This study aims to identify the circular RNAs (circRNAs) in the liver of whitespotted bamboo shark (Chiloscyllium plagiosum) and to explore the effect of the overexpression of circRNAs on the proliferation and migration of hepatocellular carcinoma HepG2 cells. We conducted high-throughput sequencing for prediction of the circRNAs and then designed forward and reverse primers to verify them. Further, we constructed overexpression vectors for transient transfection of circRNAs into HepG2 cells. Finally, we employed CCK-8 assay and scratch assay to measure the proliferation and migration of the treated HepG2 cells. A total of 4 558 circRNAs were predicted, among which 14 circRNAs were confirmed. The qRT-PCR showed that circRNA 13-566, circRNA 4-475, circRNA 5-402, circRNA 294-177, and circRNA 30-219 were transiently overexpressed in HepG2 cells. The overexpression of these five circRNAs inhibited the proliferation and migration of HepG2 cells to varying degrees, and circRNA 4-475 and circRNA 294-177 had especially notable effect. This study provided a basic database of circRNA genes that particularly active in whitespotted bamboo shark liver and demonstrated with functional studies of these circRNAs potentially involved in normal and malignant liver cells.
Animals ; High-Throughput Nucleotide Sequencing ; Liver ; RNA, Circular/genetics* ; Sharks/genetics* ; Sincalide/genetics*

The potential effects of recombinant shark hepatical stimulator analogue (r-sHSA) in liver disease have been revealed in our previous studies. In order to further evaluate its clinic application, we carried out high cell-density fermentation in 5 L fermentor to get enough products. Based on the trials in shaking flask, we optimized the parameters for 5 L fermentor, including medium composition, medium supplement, inducer concentration and induction time, etc. In detail, the improved LB medium (0.97% glycerol, 0.91% yeast extract, 0.72% tryptone, 0.782% KH2PO4, 0.267% K2HPO4.3H2O, 0.062% MgSO4.7H2O, 0.5% NaCl, pH 7.0) is chosen to cultivate the engineering bacteria with the constant fermentation condition (pH 7.0, and the dissolved oxygen concentration is about 25%-30%). When bacterial culture reaches exponential phase, the modified feeding medium (620 g/L glycerol, 94.8 g/L tryptone, 3.3 mL/L trace elements, and 7.5 g/L MgSO4.7H2O) is then supplied through the method of exponential fed-batch mode. After the optical density (OD600) of engineering bacterial culture reaches to 23, the ultimately concentration of 0.5 mmol/L IPTG is added to induce the expression of r-sHSA for 6 h. Results show that the amount of r-sHSA production is (2.662 +/- 0.041) g/L, which is about 13.7 folds of the one optimized before.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Liver ; chemistry ; Peptides ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Sharks ; metabolism

AIMTo investigate the protective effects of shark hepatic stimulator substance (sHSS) against acute hepatic injury induced by acetaminophen (AAP) in mice.

METHODSAcute hepatic injury model of Balb/c mice was induced by a single intraperitoneal injection of AAP (200 mg.kg-1, i.p.). Serum ALT and AST activities were analyzed. The changes of microstructure and ultrastructure of hepatocyte were observed under optical and electronic microscope. The hepatocyte apoptosis was analyzed by flow cytometer and the expression level of Fas mRNA was determined by RT-PCR.

RESULTSThe activities of serum ALT and AST were significantly decreased and both necrosis and inflammatory infiltration were improved in the mice treated with sHSS 3.0 and 1.5 mg.kg-1. sHSS (3.0 mg.kg-1) prevented the ultrastructural changes of hepatocytes caused by AAP, decreased the percentage of apoptotic cells, and downregulated the expression level of Fas mRNA.

CONCLUSIONsHSS protected hepatocytes from AAP-induced injury, which might be associated with its protection of the mitochondria and inhibition of apoptosis and expression of Fas mRNA in hepatocytes.

Acetaminophen ; Animals ; Apoptosis ; drug effects ; Chemical and Drug Induced Liver Injury ; etiology ; pathology ; Female ; Growth Substances ; isolation & purification ; pharmacology ; Mice ; Mice, Inbred BALB C ; Peptides ; isolation & purification ; pharmacology ; Protective Agents ; pharmacology ; RNA, Messenger ; genetics ; Random Allocation ; Sharks ; fas Receptor ; biosynthesis ; genetics