Exosome-Mediated MiR-155 Transfer Contributes to Hepatocellular Carcinoma Cell Proliferation by Targeting PTEN.

BACKGROUND Most eukaryocytes release nano vesicles (30-120 nm), named exosomes, to various biological fluids such as blood, lymph, and milk. Hepatocellular carcinoma (HCC) is one of the tumors with the highest incidence rate in primary malignant carcinoma of the liver. However, the mechanism of HCC proliferation remains elusive. In this study, we aim to explore whether HCC cell-derived exosomes affect the proliferation of cancer cells. MATERIAL AND METHODS Exosomes were isolated from HCC cells by ultracentrifugation and were visualized the phenotype by transmission electron microscopy. Cell proliferation was detected by Cell Counting Kit-8 assays and EdU (5-ethynyl-2-deoxyuridine) incorporation assays. Dual-luciferase assays were performed to validate the paired correlation of miR-155 and 3'-UTR of PTEN (gene of phosphate and tension homology deleted on chromosome 10). A xenograft mice model was constructed to verify the effect of exosome-mediated miR-155 on cell proliferation in vivo. RESULTS Our finding showed that miR-155 was enriched in exosomes released from HCC cells. The exosome-containing miR-155 transferred into new HCC targeted cells and lead to the elevation of HCC cells' proliferation. Besides, the exosomal miR-155 directly bound to 3'-UTR of PTEN leading to the reduction of relevant targets in recipient liver cells. The knockdown of PTEN attenuated the proliferation of HCC cells treated with the exosomal miR-155. Moreover, nude-mouse experiment results revealed a promotional effect of the exosomal miR-155 on HCC cell-acquired xenografts. CONCLUSIONS Our study indicated that exosomal-specific miR-155 transfers to adjacent and/or more distant cells and stimulates the proliferation of HCC cells.

cDNA cloning and expression analysis of glutaredoxin (Grx) 2 in the Pacific white shrimp Litopenaeus vannamei.

Glutaredoxin (Grx) is a class molecule oxidoreductase, which can regulate the redox state of proteins and plays a key role in antioxidant defense. However, the informations of Grx cDNA sequences and their functions are lack in decapod crustacea. In the present study, the cDNA of LvGrx 2 was cloned from the Pacific white shrimp, Litopenaeus vannamei. The open reading frame (ORF) of LvGrx 2 was 360 bp, which encoded a polypeptide of 119 amino acids. The molecular mass of the predicted protein is 12.87 kDa with an estimated pI of 8.22. Sequence alignment showed that the amino acid sequence of LvGrx 2 shares 59%, 59% and 58% identity with that of the coelacanth Latimeria chalumnae, the plateau frog Nanorana parkeri and the half-smooth tongue sole Cynoglossus semilaevis, respectively. Quantitative real-time PCR analysis revealed that LvGrx 2 were detected in a wide range of tissues, with highest expression in gill, hepatopancrea and intestine, and weakest expression in muscle. The expression responses of LvGrx 2 were analyzed in hepatopancrea and gill after ammonia-N stress or lipopolysaccharide (LPS) injection. During ammonia-N exposure, the LvGrx 2 transcriptions in hepatopancrea and gill significantly up-regulated, and the peak value appeared after 12 h and 24 h exposure respectively. After LPS injection, expression levels of LvGrx 2 in hepatopancrea obviously increased in the early and late stages, while LvGrx 2 transcription in gill sharply up-regulated in the middle period. These results suggest that LvGrx 2 may play a vital role in shrimp defense system against environmental stress and pathogen infection. RNA interference experiment was designed to further probe roles of LvGrx 2 during ammonia-N exposure. Ammonia-N induced obvious improvement in expression levels of LvGrx 2, LvGrx 3, GPx, GST and Trx, accompanied by increases of protein carbonyl and malondialdehyde (MDA) contents. However, transcription of GPx and GST were much weaker in LvGrx 2 interfered-shrimp, and oxidative damage in both lipid and protein were more serious. These results further suggest that LvGrx 2 in shrimp participates in oxidative defence and regulation of antioxidant system.

Flow cytometic analysis of Penaeus monodon haemocyte responses to poly I:C.

This study was aimed at investigating the cellular responses of Penaeus monodon haemocytes to poly I:C stimulation using flow cytometric assay. Total haemocyte count (THC), percentages of different haemocyte subpopulations [hyaline cells (HC), semigranular cells (SGC) and granular cells (GC)], non-specific esterase activity (EA), total reactive oxygen species/reactive nitrogen species (ROS/RNS) production, nitric oxide (NO) production, apoptotic haemocyte ratio and plasmic phenoloxidase (PO) activity were determined in poly I:C-injected shrimp. Results showed that poly I:C at a low dose (5 µg shrimp-1) caused obvious increases in THC, GC proportion, ROS/RNS production and NO production, but had no significant effect on EA, apoptosis and PO activity. In the early stage of poly I:C injection at a high dose (20 µg shrimp-1), THC and GC proportion improvements could also be observed, suggesting that GC might be induced to release from hemocytopoietic or other tissues to participate in immune response, and this subpopulation might be the main cell type involved in the cellular defence against virus. In the later period, proportions of both GC and SGC reduced paralleled by THC reduction, indicating that depletion of GC and SGC was mainly contributed to the reduced count of circulating haemocyte. Obvious increases in ROS/RNS production and NO production were induced in haemocyte of shrimp under a high dose of poly I:C stimulation, but only slight rise of EA and suppression of PO activity could be observed in poly I:C-stimulated shrimp, suggesting that ROS/RNS-dependent system was vital in the immune defence of shrimp against virus. On the other hand, increase of apoptotic haemocyte ratio and THC reduction were presented after the drastic increases of ROS/RNS and NO productions, implying that the stimulated ROS/RNS might be excess and harmful, and was the major factor for the haemocyte apoptosis and depletion. THC recovered after 48 h injection, while haemocyte apoptosis also returned to the control level, suggesting that apoptosis might be contributed to eliminate damaged, weak or infected haemocytes to renew the circulating haemocytes, and it could be considered as an important defending strategy against virus.

First report of Rahnella aquatilis infection in crucian carp Carassius auratus in China.

Rahnella aquatilis infection is rare in aquaculture. Here, a Gram-negative rod-shaped bacterium was isolated from diseased crucian carp Carassius auratus in Xuzhou City, Jiangsu Province, eastern China. The isolate was tentatively named strain KCL-5, and subsequently identified as R. aquatilis by biochemical properties and molecular techniques. The results showed that the isolate KCL-5 was most closely related to the type strain ATCC33071 (= DSM4594) of R. aquatilis, which shared 99.67, 96.26 and 99.58% nucleotide sequence identities for 16S rDNA, gyrB and toxin yhaV genes, respectively. Experimental challenges were conducted which demonstrated pathogenicity of the isolate in crucian carp. Antimicrobial susceptibility testing showed that the isolated strain was susceptible to piperacillin, gentamicin, kanamycin, nalidixic acid, norfloxacin, ofloxacin, azithromycin and erythromycin. To our knowledge, this is the first report on R. aquatilis infection in crucian carp, and the first evidence of pathogenicity in fish.

Multi-target lentivirus specific to hepatocellular carcinoma: in vitro and in vivo studies.

BACKGROUND & AIMS: We aimed at investigating the effects of the targeted transduction of the Wtp53-pPRIME-miR30-shRNA gene into liver cancer cells, under the mediation of anti-alpha fetoprotein scFv-directed lentivirus, and the inhibitory effect of this system on liver cancer cells. METHODS: The result of infection was observed by fluorescence microscopy. Polymerase chain reaction and Western blotting were used to demonstrate the successful transduction and transcription of the Wtp53-pPRIME-miR30-shRNA-IGF1R gene. Cell growth was observed via the Cell-Counting Kit-8 Method, and cell apoptosis was detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling. To observe further the effects of AFP-Wtp53-pPRIME-miR30-shRNA-IGF1R therapy in animals, models of BALB-C nude mice bearing subcutaneous human hepatocellular carcinoma were established. The influence of the growth of subcutaneously transplanted tumor, expression of Wtp53 protein, apoptosis, and microvessel formation on the overall level of AFP-Wtp53 pPRIME-miR30-shRNA-IGF1R were also evaluated. RESULTS: Recombinant lentivirus was successfully constructed, and its functional plaque-forming unit titer was determined as 4.58 × 10(9)plaque-forming units/ml. A positive strand was detected by polymerase chain reaction and Western blotting. Lentiviral construction worked effectively in AFP-positive liver cancer cells. In vitro and in vivo experiments showed that the recombinant lentivirus was more efficacious in inhibiting the proliferation of Hep3B cells. CONCLUSIONS: The Wtp53-pPRIME-miR30-shRNA gene can be subjected to targeted transduction into liver cancer cells under the mediation of anti-alpha fetoprotein scFv-directed lentivirus. The Wtp53-pPRIME-miR30-shRNA system has targeting ability and lethal effects on liver cancer cells.