ABSTRACT
BACKGROUND: Ubiquitin Specific Peptidase 39 (USP39) is a 65 kDa SR-related protein involved in RNA splicing. Previous studies showed that USP39 is related with tumorigenesis of human breast cancer cells. RESULTS: In the present study, we investigated the functions of USP39 in human hepatocellular carcinoma (HCC) cell line SMMC-7721. We knocked down the expression of USP39 through lentivirus mediated RNA interference. The results of qRT-PCR and western blotting assay showed that both the mRNA and protein levels were suppressed efficiently after USP39 specific shRNA was delivered into SMMC-7721 cells. Cell growth was significantly inhibited as determined by MTT assay. Crystal violet staining indicated that colony numbers and sizes were both reduced after knock-down of USP39. Furthermore, suppression of USP39 arrested cell cycle progression at G2/M phase in SMMC-7721cells. In addition, Annexin V showed that downregulation of USP39 significantly increased the population of apoptotic cells. CONCLUSIONS: All our results suggest that USP39 is important for HCC cell proliferation and is a potential target for molecular therapy of HCC.
Subject(s)
Humans , Cell Cycle , Carcinoma, Hepatocellular/pathology , Lentivirus/genetics , RNA Interference/physiology , Cell Proliferation , Ubiquitin-Specific Proteases/metabolism , Liver Neoplasms/pathology , Neoplasm Proteins/metabolism , In Vitro Techniques , Gene Expression Regulation, Neoplastic/genetics , Cell Cycle/genetics , Blotting, Western , Apoptosis , Gene Transfer Techniques , Carcinoma, Hepatocellular/enzymology , Gene Silencing , Cell Line, Tumor , Cell Proliferation/genetics , Gene Knockdown Techniques , Real-Time Polymerase Chain Reaction , Ubiquitin-Specific Proteases/genetics , Liver Neoplasms/enzymology , Neoplasm Proteins/geneticsABSTRACT
Background: The AdEasy system is a fast-track system for generating recombinant adenoviruses using the efficient homologous recombination machinery between shuttle and adenovirus backbone plasmids in Escherichia coli BJ5183 cells. The key step is homologous recombination in BJ5183 cells, which is driven by RecA activity. However, culture time is stringently limited to reduce the damage to recombinant plasmids by RecA activity. Therefore, rapid identification of recombinant adenoviruses within the limited time-period is critical. Results: We developed a simple negative selection method to identify recombinant adenoviruses using colony PCR, which improves the efficiency of adenovirus recombination screening and packaging. Conclusions: The negative selection method to identify AdEasy adenovirus recombinants by colony PCR can identify the recombined colony within a short time-period, and maximally avoid damage to the recombinant plasmid by limiting recombination time, resulting in improved adenovirus packaging.
Subject(s)
Selection, Genetic/genetics , Adenoviridae/isolation & purification , Adenoviridae/genetics , Polymerase Chain Reaction/methods , Cloning, Molecular , Homologous RecombinationABSTRACT
A sensitive,specific, and rapid method for the detection of carbohydrate-protein interactions is demonstrated by fluorophore-assisted carbohydrate electrophoresis (FACE). The procedure is simple and the cost is low. The advantage of this method is that carbohydrate-protein interactions can be easily displayed by FACE, and the carbohydrates do not need to be purified.