating the levels of key adipogenic and myogenic proteins. More importantly, Ile damaged the mitochondrial function by vacuolation, swelling and cristae fracture in the gastrocnemius (GAS) and tibialis anterior (TA) with downregulation of mitochondrial function-related genes. Furthermore, Ile promoted myogenesis and more lipid droplet accumulation in myotubes. Compared with the control, the protein levels of myosin heavy chain (MyHC), myoblast determination protein 1 (MyoD), myogenin (MyoG), peroxisome proliferator-activated receptor gamma (PPARg) and fatty acid synthase (FAS) were upregulated in the Ile group, whereas the protein levels of adipose triglyceride lipase (ATGL) and lipoprotein lipase (LPL) were downregulated. Collectively, Ile increased muscle mass through myogenesis and intramyocellular lipid deposition. Our findings provide a new perspective for not only improving the lean juiciness of farm animals by increasing intramyocellular lipid accumulation, but also modulating myopathies under obesity.

Dual activities of ACC synthase: Novel clues regarding the molecular evolution of ACS genes.

Ethylene plays profound roles in plant development. The rate-limiting enzyme of ethylene biosynthesis is 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), which is generally believed to be a single-activity enzyme evolving from aspartate aminotransferases. Here, we demonstrate that, in addition to catalyzing the conversion of S-adenosyl-methionine to the ethylene precursor ACC, genuine ACSs widely have Cß-S lyase activity. Two N-terminal motifs, including a glutamine residue, are essential for conferring ACS activity to ACS-like proteins. Motif and activity analyses of ACS-like proteins from plants at different evolutionary stages suggest that the ACC-dependent pathway is uniquely developed in seed plants. A putative catalytic mechanism for the dual activities of ACSs is proposed on the basis of the crystal structure and biochemical data. These findings not only expand our current understanding of ACS functions but also provide novel insights into the evolutionary origin of ACS genes.

A Newly Identified LncRNA LncIMF4 Controls Adipogenesis of Porcine Intramuscular Preadipocyte through Attenuating Autophagy to Inhibit Lipolysis.

Intramuscular fat (IMF) is implicated in juiciness, tenderness, and flavor of pork. Meat quality of Chinese fat-type pig is much better than that of lean-type pig because of its higher IMF content. LncRNA is a vital regulator that contributes to adipogenesis. However, it is unknown about the regulation of lncRNA on IMF content. Here, by RNA sequence analysis of intramuscular adipocyte from Bamei pig (fat-type) and Yorkshire pig (lean-type), we found that a novel lncRNA, lncIMF4, was associated with adipogenesis. LncIMF4, abundant in adipose, differently expressed along with intramuscular preadipocyte proliferation and differentiation. Meanwhile, it is located both in cytoplasm and nucleus. Besides, lncIMF4 knockdown promoted proliferation and differentiation of porcine intramuscular preadipocytes, whereas inhibited autophagy. Moreover, lncIMF4 knockdown facilitated intramuscular adipogenesis through attenuating autophagy to repress the lipolysis. Our findings will contribute to understand better the mechanism of lncRNA controlling intramuscular adipogenesis for promoting pork quality.

Overexpression of DNMT3A promotes proliferation and inhibits differentiation of porcine intramuscular preadipocytes by methylating p21 and PPARg promoters.

Intramuscular fat (IMF), which is modulated by the number and size of intramuscular preadipocytes, plays a key role in pork quality. DNA (cytosine-5)-methyltransferase 3A (DNMT3A), an enzyme that catalyzes the transfer of methyl groups to specific CpG structures in DNA, is involved in the management of diverse intracellular processes. However, the physiological functions of DNMT3A in proliferation and differentiation of porcine intramuscular preadipocytes have not been clearly established. Here, we found that DNMT3A significantly promoted the proliferation, while inhibited the differentiation of intramuscular preadipocytes. We demonstrated that overexpression of DNMT3A promoted the expression of cell proliferation markers but significantly decreased the expression of p21 to repress cell proliferation by the methylation of p21 promoter. Moreover, overexpression of DNMT3A decreased lipid accumulation and significantly down-regulated the levels of adipogenic marker genes including PPARg (Peroxisome proliferator-activated receptor gamma), SREBP-1c (Sterol regulatory element-binding protein 1c), and aP2 (FABP4, fatty acid binding protein 4) through the methylation of PPARg promoter. The blocking effect of DNMT3A on adipogenesis can be rescued by rosiglitazone treatment. Collectively, these findings illustrated the essential role of DNMT3A in the proliferation and differentiation of porcine intramuscular preadipocytes, and provide a potential target to improve pork quality.

Differences between porcine longissimus thoracis and semitendinosus intramuscular fat content and the regulation of their preadipocytes during adipogenic differentiation.

Intramuscular fat (IMF) plays an important role in pork quality. However, differences in the adipogenic regulation of IMF content between pig longissimus thoracis (LT) and semitendinosus (ST) remain unclear. Here, we found that IMF content of 180-day-old pig LT was greater than that of pig ST. Furthermore, lipid accumulation was earlier and greater in LT intramuscular preadipocytes (L-IMA) than in ST intramuscular preadipocytes (S-IMA) during differentiation. Interestingly, glucose consumption was lower in L-IMA than in S-IMA. Moreover, monounsaturated fatty acid content was greater in L-IMA than in S-IMA, whereas polyunsaturated fatty acid content was lower. Levels of the expression of key adipogenic genes were higher in L-IMA than S-IMA. Compared with S-IMA, adipogenic signals were more activated in L-IMA after adipogenic induction. In conclusion, IMF deposition differences between pig LT and ST were due to different glucose consumption, fatty acid composition, expression of key adipogenic genes and level of activating adipogenic signals between S-IMA and L-IMA during adipogenesis.

Long noncoding RNA: multiple players in gene expression.

Previously considered as a component of transcriptional noise, long noncoding RNAs (lncRNAs) were neglected as a therapeutic target, however, recently increasing evidence has shown that lncRNAs can participate in numerous biological processes involved in genetic regulation including epigenetic, transcriptional, and post-transcriptional regulation. In this review, we discuss the fundamental functions of lncRNAs at different regulatory levels and their roles in metabolic balance. Typical examples are introduced to illustrate their diverse molecular mechanisms. The comprehensive investigation and identification of key lncRNAs will not only contribute to insights into diseases, such as breast cancer and type II diabetes, but also provide promising therapeutic targets for related diseases. [BMB Reports 2018; 51(6): 280-289].

Comparative Analysis of Long Noncoding RNAs Expressed during Intramuscular Adipocytes Adipogenesis in Fat-Type and Lean-Type Pigs.

The meat quality of local breed pigs is more tender and juicier than the imported varieties. The important reason is that the intramuscular fat content is high. Even through modest sequence conservation and evolution, the expression pattern and function of long noncoding RNAs (lncRNAs) seem to be conserved. In spite of that, analysis of lncRNAs associated with intramuscular fat development remains unknown to us in porcine. Here, we systematically investigated lncRNAs of intramuscular adipocytes of fat local Bamei pigs and lean Large White pigs to consider the function of lncRNAs on intramuscular fat development. We selected three piglets of both breeds separately to isolate intramuscular preadipocytes, performed RNA sequencing across four stages (0, 2, 4, and 8 d) during the intramuscular preadipocytes differentiation, and identified 1932 lncRNAs (760 novel). In addition, we have screened lnc_000414 closely related to fat synthesis. This lncRNA function as an inhibitor in the proliferation of porcine intramuscular adipocytes. These novel findings will provide new targets for improving pork quality and making pig breeding better.

CTRP6 Regulates Porcine Adipocyte Proliferation and Differentiation by the AdipoR1/MAPK Signaling Pathway.

Intramuscular fat (IMF) and subcutaneous fat (SCF), which are modulated by adipogenesis of intramuscular and subcutaneous adipocytes, play key roles in pork quality. C1q/tumor necrosis factor-related protein 6 (CTRP6), an adipokine, plays an important role in the differentiation of 3T3-L1 cells. However, the effect and regulatory mechanisms of CTRP6 on porcine adipogenesis, and whether CTRP6 has the same effect on intramuscular and subcutaneous adipocytes, are still unknown. Here, we found that CTRP6 significantly inhibited both adipocyte proliferation assessed by proliferative marker expression, but CTRP6 decreased the proliferation rate of intramuscular adipocytes (IM) to a greater extent than subcutaneous adipocytes (SC). Moreover, CTRP6 promoted the activity of the p38 signaling pathway during the proliferation of both cell types. Nevertheless, in subcutaneous adipocytes, CTRP6 also influenced the phosphorylation of extracellular regulated protein kinases1/2 (p-Erk1/2), but not in intramuscular adipocytes. Additionally, during the differentiation of intramuscular and subcutaneous adipocytes, CTRP6 increased adipogenic genes expression and the level of p-p38, while it decreased the activity of p-Erk1/2. Interestingly, the effect of CTRP6 shRNA or CTRP6 recombinant protein was attenuated by U0126 (a special p-Erk inhibitor) or SB203580 (a special p-p38 inhibitor) in adipocytes. By target gene prediction and experimental validation, we demonstrated that CTRP6 may be a target of miR-29a in porcine adipocytes. Moreover, AdipoR1was identified as a receptor of CTRP6 in intramuscular adipocytes, but not in subcutaneous adipocytes. On the basis of the above findings, we suggest that CTRP6 was the target gene of miR-29a, inhibited intramuscular and subcutaneous adipocyte proliferation, but promoted differentiation by the mitogen-activated protein kinase (MAPK) signaling pathway. These findings indicate that CTRP6 played an essentially regulatory role in fat development.