Bioengineered Artificial Extracellular Vesicles Presenting PD-L1 and Gal-9 Ameliorate New-onset Type 1 Diabetes.

An important factor in the development of Type 1 diabetes (T1D) is the deficiency of inhibitory immune checkpoint ligands, specifically programmed cell death ligand 1 (PD-L1) and Galectin-9 (Gal-9), in ß-cells. Hence, modulation of the pancreas infiltrated T lymphocytes by exogenous PD-L1 or Gal-9 is an ideal approach for treating the new-onset T1D. Herein, we genetic engineered the macrophage cells to generate artificial extracellular vesicles (aEVs) overexpressing PD-L1 and Gal-9, which could restrict the islets autoreactive T lymphocytes and protect ß-cells from destruction. Intriguingly, overexpressing Gal-9 spurred macrophage polarization to M2 phenotype with immune suppressive attribute. Alternatively, both of PD-L1 and Gal-9 presenting aEVs (PD-L1-Gal-9 aEVs) favorably adhere to T cells via the interaction of programmed cell death protein 1 (PD-1)/PD-L1 or T cell immunoglobulin mucin 3 (TIM-3)/Gal-9. Moreover, PD-L1-Gal-9 aEVs prominently promoted effector T cell apoptosis and splenic regulatory T cells (Treg) cells differentiation in vitro. Virtually, PD-L1-Gal-9 aEVs efficaciously reversed the new-onset hyperglycemia in the NOD mice, prevented T1D progress, and declined the proportion and activation of CD4+ and CD8+ T cells infiltrating the pancreas notably, which together contributed to preserving the residual ß-cells survival and mitigating the hyperglycemia.

rhCNB Improves Cyclophosphamide-Induced Immunodeficiency in BALB/c Mice.

Background: This study aims to explore the immunomodulatory effect of rhCNB on mice with cyclophosphamide (CTX)-induced immunodeficiency through TLR4/MAPK pathway. Methods: BALB/c mice were randomly divided into three groups: a negative control group, an immunosuppression model group, and a rhCNB treatment group. Tail vein injection of cyclophosphamide (40 mg/kg) was used to establish a mouse immunosuppression model. Intraperitoneal injection of rhCNB (20 mg/kg) was administered to the treatment group, whereas equal quantities of normal saline were given to the control group and model group. Perform peripheral blood routine of CD4, CD8, and CD19 lymphocyte subsets and peripheral blood Th1/Th2 cell subsets 24 hours after the last administration. RT-PCR was used to detect mRNA levels of TLR4, P38, JNK, T-bet, and GATA3, the spleen immune organ index was measured, and the histopathological status of the spleen and thymus was observed. Results: The results showed that compared with the control group, WBC, PLT, LYM, NEU, immune organ index, CD4+/CD8+ and CD19+ subgroup ratio, and peripheral blood Th1/Th2 cell subgroups decreased in the model group. The mRNA levels of TLR4, P38, JNK, T-bet, and GATA3 decreased compared with the model group, while they increased in the treatment group. Conclusions: rhCNB has an immunomodulatory effect by regulating the expression of Th1/Th2 cytokine balance through the TLR4/MAPK signaling pathway and promoting the differentiation and proliferation of lymphocytes, thereby improving the immune function.

CircNDST1 Regulates Bovine Myoblasts Proliferation and Differentiation via the miR-411a/Smad4 Axis.

Circular RNA (circRNA) is endogenous noncoding RNA found throughout the eukaryotic genome. It regulates several biological activities at the transcription or post-transcription level. However, the underlying function of circRNA in bovine skeletal muscle development remains unknown. Here, we identified a novel circRNA, circNDST1, and investigated its function and mechanism on the proliferation and differentiation of bovine myoblasts. At the molecular and cellular levels, circNDST1 could promote bovine myoblasts proliferation and inhibit differentiation. Mechanistically, circNDST1 is expressed in the cytoplasmic of myoblast and was enriched by protein Ago2. circNDST1 acts as a competing endogenous RNA that sponges miR-411a and alleviates the inhibitory effect on its target gene, Smad4. miR-411a and Smad4 were also involved in regulating bovine myoblast proliferation and differentiation. These findings suggest that circNDST1 functions as a competing endogenous RNA and regulates bovine myoblast proliferation and differentiation through the miR-411a/Smad4 axis.

A T-cell-dependent antibody response (TDAR) method in BALB/c mice based on a cytometric bead array.

Most current methods to assess T-cell-dependent antibody responses (TDAR) are semi-quantitative and based on measures of antibody titer generated against a standard antigen like keyhole limpet hemocyanin (KLH). The precision, sensitivity, and convenience of TDAR assays might be improved by applying rapid, sensitive, specific cytometric bead assays (CBA). In the study here, KLH antigen was covalently coupled onto the surface of cytometric beads using immune microsphere technology, and IgM antibody capture spheres were prepared for use in pretreatment processing of samples. The working parameters associated with this novel TDAR-CBA system were optimized in orthogonal experiments. The optimal concentration of the KLH coating solution in this system was 160 µg/ml, that of the anti-KLH IgG capture spheres 6.0 × 105/ml, and the optimal dilution of fluorescein isothiocyanate (FITC)-conjugated Affini-Pure Goat Anti-Mouse IgG (H + L) was 60 µg/ml. Repeated tests indicated that this approach yielded good linearity (r2 = 0.9937) method, with a within-run precision of 3.1-4.9%, and a between-run precision of 4.4-4.9%. This new approach had a limit of detection of 113.43 ng/ml (linear range = 390.63-50 000), and an interference rate of just 0.04-3.51%. Based on these findings, it seems that a new mouse TDAR assay based on CBA can be developed that would appear to be more sensitive, accurate, and precise than the current TDAR assay approaches based on traditional ELISA.

PML Body Component Sp100A Restricts Wild-Type Herpes Simplex Virus 1 Infection.

Sp100 (speckled protein 100 kDa) is a constituent component of nuclear structure PML (promyelocytic leukemia) bodies, playing important roles in mediating intrinsic and innate immunity. The Sp100 gene encodes four isoforms with distinct roles in the transcriptional regulation of both cellular and viral genes. Since Sp100 is a primary intranuclear target of infected-cell protein 0 (ICP0), an immediate early E3 ligase encoded by herpes simplex virus 1 (HSV-1), previous investigations attempting to analyze the functions of individual Sp100 variants during HSV-1 infection mostly avoided using a wild-type virus. Therefore, the role of Sp100 under natural infection by HSV-1 remains to be clarified. Here, we reappraised the antiviral capacity of four Sp100 isoforms during infection by a nonmutated HSV-1, examined the molecular behavior of the Sp100 protein in detail, and revealed the following intriguing observations. First, Sp100 isoform A (Sp100A) inhibited wild-type HSV-1 propagation in HEp-2, Sp100-/-, and PML-/- cells. Second, endogenous Sp100 is located in both the nucleus and the cytoplasm. During HSV-1 infection, the nuclear Sp100 level decreased drastically upon the detection of ICP0 in the same subcellular compartment, but cytosolic Sp100 remained stable. Third, transfected Sp100A showed subcellular localizations similar to those of endogenous Sp100 and matched the protein size of endogenous cytosolic Sp100. Fourth, HSV-1 infection induced increased secretion of endogenous Sp100 and ectopically expressed Sp100A, which copurified with extracellular vesicles (EVs) but not infectious virions. Fifth, the Sp100A level in secreting cells positively correlated with its level in EVs, and EV-associated Sp100A restricted HSV-1 in recipient cells. IMPORTANCE Previous studies show that the PML body component Sp100 protein is immediately targeted by ICP0 of HSV-1 in the nucleus during productive infection. Therefore, extensive studies investigating the interplay of Sp100 isoforms with HSV-1 were conducted using a mutant virus lacking ICP0 or in the absence of infection. The role of Sp100 variants during natural HSV-1 infection remains blurry. Here, we report that Sp100A potently and independently inhibited wild-type HSV-1 and that during HSV-1 infection, cytosolic Sp100 remained stable and was increasingly secreted into the extracellular space, in association with EVs. Furthermore, the Sp100A level in secreting cells positively correlated with its level in EVs and the anti-HSV-1 potency of these EVs in recipient cells. In summary, this study implies an active antiviral role of Sp100A during wild-type HSV-1 infection and reveals a novel mechanism of Sp100A to restrict HSV-1 through extracellular communications.

miR-152 Regulates Bovine Myoblast Proliferation by Targeting KLF6.

Though miRNAs have been reported to regulate bovine myoblast proliferation, but many miRNAs still need to be further explored. Specifically, miR-152 is a highly expressed miRNA in cattle skeletal muscle tissues, but its function in skeletal muscle development is unknown. Herein, we aimed to investigate the role of miR-152 in regulating bovine myoblast proliferation. Functionally, RT-qPCR, Western blotting, EdU assay, and flow cytometry detection results showed that miR-152 inhibited bovine myoblast proliferation. Mechanistically, we demonstrated transcription factor KLF6 was a target gene of miR-152 by means of bioinformatics software prediction and dual-luciferase report analysis, which had been demonstrated to be favorable for myoblast proliferation. Collectively, our research suggested that miR-152 inhibits bovine myoblast proliferation via targeting KLF6.

MEIS1 and its potential as a cancer therapeutic target (Review).

Meis homeobox 1 (Meis1) was initially discovered in 1995 as a factor involved in leukemia in an animal model. Subsequently, 2 years later, MEIS1, the human homolog, was cloned in the liver and cerebellum, and was found to be highly expressed in myeloid leukemia cells. The MEIS1 gene, located on chromosome 2p14, encodes a 390­amino acid protein with six domains. The expression of homeobox protein MEIS1 is affected by cell type, age and environmental conditions, as well as the pathological state. Certain types of modifications of MEIS1 and its protein interaction with homeobox or pre­B­cell leukemia homeobox proteins have been described. As a transcription factor, MEIS1 protein is involved in cell proliferation in leukemia and some solid tumors. The present review article discusses the molecular biology, modifications, protein­protein interactions, as well as the role of MEIS1 in cell proliferation of cancer cells and MEIS1 inhibitors. It is suggested by the available literature MEIS1 has potential to become a cancer therapeutic target.

CircRILPL1 promotes muscle proliferation and differentiation via binding miR-145 to activate IGF1R/PI3K/AKT pathway.

Many novel non-coding RNAs, such as microRNAs (miRNAs) and circular RNAs (circRNAs), are involved in various physiological and pathological processes. The PI3K/AKT signaling pathway is important for its role in regulating skeletal muscle development. In this study, molecular and biochemical assays were used to confirm the role of miRNA-145 (miR-145) in myoblast proliferation and apoptosis. Based on sequencing data and bioinformatics analysis, we identified a new circRILPL1, which acts as a sponge for miR-145. The interactions between circRILPL1 and miR-145 were examined by bioinformatics, a luciferase assay, and RNA immunoprecipitation. Mechanistically, knockdown or exogenous expression of circRILPL1 in the primary myoblasts was performed to prove the functional significance of circRILPL1. We investigated the inhibitory effect of miR-145 on myoblast proliferation by targeting IGF1R to regulate the PI3K/AKT signaling pathway. A novel circRILPL1 was identified that could sponge miR-145 and is related to AKT activation. In addition, circRILPL1 was positively correlated with muscle proliferation and differentiation in vitro and could inhibit cell apoptosis. The newly identified circRILPL1 functions as a miR-145 sponge to regulate the IGF1R gene and rescue the inhibitory effect of miR-145 on the PI3K/AKT signaling pathway, thereby promoting myoblast growth.

Compound TDB (Tricyclic decyl benzoxazole) induces autophagy-dependent apoptosis in the gastric cancer cell line MGC-803 by regulating PI3K/AKT/mTOR.

OBJECTIVE: Gastric cancer is a potential malignant tumor. Extensive research has shown that apoptosis and autophagy are important mechanisms of cancer pathogenesis. This study aimed to explore the role and mechanism of TDB in apoptosis and autophagy in MGC-803 cells. METHODS: In cell experiments, the proliferation, apoptosis and autophagy of MGC-803 cells were evaluated by the MTT assay, TUNEL, flow cytometry, MDC, and TEM. Through molecular experiments, the TDB-induced apoptosis and autophagy effects were evaluated by examining the levels of Cleaved-PARP/PARP, Cleaved-caspase3/procaspase3, Beclin-1, p62 and the ratio of LC3-II/LC3-I. At the animal level, the anti-tumor effect of TDB in vivo was evaluated by assessing tumor volume and bioluminescence value. RESULTS: Regarding mechanism, TDB induces apoptosis and autophagy through PI3K/AKT/mTOR. At the same time, more importantly, TDB promotes 3-methyladenine or autophagy activator rapamycin-mediated. The induced proliferation inhibition and pro-apoptosis effect, which inhibit autophagy and induce an increase in apoptosis. CONCLUSION: TDB may up-regulate PARP, Cleaved Caspase-3, Beclin1 and LC3B and down-regulate the expression of P62 and other apoptosis and autophagy genes through the activation of PI3K/AKT/mTOR pathway signalling proteins, leading to autophagy-dependent apoptosis. At the animal level, TDB has good anti-tumor efficacy in vivo. In summary, TDB has potential anti-tumor efficacy in vivo and in vitro.

Novel copy number variation of the BAG4 gene is associated with growth traits in three Chinese sheep populations.

Copy number variation (CNV) as an important source of genetic phenotypic and variation is related to complex phenotypic traits. The aim of this study was to investigate the potential associations of BAG4 (Bcl-2-associated athanogene 4) copy numbers variations with sheep growth traits in three Chinese sheep breeds (CKS, STHS, and HS). BAG4 is located within the stature and udder attachment quantitative trait loci (QTL) in sheep. Expression profiling revealed that the BAG4 gene was widely expressed in the tissues of sheep. The distribution of BAG4 gene copy number showed that the loss of copy number was more dominant in CKS and HS which was different from that in STHS. Statistical analysis revealed that the BAG4 CNV was significantly associated with body height in CKS (p < 0.05), with body slanting length in HS (p < 0.05), and with body height and hip cross height in STHS (p < 0.05). The χ2 values showed significant differences in the BAG4 CNV distribution frequency between varieties. In conclusion, the results establish the association between BAG4 CNV and sheep traits and suggest that BAG4 CNV may be a promising marker for the molecular breeding of Chinese sheep.

Feng-Liao-Chang-Wei-Kang Combined with 5-Fluorouracil Synergistically Suppresses Colitis-Associated Colorectal Cancer via the IL-6/STAT3 Signalling Pathway.

BACKGROUND: Colitis-associated colorectal cancer (CAC) develops from active colonic inflammation, which is characterized by the production of proinflammatory cytokines that can induce mutations. IL-6 is produced by multiple cell types located within the tumor microenvironment including tumor-infiltrating immune cells, stromal cells, and the tumor cells themselves. The aim of our study was to explore the mechanism of Feng-Liao-Chang-Wei-Kang (FLCWK) and 5-fluorouracil (5-FU) in treating CAC. METHOD: HCT116 cells were treated with 5-FU in the absence or presence of FLCWK. Cell proliferation was assayed by MTT assays. Apoptosis and the cell cycle phases were detected by flow cytometry. Western blotting and Q-PCR assays were used to detect the expression levels of proteins and genes related to the IL-6/STAT3 signalling pathway. A mouse model for CAC was established by treating animals with 12.5 mg/kg azoxymethane (AOM) followed by 3 cycles of 2.5% dextran sodium sulphate (DSS). The associated pathological changes were determined after haematoxylin and eosin (H&E) staining. The expression of related proteins and genes in various tissues was examined using immunofluorescence techniques. RESULTS: FLCWK enhanced the ability of 5-FU to promote apoptosis by inhibiting the proliferation of HCT116 cells and blocking the IL-6/STAT3 pathway. FLCWK combined with 5-FU reduced the number and size of colon tumors in mice with CAC and significantly increased their survival rate. In the CAC model, FLCWK synergized with 5-FU to inhibit the phosphorylation of STAT3, preventing IL-6/STAT3 signal transduction and thus further inducing apoptosis and inhibition of colon cancer cell proliferation. CONCLUSION: FLCWK can inhibit the activation of STAT3 by reducing the production of IL-6, thereby increasing the occurrence of colitis-related colorectal cancer with 5-FU.

lncRNA IGF2 AS Regulates Bovine Myogenesis through Different Pathways.

The role of long non-coding RNA (lncRNA) in the regulation of bovine skeletal muscle development remains poorly understood. The present study investigated the function and regulatory mechanism of a novel lncRNA, insulin-like growth factor 2 antisense transcript (IGF2 AS), in bovine myoblast proliferation and differentiation. Gain or loss of IGF2 AS was performed using an expression plasmid or small interfering RNA (siRNA), respectively. Bovine myoblasts were used to investigate the biological function and mechanisms of IGF2 AS in vitro. Results were conjointly analyzed by celluar and molecular biology experiments as well as bioinformatics. Functionally, IGF2 AS could promote proliferation and differentiation of bovine myoblasts. The preliminary mechanism suggests, on the one hand, that IGF2 AS could complement the IGF2 gene intron region and affect the stability and expression of IGF2 mRNA. On the other hand, RNA pull-down and immunoprecipitation assays demonstrated that IGF2 AS could directly bind to the interleukin enhancer binding factor 3 (ILF3) protein and maybe partly though it to regulate myogenesis. In conclusion, the novel identified lncRNA IGF2 AS promoted proliferation and differentiation of bovine myoblasts through various pathways.

circRNA Profiling Reveals an Abundant circFUT10 that Promotes Adipocyte Proliferation and Inhibits Adipocyte Differentiation via Sponging let-7.

Adipose development is regulated by a series of complex processes, and non-coding RNAs (ncRNAs), including circular RNAs (circRNAs), play important roles in regulating proliferation and differentiation of adipocytes. In this study, we profiled circRNA expression in cattle fat tissue during calf and adult developmental stages and detected 14,274 circRNA candidates. Some circRNAs are differentially expressed between two developmental stages. We characterized circFUT10, named for its host gene FUT10, a highly expressed and abundant circRNA. Luciferase screening, an RNA-binding protein immunoprecipitation (RIP) assay, quantitative real-time PCR, and western blotting assays indicated that circFUT10 directly binds let-7c/let-e, and PPARGC1B (peroxisome proliferator-activated receptor γ coactivator 1-ß) is identified as a target of let-7c. Flow cytometry, EdU (5-ethynyl-2'-deoxyuridine) incorporation, a CCK-8 (cell counting kit-8) assay, oil red O staining, and western blotting assays demonstrated that circFUT10 promotes adipocyte proliferation and inhibits cell differentiation by sponging let-7c. The results demonstrate that circFUT10 binding of let-7c promotes cell proliferation and inhibits cell differentiation by targeting PPARGC1B in cattle adipocytes.

Micro-Ribonucleic Acid-216a Regulates Bovine Primary Muscle Cells Proliferation and Differentiation via Targeting SMAD Nuclear Interacting Protein-1 and Smad7.

MicroRNAs (miRNAs), belonging to a class of evolutionarily conserved small noncoding RNA of ∼22 nucleotides, are widely involved in skeletal muscle growth and development by regulating gene expression at the post-transcriptional level. While the expression feature and underlying function of miR-216a in mammal skeletal muscle development, especially in cattle, remains to be further elucidated. The aim of this study was to investigate the function and mechanism of miR-216a during bovine primary muscle cells proliferation and differentiation. Herein, we found that the expression level of miR-216a both presented a downward trend during the proliferation and differentiation phases, which suggested that it might have a potential role in the development of bovine skeletal muscle. Functionally, during the cells proliferation phase, overexpression of miR-216a inhibited the expression of proliferation-related genes, reduced the cell proliferation status, and resulted in cells G1 phase arrest. In cells differentiation stages, overexpression of miR-216a suppressed myogenic maker genes mRNA, protein, and myotube formation. Mechanistically, we found that SNIP1 and smad7 were the directly targets of miR-216a in regulating bovine primary muscle cells proliferation and differentiation, respectively. Altogether, these findings suggested that miR-216a functions as a suppressive miRNA in development of bovine primary muscle cells via targeting SNIP1 and smad7.

RNA-Seq Analysis Identifies Differentially Expressed Genes Insubcutaneous Adipose Tissuein Qaidamford Cattle, Cattle-Yak, and Angus Cattle.

In the beef industry, fat tissue is closely related to meat quality. In this study, high-throughput RNA sequencing was utilized for adipose tissue transcriptome analysis between cattle-yak, Qaidamford cattle, and Angus cattle. The screening and identification of differentially expressed genes (DEGs) between different breeds of cattle would facilitate cattle breeding. Compared to Angus cattle adipose tissue, a total of 4167 DEGs were identified in cattle-yak adipose tissue and 3269 DEGs were identified in Qaidamford cattle adipose tissue. Considering cattle-yak as a control group, 154 DEGs were identified in Qaidamford cattle adipose tissue. GO analysis indicatedthe significant enrichment of some DEGs related to lipid metabolism. The KEGG pathway database was also used to map DEGs and revealed that most annotated genes were involved in ECM-receptor interaction and the PI3K-Akt signal pathway, which are closely related to cell metabolism. Eight selected DEGs related to adipose tissue development or metabolism were verified by RT-qPCR, indicating the reliability of the RNA-seq data. The results of this comparative transcriptome analysis of adipose tissue and screening DEGs suggest several candidates for further investigations of meat quality in different cattle breeds.

Circular RNA TTN Acts As a miR-432 Sponge to Facilitate Proliferation and Differentiation of Myoblasts via the IGF2/PI3K/AKT Signaling Pathway.

Circular RNAs (circRNAs) are ubiquitous endogenous RNA found in various organisms that can regulate gene expression in eukaryotes. However, little is known about potential roles for circRNAs in muscle development. We analyzed circRNA sequencing data of bovine skeletal muscle tissue and found differential expression of circTitin (circTTN) in fetal and adult bovine muscle tissue. We then further studied the role of circTTN in bovine muscle development. Overexpression and inhibition of circTTN together elicited its promoting roles in proliferation and differentiation of bovine primary myoblasts. Mechanistically, circTTN showed interaction with miR-432 by luciferase screening and RNA immunoprecipitation (RIP) assays. Additionally, miR-432 is a regulator of insulin-like growth factor 2 (IGF2), as indicated by luciferase activity, quantitative real-time PCR, and western blotting assays. Increased miR-432 expression inhibited the expression of IGF2, but this effect was remitted by circTTN. Conclusively, our results showed that circTTN promoted proliferation and differentiation of bovine primary myoblasts via competitively combining with miR-432 to activate the IGF2/phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway.

Improvement and optimization of a T-cell-dependent antibody response (TDAR) method for BALB/c mice using keyhole limpet hemocyanin (KLH) as specific antigen.

Although T-cell-dependent antibody response (TDAR) assays with keyhole limpet hemocyanin (KLH) as specific antigen have many advantages, most experiments produce qualitative results based on antibody titers. It was hypothesized that if experimental conditions (like antigen coating concentration, serum dilution, and detecting [here, horseradish peroxidase-goat anti-mouse IgG] antibody dilution) could be optimized, the resulting measured value (here, optical density) could be used to directly analyze and evaluate the experimental results. This means specifically that the assay OD values could be used for approximate quantitative statistical analysis; it does not require a further conversion of the data into qualitative forms or require obtaining further titer data from additional experiments. As such, the use of this "improved" assay would: greatly reduce the complexity of experimental operations; improve overall sensitivity and practicality of traditional TDAR assays; and, allow for direct assessing of any immunosuppression caused by a test drug in a host. Here, KLH-immunized serum was obtained from BALB/c mice, and the means to detect serum anti-KLH antibodies by an indirect ELISA were optimized. The results indicated that in this system, the optimal KLH coating concentration was 80 µg/ml, the optimal dilution range of the serum (at immunization dose of 5 mg KLH/kg) was 1:200-1:800, and the optimal dilution of HRP-goat anti-mouse IgG antibody was 1:16,000. Methodology verification was performed and a regression model of y = 144.16x + 0.9815 (R2 = 0.9571, indicating very good linearity) was obtained. Intragroup precision was 7.51-9.40%; the intergroup coefficient of variation was 9.83-14.22%. The lower limit of detection was 0.1385. The present results showed this indirect ELISA exhibited very good linearity, accuracy, and precision.

miR-148a-3p regulates proliferation and apoptosis of bovine muscle cells by targeting KLF6.

Skeletal muscle development is regulated by a series of regulatory factors, and also including noncoding RNA, especially microRNAs (miRNAs). Recently, miR-148a has been found to be involved in murine C2C12 differentiation by targeting ROCK1. However, the function of miR-148a-3p for the proliferation and apoptosis of bovine muscle cells has not been determined. In this study, we found that miR-148a-3p was highly expressed in fetal bovine skeletal muscle and exhibited a decreasing trend in muscle cells during its growth phase. Functional studies indicated that gain of miR-148a-3p inhibited the proliferation of bovine muscle cells and promoted apoptosis. Conversely, interference with miR-148a-3p inhibitor promoted muscle cell proliferation and inhibited its apoptosis. Mechanistically, KLF6 was confirmed as a new potential target gene of miR-148a-3p by TargetScan software prediction and the dual-luciferase assay verification. Additionally, after a gain or loss of KLF6, the function of KLF6 for muscle cell proliferation and apoptosis was opposite to that of miR-148a-3p. Collectively, these findings proposed a novel avenue whereby miR-148a-3p impeded bovine myoblast cell proliferation and promoted apoptosis through the posttranscriptional downregulation of KLF6.

miR-483 inhibits bovine myoblast cell proliferation and differentiation via IGF1/PI3K/AKT signal pathway.

MicroRNAs (miRNAs) have been established to regulate skeletal muscle development in mammals. However, few studies have been conducted on the regulation of proliferation and differentiation of bovine myoblast cells by miRNAs. The aim of our study was to explore the function of miR-483 in cell proliferation and differentiation of bovine myoblast. Here, we found that miR-483 declined in both proliferation and differentiation stages of bovine myoblast cells. During the proliferation phase, the overexpression of miR-483 downregulated the cell cycle-associated genes cyclin-dependent kinase 2 (CDK2), proliferating cell nuclear antigen (PCNA) messenger RNA (mRNA), and the protein levels. At the cellular level, cell cycle, cell counting kit-8, and 5-ethynyl-2´-deoxyuridine results indicated that the overexpression of miR-483 block cell proliferation. During differentiation, the overexpression of miR-483 led to a decrease in the levels of the myogenic marker genes MyoD1 and MyoG mRNA and protein. Furthermore, the immunofluorescence analysis results showed that the number of MyHC-positive myotubes was reduced. In contrast, the opposite experimental results were obtained concerning both proliferation and differentiation after the inhibition of miR-483. Mechanistically, we demonstrated that miR-483 target insulin-like growth factor 1 (IGF1) and downregulated the expression of key proteins in the PI3K/AKT signaling pathway. Altogether, our findings indicate that miR-483 acts as a negative regulator of bovine myoblast cell proliferation and differentiation.

Linc-smad7 promotes myoblast differentiation and muscle regeneration via sponging miR-125b.

Long noncoding RNAs (lncRNAs) are involved in the regulation of skeletal muscle development. In the present study, differentially expressed lncRNAs were identified from RNA-seq data derived from myoblasts and myotubes. We conducted studies to elucidate the function and molecular mechanism of action of Linc-smad7 during skeletal muscle development. Our findings show that Linc-smad7 is upregulated during the early phase of myoblasts differentiation. In in vitro studies, we showed that overexpression of Linc-smad7 promoted the arrest of myoblasts in G1 phase, inhibited DNA replication, and induced myoblast differentiation. Our in vivo studies suggest that Linc-smad7 stimulates skeletal muscle regeneration in cardiotoxin-induced muscle injury. Mechanistically, Linc-smad7 overexpression increased smad7 and IGF2 protein levels. On the contrary, overexpression of miR-125b reduced smad7 and IGF2 protein levels. Results of RNA immunoprecipitation analysis and biotin-labeled miR-125b capture suggest that Linc-smad7 could act as a competing endogenous RNA (ceRNA) for miRNA-125b. Taken together, our findings suggest that the novel noncoding regulator Linc-smad7 regulates skeletal muscle development.