HBV HBx-Downregulated lncRNA LINC01010 Attenuates Cell Proliferation by Interacting with Vimentin

Hepatitis B virus (HBV) infection is closely related to hepatocellular carcinoma (HCC) development. To investigate the mechanism of HBV causing HCC, we previously analyzed the transcription of the HBV-transgenic cell line HepG2-4D14 and parental HepG2 cells and identified a subset of long noncoding RNAs (lncRNAs) differentially expressed between them. In this study, we focus on lncRNA LINC01010, as it is significantly downregulated in HepG2-4D14 cells and in liver tissues of HCC patients, and positively correlated with survival. We found that HBV-encoded HBx can reduce the transcription of LINC01010. Functional analysis showed that the overexpression of LINC01010 inhibits proliferation, migration and invasion of HepG2 cells while the knockdown of LINC01010 promotes these processes. By taking the approach of RNA immunoprecipitation (RIP) and mass spectrometry, we identified that LINC01010 can interact with vimentin. Further studies demonstrated that LINC01010 negatively affects the vimentin network extension and causes more rapid subunit exchange and lower stability of vimentin filaments. In addition, LINC01010 can reduce the amount of insoluble vimentin within cells, which suggests that LINC01010 interfers with vimentin polymerization. These data indicate that LINC01010 can inhibit the assembly of vimentin filament. Thus, we revealed that HBV HBx-downregulated LINC01010, which suppresses cell proliferation and migration by negatively regulating the formation of vimentin filament. Taken together, LINC01010 is a potential tumor suppressor that may restrain HBV-related HCC development.

Potential limitations of the Sleeping Beauty transposon use in gene expression studies

MCPIP2 is the least known member of the MCPIP family of proteins. Recently we have found that it is a new RNase involved in transcript turnover. However, the full spectrum of its cellular targets is still unidentified. To discover transcripts which are regulated by this protein we have employed Sleeping Beauty transposons. This tool allows for rapid generation of a stable transgenic cell line with inducible expression of the desired gene. In this study, we analysed how the Sleeping Beauty system itself influences expression of chosen genes, namely IL-6, Regnase-1 and VEGF. We found that the system alone may influence expression of IL-6. Our results indicate that Sleeping Beauty transposons should be used with caution in studies that are focused on changes in the transcript level.

The impact of chromatin dynamics on Cas9-mediated genome editing in human cells

ABSTRACTIn order to efficiently edit eukaryotic genomes, it is critical to test the impact of chromatin dynamics on CRISPR/Cas9 function and develop strategies to adapt the system to eukaryotic contexts. So far, research has extensively characterized the relationship between the CRISPR endonuclease Cas9 and the composition of the RNADNA duplex that mediates the system’s precision. Evidence suggests that chromatin modifications and DNA packaging can block eukaryotic genome editing by custom-built DNA endonucleases like Cas9; however, the underlying mechanism of Cas9 inhibition is unclear. Here, we demonstrate that closed, gene-silencing-associated chromatin is a mechanism for the interference of Cas9-mediated DNA editing. Our assays use a transgenic cell line with a drug-inducible switch to control chromatin states (open and closed) at a single genomic locus. We show that closed chromatin inhibits editing at specific target sites, and that artificial reversal of the silenced state restores editing efficiency. These results provide new insights to improve Cas9-mediated editing in human and other mammalian cells.

22 PRODUCTION OF PORCINE TRANSGENIC CELL LINE INSERTED WITH SV40LT, EGFRvIII Gene, AND INDUCIBLE CreERT SYSTEM

Transgenic pigs are currently believed to be an important model for biomedical research, including for disease models, pharmaceutical toxicity testing, and regenerative medicine. However, production efficiency of animal disease models using somatic cell NT (SCNT) is very low. One of the main reasons is probably characteristics of the transgene. In this study, we introduce SV40LT oncogene into the fibroblast cells in order to establish immortalized transgenic cell line for producing the pig model of human brain cancer. We evaluated the effect of SV40LT oncogene on transgenic SCNT embryo development. As a results, the cleavage rates (73.8 ± 4.0 and 48.6 ± 2.4 in the normal and SV40LT group, respectively; P < 0.05) and blastocyst formation rates (19.5 ± 1.2 and 5.6 ± 1.8 in the normal and SV40LT group, respectively; P < 0.05) of transgenic SCNT embryos was significantly lower than the case of using normal cells. In addition, we evaluated the transgenic SCNT embryo development of the donor cell transfected with SV40LT and HrasV12 genes (SV40LT+HrasV12 group). As a results, there was no significant difference between the groups in the cleavage rates, but blastocyst formation rates of transfected SCNT embryos (SV40LT+HrasV12 group) was significantly lower than the case of using normal cells (19.5 ± 1.2 and 6.2 ± 1.8 in the normal and SV40LT+HrasV12 group, respectively; P < 0.05). Genes SV40LT or HrasV12 showed a negative effect on SCNT cloned embryo development. Therefore, a Cre/loxP inducible system was applied to producing donor cells transfected with EGFRvIII and SV40LT gene. As a result, the cleavage rates (73.8 ± 4.0 and 68.6 ± 6.6 in the normal and Cre/loxP-EGFRvIII-SV40LT group, respectively; P < 0.05) and blastocyst formation rate (19.5 ± 1.2 and 23.0 ± 3.7 in the normal and Cre/loxP-EGFRvIII-SV40LT group, respectively; P < 0.05) were improved to the same level, when used as a donor cell to a normal cell. In conclusion, these results indicated that harmful effects of transgenic SCNT embryo development caused by the characteristics of the inserted genes can be overcome through the inducible system. Further studies are needed to experiment with mRNA expression of apoptotic gene and target gene in 4- to 8-cell embryos and blastocysts. This work was supported, in part, by a grant from the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ011077, PJ011288), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean government (NRF-2013R1A2A2A04008751), Republic of Korea.

Small Heat Shock Proteins Can Confer Tolerance to Nanomaterial-induced Toxicity

The expression of a small heat shock protein (sHSP) in plants and its possible function in conditions related to nanomaterial exposure were examined. Multiwalled carbon nanotubes (MWCNTs) and silver nanoparticles (AgNPs) induced toxicity that was indicated by the bending and curling of carrot leaf tissues. Both nanomaterials induced the expression of a small heat shock protein in carrot, DcHsp17.7, but reduced the level of a constitutive heat shock cognate 70. To examine the possible function of DcHsp17.7, the coding gene was heterologously expressed in Escherichia coli. Both nanomaterials reduced the viability of E. coli cell lines. However, the transgenic cell line heterologously expressing DcHsp17.7 showed higher levels of cell viability, compared with vector controls, when exposed to MWCNTs and, more notably, to AgNPs. To the best of our knowledge, this is the first study reporting the influence of nanomaterials on the expression of a plant sHSP and its possible function in conferring tolerance to nanomaterial stress.