Luxi Chen

Up-regulated miR-145 expression inhibits porcine preadipocytes differentiation by targeting IRS1.

Generally, most miRNAs that were up-regulated during differentiation promoted adipogenesis, but our research indicated that up-regulation of miR-145 in porcine preadipocytes did not promote but inhibit adipogenesis. In this study, miR-145 was significantly up-regulated during porcine dedifferentiated fat (DFAT) cells differentiation. In miR-145 overexpressed DFAT cells, adipogenesis was inhibited and triglycerides accumulation was decreased after hormone stimulation (P<0.05). Furthermore, up-regulation of miR-145 expression repressed induction of mRNA levels of adipogenic markers, such as CCAAT/enhancer-binding protein α (C/EBPα), and peroxisome proliferator-activated receptor γ2 (PPARγ2). These effects caused by miR-145 overexpression were mediated by Insulin receptor substrate 1 (IRS1) as a mechanism. These data suggested that induced miR-145 expression during differentiation could inhibit adipogenesis by targeting IRS1, and miR-145 may be novel agent for adipose tissue engineering.

MicroRNA-344 inhibits 3T3-L1 cell differentiation via targeting GSK3β of Wnt/β-catenin signaling pathway

Differentiation of 3T3‐L1 cells into adipocytes involves a highly orchestrated series of complex events in which microRNAs might play an essential role. In this study, we found that the overexpression of microRNA‐344 (miR‐344) inhibits 3T3‐L1 cell differentiation and decreases triglyceride accumulation after MDI stimulation. We demonstrated that miR‐344 directly targets the 3′ UTR of GSK3β (Glycogen synthase kinase 3 beta). Knockdown of GSK3β with siRNA results in inhibiting 3T3‐L1 differentiation, while its overexpression restores the effect of miR‐344. In addition, miR‐344 elevates the level of active β‐catenin, which is the downstream effector of GSK3β in the Wnt/β‐catenin signaling pathway. These data indicate that miR‐344 inhibits adipocyte differentiation via targeting GSK3β and subsequently activating the Wnt/β‐catenin signaling pathway.

Activating transcription factor 4 regulates adipocyte differentiation via altering the coordinate expression of CCATT/enhancer binding protein β and peroxisome proliferator-activated receptor γ.

Adipose tissue is crucial for energy homeostasis and is a topic interest with respect to investigating the regulation of adipose tissue formation for the ever‐increasing health concerns of obesity and type 2 diabetes. Adipocyte differentiation is tightly regulated by the characteristic sequential expression change of adipocyte genes, including members of the CCATT/enhancer binding protein (C/EBP) family of transcription factors, peroxisome proliferator‐activated receptor γ and tribbles homolog 3. In the present study, we demonstrate that C/EBPβ and peroxisome proliferator‐activated receptor γ (but not tribbles homolog 3) are targeted for activation by activating transcription factor 4 (ATF4), a member of cAMP response element‐binding/activator transcription factor family. Importantly, overexpression of ATF4 in 3T3‐L1 cells enhanced adipogenesis, whereas small‐interfering ATF4 blocked conversion of preadipocytes to adipocytes. These findings were accomplished by altering the coordinate expression of adipogenic transcription factors. Taken together, our results suggest that ATF4 is a positive regulator of adipocyte differentiation. This notion is also supported by the results of the present study showing that the expression of ATF4 is induced during adipocyte differentiation. Thus, ATF4 could be an important regulator of energy homeostasis.

Integrated miRNA and mRNA transcriptomes of porcine alveolar macrophages (PAM cells) identifies strain-specific miRNA molecular signatures associated with H-PRRSV and N-PRRSV infection

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant viral diseases in swine, which causes large economic losses to the swine industry worldwide. There is considerable strain variation in PRRSV and two examples of this are the highly virulent Chinese-type PRRSV (H-PRRSV) and the classical North American type PRRSV (N-PRRSV), both with different pathogenesis. These differences may be due in part to genetic and phenotypic differences in virus replication, but also interaction with the host cell. MicroRNAs (miRNAs) are crucial regulators of gene expression and play vital roles in virus and host interactions. However, the regulation role of miRNAs during PRRSV infection has not been systematically investigated. In order to better understand the differential regulation roles of cellular miRNAs in the host response to PRRSV, miRNA expression and a global mRNA transcriptome profile was determined in primary cells infected with either H-PRRSV or N-PRRSV as multiple time points during the viral lifecycle. miRNA-mRNA interactome networks were constructed by integrating the differentially expressed miRNAs and inversely correlated target mRNAs. Using gene ontology and pathway enrichment analyses, cellular pathways associated with deregulated miRNAs were identified, including immune response, phagosome, autophagy, lysosome, autolysis, apoptosis and cell cycle regulation. To our knowledge, this is the first global analysis of strain-specific host miRNA molecular signatures associated with H- and N-PRRSV infection by integrating miRNA and mRNA transcriptomes and provides a new perspective on the contribution of miRNAs to the pathogenesis of PRRSV infection.

HMGB2 regulates satellite-cell-mediated skeletal muscle regeneration through IGF2BP2

ABSTRACT Although the mechanism underlying modulation of transcription factors in myogenesis has been well elucidated, the function of the transcription cofactors involved in this process remains poorly understood. Here, we identified HMGB2 as an essential nuclear transcriptional co-regulator in myogenesis. HMGB2 was highly expressed in undifferentiated myoblasts and regenerating muscle. Knockdown of HMGB2 inhibited myoblast proliferation and stimulated its differentiation. HMGB2 depletion downregulated Myf5 and cyclin A2 at the protein but not mRNA level. In contrast, overexpression of HMGB2 promoted Myf5 and cyclin A2 protein upregulation. Furthermore, we found that the RNA-binding protein IGF2BP2 is a downstream target of HMGB2, as previously shown for HMGA2. IGF2BP2 binds to mRNAs of Myf5 or cyclin A2, resulting in translation enhancement or mRNA stabilization, respectively. Notably, overexpression of IGF2BP2 could partially rescue protein levels of Myf5 and cyclin A2, in response to HMGB2 decrease. Moreover, depletion of HMGB2 in vivo severely attenuated muscle repair; this was due to a decrease in satellite cells. Taken together, these results highlight the previously undiscovered and crucial role of the HMGB2–IGF2BP2 axis in myogenesis and muscle regeneration. Summary: HMGB2 promotes myoblast proliferation and muscle regeneration through IGF2BP2, which regulates the protein production of cyclin A2 and Myf5.

DRACO inhibits porcine reproductive and respiratory syndrome virus replication in vitro

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Current vaccination strategies and antiviral drugs against PRRSV are still inadequate. Therefore, there is an urgent need for new antiviral strategies to control PRRSV. Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer (DRACO) is a synthetic construct consisting of a dsRNA detection domain, an apoptosis induction domain, and a transduction tag. It has been shown to have broad-spectrum antiviral activity, but there have been no reports regarding its effect on PRRSV. Here, we demonstrate that DRACO exhibits robust antiviral activity against PRRSV infection by suppressing virus RNA and protein synthesis in both Marc-145 cells and porcine alveolar macrophages (PAMs). In addition, DRACO still exhibited strong anti-PRRSV activity when viral replication was enhanced by knockdown of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) in Marc-145 cells. Furthermore, in PAMs, DRACO was capable of inducing IL-6 expression and reducing Hsp70 expression, which might contribute to the inhibition of PRRSV infection. Collectively, our results imply that DRACO holds promise as a novel anti-PRRSV therapeutic drug.

ATF4 regulates SREBP1c expression to control fatty acids synthesis in 3T3-L1 adipocytes differentiation

Activating transcription factor 4 (ATF4), which is highly expressed in 3T3-L1 adipocytes after adipogenic induction, is essential for adipocytes differentiation. ATF4 also plays a vital role in regulating fatty acids biosynthesis, whereas the detailed mechanism of this process is still unclear. Here we demonstrated that siRNA-based ATF4 depletion in 3T3-L1 adipocytes significantly reduced the accumulation of fatty acids and triglycerides. Moreover, SREBP1c protein, which is an important transcription factor of lipogenesis, appreciably decreased while Srebp1c mRNA increased. Then we identified that ATF4 could maintain SREBP1c protein stability by directly activating the expression of USP7 which deubiquitinates SREBP1c and increases its protein content in cell. Besides, USP7 could restore the synthesis of fatty acids and triglycerides in the absence of ATF4. On the other hand, we found that ATF4 might inhibit the transcription of Srebp1c through TRB3, which is repressed by IBMX and DEX during early adipogenesis. Thus, our data indicate that ATF4 regulates SREBP1c expression to control fatty acids synthesis.

miR-709 modulates LPS-induced inflammatory response through targeting GSK-3β.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs which modulate gene expression at the post-transcriptional level by either translational inhibition or mRNA degradation. MicroRNAs play important roles in both innate and adaptive immune response, including TLR-triggered immune response. In this study, we found that the expression of miR-709 was up-regulated in primary macrophage and RAW264.7 cells during the stimulation of LPS. Overexpression of miR-709 in RAW264.7 cells led to reduced production and gene expression of inflammatory cytokines (IL-6, TNF-α, IL-1β) during activation by LPS, whereas knockdown of miR-709 had completely opposite effects. We used bioinformatics and experimental techniques to demonstrate that GSK-3β is a direct target of miR-709. miR-709 mimics decreased GSK-3β protein but not mRNA level. We also found that miR-709 regulated the LPS-induced inflammatory response by targeting GSK-3β and elevating β-catenin. In conclusion, our data revealed a novel role for miR-709 in regulation of inflammatory response by targeting GSK-3β.

An integrated analysis revealed different microRNA-mRNA profiles during skeletal muscle development between Landrace and Lantang pigs

Pigs supply vital dietary proteins for human consumption, and their economic value depends largely on muscle production. MicroRNAs are known to play important roles in skeletal muscle development. However, their relationship to distinct muscle production between pig breeds remains unknown. Here, we performed an integrated analysis of microRNA-mRNA expression profiles for Landrace (LR, lean) pigs and the Chinese indigenous Lantang pig (LT, lard-type) during 8 stages of skeletal muscle developmental, including at 35, 49, 63, 77 dpc (days post coitum) and 2, 28, 90, 180 dpn (days postnatal). As differentially expressed-miRNA expression profiles can be well classified into two clusters by PCA analysis, we grouped the embryonic stages as G1 and the postnatal stages as G2. A total of 203 genes were predicted miRNA targets, and a STEM analysis showed distinct expression patterns between G1 and G2 in both breeds based on their transcriptomic data. Furthermore, a STRING analysis predicted interactions between 22 genes and 35 miRNAs, including some crucial myogenic factors and myofibrillar genes. Thus, it can be reasonably speculated that myogenic miRNAs may regulate myofibrillar genes in myofiber formation during embryonic stages and muscle hypertrophy during postnatal stages, leading to distinct differences in muscle production between breeds.

Transcriptome Landscape of Porcine Intramuscular Adipocytes during Differentiation

The adipocyte differentiation process, controlled by a tightly regulated transcriptional cascade, contributes partly to determine intramuscular adipose tissue (IMAT) mass, which is associated with meat quality in food animals, as well as obesity and related metabolic complications in human. Thus, this study aimed to characterize genes critical for intramuscular preadipocyte differentiation. Primary intramuscular preadipocytes were isolated from pigs, and mRNA profiles were performed at several key points (0 h, 4 h, 8 h, 1 day, 2 days, and 6 days) during adipogenesis using microarrays. By gene functional analysis, we identified numerous differentially expressed genes among distinct stages of intramuscular preadipocyte differentiation, which included numbers of transcription factors in the early stages. We obtained 4 clusters of differential gene expression pattern, including crucial candidate genes associated with adipogenesis of intramuscular adipocytes. Further, we demonstrated that POSTN and FGFR4 suppressed, whereas AKR1CL1 promoted, the expression of adipogenic marker PPARγ and C/EBPα. Taken together, our data delineated the transcriptome landscape during porcine intramuscular preadipocyte differentiation, which provided a valuable resource for finding the genes responsible for IMAT formation.