MiRNA-Seq reveals key MicroRNAs involved in fat metabolism of sheep liver.

There is a genetic difference between Hu sheep (short/fat-tailed sheep) and Tibetan sheep (short/thin-tailed sheep) in tail type, because of fat metabolism. Previous studies have mainly focused directly on sheep tail fat, which is not the main organ of fat metabolism. The function of miRNAs in sheep liver fat metabolism has not been thoroughly elucidated. In this study, miRNA-Seq was used to identify miRNAs in the liver tissue of three Hu sheep (short/fat-tailed sheep) and three Tibetan sheep (short/thin-tailed sheep) to characterize the differences in fat metabolism of sheep. In our study, Hu sheep was in a control group, we identified 11 differentially expressed miRNAs (DE miRNAs), including six up-regulated miRNAs and five down-regulated miRNAs. Miranda and RNAhybrid were used to predict the target genes of DE miRNAs, obtaining 3,404 target genes. A total of 115 and 67 GO terms as well as 54 and 5 KEGG pathways were significantly (padj < 0.05) enriched for predicted 3,109 target genes of up-regulated and 295 target genes of down-regulated miRNAs, respectively. oar-miR-432 was one of the most up-regulated miRNAs between Hu sheep and Tibetan sheep. And SIRT1 is one of the potential target genes of oar-miR-432. Furthermore, functional validation using the dual-luciferase reporter assay indicated that the up-regulated miRNA; oar-miR-432 potentially targeted sirtuin 1 (SIRT1) expression. Then, the oar-miR-432 mimic transfected into preadipocytes resulted in inhibited expression of SIRT1. This is the first time reported that the expression of SIRT1 gene was regulated by oar-miR-432 in fat metabolism of sheep liver. These results could provide a meaningful theoretical basis for studying the fat metabolism of sheep.

Calycosin-7-glucoside promotes mitochondria-mediated apoptosis in hepatocellular carcinoma by targeting thioredoxin 1 to regulate oxidative stress.

Thioredoxin1 (TRX1) is a key protein that regulates redox and is considered to be a key target for cancer therapy. Flavonoids have been proven to have good antioxidant and anticancer activities. This study aimed to investigate whether the flavonoid calycosin-7-glucoside (CG) exerts an anti-hepatocellular carcinoma (HCC) role by targeting TRX1. Different doses of CG were used to treat HCC cell lines Huh-7 and HepG2 to calculate the IC50. On this basis, the effects of low, medium and high doses of CG on cell viability, apoptosis, oxidative stress and TRX1 expression of HCC cells were investigated in vitro. Also, HepG2 xenograft mice were used to evaluate the role of CG on HCC growth in vivo. The binding mode of CG and TRX1 was explored by molecular docking. Then si-TRX1 was used to further discover the effects of TRX1 on CG inhibition of HCC. Results found that CG dose-dependent decreased the proliferation activity of Huh-7 and HepG2 cells, induced apoptosis, significantly activated oxidative stress and inhibited TRX1 expression. In vivo experiments also showed that CG dose-dependent regulated oxidative stress and TRX1 expression, and promoted the expression of apoptotic proteins to inhibit HCC growth. Molecular docking confirmed that CG had a good binding effect with TRX1. Intervention with TRX1 significantly inhibited the proliferation of HCC cells, promoted apoptosis, and further promoted the effect of CG on the activity of HCC cells. In addition, CG significantly increased ROS production, reduced mitochondrial membrane potential, regulated the expression of Bax, Bcl-2 and cleaved-caspase-3, and activated mitochondria-mediated apoptosis. And si-TRX1 enhanced the effects of CG on mitochondrial function and apoptosis of HCC, suggesting that TRX1 participated in the inhibitory effect of CG on mitochondria-mediated apoptosis of HCC. In conclusion, CG exerts anti-HCC activity by targeting TRX1 to regulate oxidative stress and promote mitochondria-mediated apoptosis.

Transcriptome Comparison Reveals the Difference in Liver Fat Metabolism between Different Sheep Breeds.

Hu sheep and Tibetan sheep are two commonly raised local sheep breeds in China, and they have different morphological characteristics, such as tail type and adaptability to extreme environments. A fat tail in sheep is the main adipose depot in sheep, whereas the liver is an important organ for fat metabolism, with the uptake, esterification, oxidation, and secretion of fatty acids (FAs). Meanwhile, adaptations to high-altitude and arid environments also affect liver metabolism. Therefore, in this study, RNA-sequencing (RNA-seq) technology was used to characterize the difference in liver fat metabolism between Hu sheep and Tibetan sheep. We identified 1179 differentially expressed genes (DEGs) (Q-value < 0.05) between the two sheep breeds, including 25 fat-metabolism-related genes. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, 16 pathways were significantly enriched (Q-value < 0.05), such as the proteasome, glutamatergic synapse, and oxidative phosphorylation pathways. In particular, one of these pathways was enriched to be associated with fat metabolism, namely the thermogenesis pathway, to which fat-metabolism-related genes such as ACSL1, ACSL4, ACSL5, CPT1A, CPT1C, SLC25A20, and FGF21 were enriched. Then, the expression levels of ACSL1, CPT1A, and FGF21 were verified in mRNA and protein levels via qRT-PCR and Western blot analysis between the two sheep breeds. The results showed that the mRNA and protein expression levels of these three genes were higher in the livers of Tibetan sheep than those of Hu sheep. The above genes are mainly related to FAs oxidation, involved in regulating the oxidation of liver FAs. So, this study suggested that Tibetan sheep liver has a greater FAs oxidation level than Hu sheep liver. In addition, the significant enrichment of fat-metabolism-related genes in the thermogenesis pathway appears to be related to plateau-adaptive thermogenesis in Tibetan sheep, which may indicate that liver- and fat-metabolism-related genes have an impact on adaptive thermogenesis.

Derivate isocorydine inhibits cell proliferation in hepatocellular carcinoma cell lines by inducing G2/M cell cycle arrest and apoptosis.

We have previously demonstrated that isocorydine (ICD) can be served as a potential antitumor agent in hepatocellular carcinoma (HCC). A novel derivate of isocorydine (d-ICD) could significantly improve its anticancer activity in tumors. However, the molecular mechanisms of d-ICD on HCC cells remain to be unclear. In this study, we observed that d-ICD inhibited cell proliferation and induced apoptosis of HCC cells in a concentration-dependent manner. We found d-ICD induced G2/M cycle arrest of HCC cells via DNA damage 45 alpha (GADD45A) and p21 pathway in vitro and in vivo. In d-ICD-treated cells, cell cycle-related proteins cyclin B1 and p-CDC2 were upregulated and p-cyclin B1, CDC2, and E2F1 were inhibited. p21 expression can be reversed by knockdown of GADD45A in d-ICD-treated HCC cells. Enforced expression of CCAAT/enhancer-binding protein ß (C/EBPß) in combination with d-ICD enhanced the p21 expression in HCC cells. Furthermore, the luciferase reporter assay showed that upregulation of GADD45A by C/EBPß was achieved through the increase of GADD45A promoter activity. These findings indicate that d-ICD inhibits cell proliferation and induces cell cycle arrest through activation of C/EBPß-GADD45A-p21 pathway in HCC cells. d-ICD might be a promising chemotherapeutic agent for the treatment of HCC.

Isocorydine targets the drug-resistant cellular side population through PDCD4-related apoptosis in hepatocellular carcinoma.

Isocorydine (ICD), an anticancer agent under current evaluation, decreased the percentage of side population (SP) cells significantly in hepatocellular carcinoma (HCC) cell lines. ICD treatment sensitized cancer cells to doxorubicin (DXR), a conventional clinical chemotherapeutic drug for HCC. We found that ICD decreased the percentage of SP cells in HCC cell lines by preferentially killing SP cells. In the early stage of treatment, ICD inhibited SP cell growth by arresting cells in G2/M; later, it induced apoptosis. Our xenograft model confirmed that ICD selectively reduced the size and weight of SP-induced tumor masses in vivo. Furthermore, it was found that programmed cell death 4 (PDCD4), a tumor suppressor gene, was relatively low when expressed in SP cells compared with non-SP cells, and its expression level was remarkably elevated when cells were treated with ICD. Taken together, these data suggest that ICD is a drug that may target the SP cells of HCC.

Isocorydine inhibits cell proliferation in hepatocellular carcinoma cell lines by inducing G2/m cell cycle arrest and apoptosis.

The treatment of human hepatocellular carcinoma (HCC) cell lines with (+)-isocorydine, which was isolated and purified from Papaveraceae sp. plants, resulted in a growth inhibitory effect caused by the induction of G2/M phase cell cycle arrest and apoptosis. We report that isocorydine induces G2/M phase arrest by increasing cyclin B1 and p-CDK1 expression levels, which was caused by decreasing the expression and inhibiting the activation of Cdc25C. The phosphorylation levels of Chk1 and Chk2 were increased after ICD treatment. Furthermore, G2/M arrest induced by ICD can be disrupted by Chk1 siRNA but not by Chk2 siRNA. In addition, isocorydine treatment led to a decrease in the percentage of CD133(+) PLC/PRF/5 cells. Interestingly, isocorydine treatment dramatically decreased the tumorigenicity of SMMC-7721 and Huh7 cells. These findings indicate that isocorydine might be a potential therapeutic drug for the chemotherapeutic treatment of HCC.