Porcine skeletal muscle development regulated by MicroRNA: a review / 生物工程学报

The growth and development of skeletal muscle is an important factor affecting pork production and quality, which is elaborately regulated by many genetic and nutritional factors. MicroRNA (miRNA) is a non-coding RNA with a length of about 22 nt, which binds to the 3'UTR sequence of the mRNA of the target genes, and consequently regulates its post-transcriptional expression level. In recent years, a large number of studies have shown that miRNAs are involved in various life processes such as growth and development, reproduction, and diseases. The role of miRNAs in the regulation of porcine skeletal muscle development was reviewed, with the hope to provide a reference for the genetic improvement of pigs.

Exosome-mediated regulatory mechanisms in skeletal muscle: a narrative review / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Skeletal muscle plays a paramount role in physical activity, metabolism, and energy balance, while its homeostasis is being challenged by multiple unfavorable factors such as injury, aging, or obesity. Exosomes, a subset of extracellular vesicles, are now recognized as essential mediators of intercellular communication, holding great clinical potential in the treatment of skeletal muscle diseases. Herein, we outline the recent research progress in exosomal isolation, characterization, and mechanism of action, and emphatically discuss current advances in exosomes derived from multiple organs and tissues, and engineered exosomes regarding the regulation of physiological and pathological development of skeletal muscle. These remarkable advances expand our understanding of myogenesis and muscle diseases. Meanwhile, the engineered exosome, as an endogenous nanocarrier combined with advanced design methodologies of biomolecules, will help to open up innovative therapeutic perspectives for the treatment of muscle diseases.

Effect of over-expressed miR-155 on inhibiting C2C12 myogenic differentiation / 生物工程学报

To clarify the function and molecular mechanism of miR-155 in myogenic differentiation of C2C12, we constructed adenovirus over-expression vector of miR-155, then C2C12 cells were infected by adenovirus and induced myogenic differentiation. First, we observed the morphology of C2C12 after differentiation. Then the mRNA and protein expressions of myogenic markers (MyoD, MyoG and MyHC) were detected by qPCR and western blotting. Subsequently, the dual luciferase reporter gene assay was carried out to validate putative target gene (TCF4) of miR-155. Meanwhile, mRNA level of TCF4 was analyzed after over-expressing miR-155. The results show that over-expressed miR-155 reduced myotubes formation. Moreover, the mRNA and protein expression of MyoG and MyHC decreased significantly (P < 0.01). Further research demonstrated miR-155 bound the one (4532-4538) of three putative sites (1487-1493,1516-1522, 4532-4583) of TCF4 by luciferase reporter gene assay and the mRNA level of TCF4 decreased notably (P < 0.05). The data suggest that miR-155 inhibited myogenic differentiation of C2C12 through targeted TCF4.

BAMBI inhibits porcine preadipocyte differentiation by facilitating ERK1/2 phosphorylation / 生物工程学报

To study the role of BAMBI in adipogenesis, we constructed lentivirus interfering vector targeting on porcine BAMBI, packaged and infected the porcine preadipocyte. The differentiation state of preadipocyte was detected by Oil Red O staining and Oil Red O extraction assay and the expression levels of adipogenic marker genes were detected by Real-time qPCR and Werstern bloting. Results show that BAMBI expression was significant decreased after lentivirus infection, which was repressed more than 60% by shRNA2. Moreover, knockdown BAMBI increased the lipid accumulation of porcine preadipocyte and improved the expression of PPARγ (peroxisome proliferator-activated receptorγ) and ap2 (adipocyte protein 2). In summary, these data indicated that BAMBI inhibited adipocyte differentiation by facilitating the phosphorylation of ERK1/2.

Effects of retinol binding protein 4 knockdown on the PI3K/Akt pathways in porcine adipocytes / 生物工程学报

Retinol-binding protein 4 (RBP4) is adipocyte-derived secreted adipokines and elevated RBP4 expression level was closely related to insulin resistance and type II diabetes mellitus. However, the exact mechanisms are unknown. To clarify the mechanism, RBP4 lentivirus particles were packaged to infect porcine preadipocytes. Then porcine preadipocytes were activated by insulin or induced model of insulin resistance. RBP4 interference efficiency and the gene expression of each treatment groups in PI3K/Akt pathways were examined by QRT-PCR and Western blotting. The result shows that RBP4 mRNA and protein expressions were suppressed more than 60% (P < 0.01). Furthermore, no matter under insulin stimulation or insulin resistance, RBP4 knockdown significantly increased the mRNA expressions of AKT2, PI3K, GLUT4 and IRS1 compared with the control. The protein phosphorylate levels of AKT2, PI3K, IRS1 arised, meanwhile enhanced the AKT2, PI3K, GLUT4 total protein expressions. Collectively, knockdown of RBP4 increased the insulin sensitivity through upregulated PI3K/Akt pathways related factors' expression and phosphorylation in porcine adipocytes. This research will provide a new idea to treat insulin resistance related diseases.

Over-expressed MiR-103 promotes porcine adipocyte differentiation / 生物工程学报

To clarify the function of miR-103 in the differentiation of porcine preadipocyte, we carried out real-time PCR to detect the expression pattern of miR-103 during adipogenesis, and clarified its expression tendency through cell differentiation. Then we used adenovirus that overexpressed miR-103 to infect porcine preadipocyte. Subsequently, mRNA and protein expression of adipogenesis marker--PPARgamma and aP2 was analyzed by real-time PCR and Western blotting. At last, Oil-Red O staining was used to detect lipids accumulation in the 8th day after adipogenic inducement. The expression of miR-103 increased during adipocyte differentiation; compared with the control, the preadipocyte infected by pAd-miR-103 had an elevated expression level of adipocyte marker gene PPARgamma, aP2, and obvious lipid droplet was seen in the 8th day after adipogenic inducement. These results showed that miR-103 can enhance adipogenesis in primary cultured porcine adipocytes.

Hyperlipidemia in hepatic MED1 deficient mice in response to fasting / 生物工程学报

MED1 is a key transcription co-activator subunit of the Mediator complex that is essential for RNA polymerase II-dependent transcription. MED1 functions as a co-activator for PPARs and other nuclear receptors and transcription factors, and plays an important role in lipid metabolism. To examine how MED1 might affect plasma lipids, plasma triglyceride, cholesterol levels, and lipoprotein profiles, were measured in MED1(deltaLiv) mice fasted for 24, 48 and 72 hours. Histological changes in liver sections from MED1(deltaLiv) mice after 72 hours of fasting were also examined using H&E staining. There was no fat accumulation in livers of MED1(deltaLiv) mice compared to MED1(fl/fl) and PPARalpha -/- control mice after 72 hours of fasting. Compared with MEDl(fl/fl) mice, plasma triglycerides in MED1(deltaLiv) mice were significantly increased after 24, 48 and 72 hours of fasting, and plasma cholesterol was significantly increased after 48 and 72 hours of fasting. Lipoprotein profiles were similar in fed MED1(fl/fl) and MED1(deltaLiv) mice. However, very low density lipoprotein (VLDL) was significantly increased in MED1(deltaLiv) mice after 24 hours of fasting. We conclude that, hyperlipidemia in MED1(deltaLiv) mice in response to fasting is due to the accumulation of VLDL, which suggests that MED1 plays a pivotal role in the regulation of plasma triglyceride and cholesterol levels.

Role of estrogen-related receptor alpha in adipocyes lipolysis / 生物工程学报

Estrogen-related receptor a (ERRalpha) is a key regulator for energy metabolism and adipogenesis. However, its role in lipolysis is unknown. To study the function of ERRalpha in lipolysis, primary cultured differentiated porcine adipocytes were treated by a specific inverse agonist XCT790 or infected with adenoviral vector expressed ERRalpha for 48 h, in the absence and/or presence of specific protein kinase A (PKA) inhibitor or extracellular signal-related kinase (ERK) inhibitor. Then, we measured the triglyceride (TG) content and the glycerol release into the culture media to analysis the effect of ERRalpha on lipolysis; Further, we analyzed the expression of PPARgamma, perilipin A, p-perilipin A, HSL and ATGL with Western blotting. Here, we found that ERRalpha significantly increased adipocytes differentiation, TG accumulation and glycerol release. Separately or simultaneously block the PKA and ERK pathway do not significantly altered the effect of ERRalpha on glycerol release. ERRalpha significantly up-regulated the proteins expression of PPARgamma, perilipin A, HSL and ATGL, while the p-perilipin A protein level was not significantly changed. These findings imply that ERRalpha could increase lipolysis via up-regulating HSL and ATGL, thereby to supply more FFA as substrate for a larger turnover of cellular triglyceride pool during adipocytes differentiation.

Effect and underlying mechanism of resveratol on porcine primary preadipocyte apoptosis / 生物工程学报

We demonstrated the effect of resveratrol on porcine primary preadipocytes apoptosis, to study the intracellular molecular mechanism. Porcine primary preadipocyte was treated with different concentration of resveratrol (0 micromol/L, 50 micromol/L, 100 micromol/L, 200 micromol/L, 400 micromol/L). We used optical microscope and fluorescence microscope to observe morphological changes during apoptosis after Hoechst 33258 Fluorescent dyes staining; and RT-PCR and Western blotting to measure the expression of apoptosis-associated gene sirt1, caspase-3, bcl-2, bax, p53, NF-kappaB. Primary preadipocyte apoptosis was apparent, accompanied by reduced cell volume, chromatin condensation, and nuclear shrinkage. Compared to the control and low concentration group, high dose group (200 micromol/L) significantly increased the ratio of primary preadipocyte apoptosis. The expression of sirt1, caspase-3, and bax was up-regulated markedly in response to resveratrol; in contrast, apoptotic inhibitor bcl-2, p53, NF-kappaB down-regulated. We further proved fact that resveratrol can specifically promote the activity of sirt1; moreover, activated sirt1 modulates the activity of caspase-3 and bcl-2 family, involving in transcriptional regulation of p53 and NF-kappaB through antagonizing factor-induced acetylation. Taken together, our data established resveratrol as new regulator in porcine primary preadipocyte apoptosis via activating the expression of sirt1, modulating activity of apoptotic-associated factor.

Construction of SOCS3 recombinant adenovirus and its expression in porcine primary adipocytes / 生物工程学报

In order to construct recombinant adenovirus vector expressing Suppressor of cytokine signaling 3 (SOCS3) and obtain infectious adenoviral particles, SOCS3 gene was amplified from plasmid pcDNA3-SOCS3 and subcloned into the adenovirus shuttle plasmid pAdTrack-CMV. After sequence confirmation, the recombinant shuttle plasmid pAdTrack-CMV-SOCS3 was linearized by Pme I, and then transformed into BJ5183 competent cell, the recombinant plasmid pAd-SOCS3 was obtained by homologous recombination between pAdTrack-CMV-SOCS3 and the adenoviral backbone plasmid pAdEasy-1 in BJ5183. The pAd-SOCS3 was linearized by Pac I and transfected into HEK293 cells via liposome. The recombinant adenovirus was packaged and amplified in HEK293 cells. After purifying, virus titer was determined by tissue culture infectious dose 50 (TCID50). Using the recombinant adenoviruses to infect porcine primary adipocytes, the expression of green fluorescent protein (GFP) was observed by fluorescent microscopy, and SOCS3 gene was identified by RT-PCR and Western blotting. Restriction enzyme and PCR analysis demonstrated that the recombinant adenovirus vector was constructed correctly, and the virus titer reached 1.2x10(9) PFU/mL. The result of RT-PCR and Western blotting showed that SOCS3 mRNA and protein expression was remarkably increased in porcine primary adipocytes infected with recombinant adenovirus. In conclusion, this study successfully constructed the recombinant adenovirus containing SOCS3 gene, and can be helpful for further research on the function of SOCS3.

Over-expression of FoxO1 inhibits the differentiation of porcine skeletal muscle myoblast / 生物工程学报

The Forkhead box O1 (FoxO1) transcription factor governs muscle growth, metabolism and cell differentiation. However, its role in myoblast differentiation is unclear. To study the biological function of FoxO1 during differentiation in porcine primary myoblast, we constructed stably FoxO1 over-expressed porcine myoblast mediated by liposome and adopted morphological observation, quantitative real-time RT-PCR and Western blotting methods to analyze FoxO1 and early and late myogenic regulation factors MyoD and myogenin expression. During differentiation the mRNA level of FoxO1 was significantly increased. However, the total protein did not change but the phosphorylation of FoxO1 was upregulated. Furthermore, overexpression of FoxO1 in porcine myoblast decreased MyoD and myogenin mRNA, whereas MyoD protein changed little and myogenin was significantly suppressed (P < 0.05). These results indicated that FoxO1 delays and negatively regulates the porcine myoblast differentiation. Moreover, FoxO1 may play a critical role in muscle fiber-type specification through the inhibition of myogenic regulation factors.

Chronic high dose of insulin stimulates lipolysis in porcine adipocytes / 生物工程学报

To explore the effect of chronic high dose of insulin on lipolysis in porcine adipocytes and the underlying molecular regulation mechanisms, we cultured primary porcine adipocytes and incubated them with different concentrations of insulin (0, 200, 400, 800, 1600 nmol/L) for 24-96 h in the absence or presence of specific protein kinase A (PKA) inhibitor or extracellular signal-related kinase (ERK) inhibitor. Then, we measured the glycerol release into the culture media as an indicator of the lipolysis, and observed the lipid accumulation morphology by phase-contrast microscopy. Further, we analyzed the gene expressions of perilipin A and peroxisome proliferator-activated receptor-gamma 2 (PPAR gamma 2) with semi-quantitative RT-PCR and Western blotting, respectively. The results showed that chronic high dose of insulin stimulated lipolysis in differentiated porcine adipocytes in a dose- and time-dependent manner, and significantly attenuated the lipolytic response to isoprenaline. Meanwhile, the protein and mRNA expressions of PPAR gamma 2 and perilipin A were significantly reduced. In addition, both PKA and ERK inhibitors significantly suppressed insulin-stimulated lipolysis, however, only ERK inhibitor reversed the insulin-induced down-regulation of perilipin A. These findings imply that chronic high dose of insulin stimulates lipolysis in porcine adipocytes by repressing perilipin A, which is involved in ERK pathway.

Cloning, expression of the porcine estrogen-related receptor alpha gene and its effect on lipid accumulation in mature adipocytes / 生物工程学报

Estrogen-related receptor alpha (ERR alpha) is an orphan nuclear receptor and functions as a key regulator of energy metabolism in high energy demand tissues. However, its role in white adipose tissue is largely unknown. In this study, we aim to clone the ORF sequence of pig ERR alpha with touch down-PCR, analyze the expression pattern of ERR alpha protein and its subcellular localization with Western blotting and cell immunofluorescence method respectively, and identify the effect of ERR alpha on lipid accumulation in mature porcine adipocytes with its specific inhibitor XCT790. The results showed that the ERR alpha ORF sequence is 1269 bp (GenBank Accession No. FJ446485, not published), and encode 422 amino acids. The homologies of ERR alpha nucleotide and amino acids sequences are high in different species. ERR alpha protein is highly expressed in pig white adipose tissue, kidney and heart, while remarkably lower in spleen. Cell immunofluorescence results indicated that ERR alpha protein distribute widely in adipocytes nucleus and cytoplasm. XCT790 significantly inhibited the expression of ERR alpha and lipid accumulation in porcine mature adipocyte. This study will provide new target and theoretical reference for fat deposition control.

Sequential expression of Wnt/beta-catenin signal pathway related genes and adipocyte transcription factors during porcine adipose tissue development / 生物工程学报

To evaluate the influence of Wnt/beta-catenin signal pathway on the porcine adipose tissue development and explore the mechanism, we detected the mRNA expression of Wnt/beta-catenin signal pathway related genes: beta-catenin, GSK3beta, Fzl and adipogenic transcription factors: PPARy, C/EBPalpha and early differentiation marker gene LPL with semi-quantitative (SQ) RT-PCR method. Immunohistochemical method (IHC) was applied to qualitatively measure the sequential expression of beta-catenin protein. The results of SQ RT-PCR showed that beta-catenin highly expressed at the first day after birth, then decreased to a low plateau after 60 days, the expression of GSK3beta and Fzl also decreased with the development process of the porcine adipose tissue development. However, the sequential expression of PPARgamma, C/EBPalpha, LPL appeared to be an opposite manner and kept at a high level after 60 days. The result of IHC showed that the expression of beta-catenin protein was strong in nucleus and cytoplasm at the first day after birth, then tended to decline with the process of adipose tissue development and could be only found in cytoplasm after 30-day old. These results suggest that beta-catenin plays an important role in the undifferentiated state maintenance of preadipocytes and the inhibition of porcine adipose tissue development, the mechanism maybe due to its regulation function on the adipogenic transcription factors PPARy, C/EBPalpha and early differentiation marker gene LPL.

Regulation of SOCS-3, OB, GLUT4 and PPARgamma gene expression by insulin and dexamethasone in porcine primary adipocyte / 生物工程学报

Swine is an ideal model for diabetes studies. Insulin and insulin resistance are closely related with diabetes. To investigate the effect of SOCS-3 in insulin resistance, porcine primary adipocyte was treated with insulin (100 nmol/L) and dexamethasone (300 nmol/L) to induce insulin resistance. The simi-quantitative PCR results suggested that insulin increased GLUT4, PPARgamma and SOCS-3 gene expression in primary culture porcine adipocytes and no change of OB gene expression. Under insulin resistance conditions, SOCS-3 and OB gene expression were up-regulated, whereas GLUT4 and PPARgamma gene expression were down-regulated in primary porcine adipocytes. The overexpression of PPARgamma gene resulted in the increase of GLUT4 expression by insulin. Different expression levels of SOCS-3 determined the inhibitory effects of insulin signaling. Induction of insulin resistance by dexamethasone was not only due to inhibition of glucose transportation, but also repression of insulin signaling. SOCS-3 might be a potential gene to block the insulin resistance.

Effects of leptin on porcine primary adiocytes lipolysis and mRNA expression of key lipolytic enzymes / 生物工程学报

Leptin, a cytokine predominantly secreted from fat tissue, plays an important role in regulating organism energy balance. Leptin can stimulate lipolysis, but the mechanism is unclear. In order to study the molecular mechanism of leptin stimulating lipolysis, we systemically studied the mRNA expression of key lipolytic enzymes. Morphological observation, Oil Red O staining and RT-PCR were used to identify pig primary adipocytes; commercial kits were used to measure the glycerol and FFA release; Semiquantitative RT-PCR was used to detect the mRNA expression of key lipolytic enzymes. The results showed that 100 nmol/L leptin up-regulated the mRNA expression of ATGL, TGH-2, HSL, MGL and LPL (P<0.01), but down-regulated the Perilipin mRNA expression (P<0.01). At the same time, leptin promoted the glycerol release in a dose dependent manner (P<0.01), but had no effect on the FFA release (P>0.05). These indicate that leptin may mainly stimulate lipolysis in pig primary adipocytes by up-regulating the expression of ATGL, MGL, LPL and down-regulating the expression of Perilipin. The unchanged FFA release may be resulted from Leptin promoting UCPs mRNA expression and increasing FFA expenditure.

Effects of Arachidonic Acid on Growth and Differentiation of Rat Preadipocytes / 中国生物化学与分子生物学报

To study effects of arachidonic acid (AA) on the growth and differentiation of rat adipocytes, a cells culture system of rat primary preadipocytes was established. The cells treated by different concentration of AA supplemented based on DMEM medium containing 10% fetal bovine serum. Cell proliferation was measured by trypan blue exclusion and methyl thiazolyl tetrazolium (MTT) assay method. Hoechst33342 fluorescence staining observed AA induced morphological changes. Oil Red O staining extraction assay assess the degree of adipogenesis and differentiation, and cyclooxygenases-2(COX-2) mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that rat preadipocytes treated with 120 μmol/L AA for 24-72 hours remarkably promoted the cells proliferation compared with control, 160 μmol/L AA treated for 48 hours could induce apoptosis of preadipocytes. 40, 80 μmol/L AA decreased the fat content in cells at 72 hours, and 40 μmol/L AA significantly up-regulated the expression of COX-2 mRNA at 24 hours. This results indicate that AA regulate adipocytes proliferation and differentiation depended on treatment time and concentration. 40-80 μmol/L AA maybe useful to control body fat, which may be associated with the increase of COX-2 mRNA.