Xiaohua Tan

Cellular MicroRNA Let-7a Suppresses KSHV Replication through Targeting MAP4K4 Signaling Pathways

Background Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is the etiologic agent of KS, the most common AIDS-related malignancy. The majority of KS tumor cells harbor latent KSHV virus but only a small percentage undergoes spontaneous lytic replication. Viral reactivation from latency is crucial for the pathogenesis and development of KS, but the cellular mechanisms underlying the switch between viral latency and replication are not well understood. Methods The level of let-7 miRNAs and MAP4K4 in KSHV infected 293T cells were quantified by real-time PCRs. Let-7 expression was silenced by the miRNA sponge technique. In let-7a transfected 293T cells, the expression of MAP4K4 was measured by real-time PCR and western blot. Luciferease expression was employed to examine the effect of let-7a on the 3’-untranslated region (UTR) of the MAP4K4 gene in 293T cells. Real-time PCR was used to quantify the KSHV copy numbers in BC-3 cells in which the expression of let-7a and/or MAP4K4 were altered. Finally, ERK, JNK and p38 protein production and their phosphorylation status were detected by western blots in let-7a or MAP4K4 transfected BCBL-1 cells. Results The expression of microRNA let-7 was dramatically decreased in KSHV infected 293T cells, but that of MAP4K4 was increased significantly. Let-7a is physically associated with and targets the MAP4K4 3’UTR, and inhibits MAP4K4 expression at both mRNA and protein levels. MAP4K4 stimulates KSHV reactivation from latency, whereas let-7a inhibits the function of MAP4K4 by reversing the function of MAP4K4 on JNK, phospho-JNK and phospho-ERK1/2 levels. Conclusion Our results establish that let-7a specifically suppresses MAP4K4 expression, and further inhibits KSHV reactivation by interfering with the function of MAP4K4 on the MAPK pathway, highlighting let-7a as a potential treatment for KS.

ATP- binding cassette transporter A1 ( ABCA1) promotes arsenic tolerance in human cells by reducing cellular arsenic accumulation

Arsenic is a toxic element widely distributed in nature, such as water and soil. To survive this metalloid in the environment, nearly all organisms develop strategies to tolerate arsenic toxicity to some degree. Some arsenic‐resistance genes have been identified in bacteria and yeast, but for mammals, especially humans, these genes are largely unknown. The aim of the present study was to identify these genes and benefit our intervention of arsenic resistance. We first established a human arsenic‐resistant ECV‐304 (AsRE) cell line and then used suppression subtractive hybridization and microarray analysis to identify arsenic‐resistant genes in these cells. Of the significantly upregulated genes, three ATP‐binding cassette (ABC) subfamily members, namely ABCA1, ABCE1 and ABCF1, were chosen for further study with RNA interference and overexpression analyses. The 3‐(4,5‐dimethyl‐2 thiazoyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide assay was used to determine the cell survival rate and the IC50, whereas atomic fluorescence spectrophotometry was used to determine intracellular arsenic levels. We found that among the three ABC genes, only when ABCA1 gene expression was silenced did cells obviously lose their arsenic tolerance. The arsenic accumulation in ABCA1 deficiency AsRE cells was greater than that in wild type AsRE cells. Overexpression of ABCA1 in HeLa cells decreased arsenic accumulation in the cells and the cells were more resistant to As(III) than control cells transfected with empty vector. These results suggest a new functional role for ABCA1 in the development of arsenic resistance in human cells.

Effects of neuritin on the differentiation of bone marrow‑derived mesenchymal stem cells into neuron‑like cells

While the neurotrophic factor neuritin is known to be involved in neurodevelopment, the effects of this compound on cell differentiation remain unclear. The present study demonstrated that neuritin treatment induced the differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) into neuron-like (NL) cells. For these analyses, rBM-MSCs were incubated with 0.5 µg/ml neuritin for 24 h. Following induction, 27% of the rBM-MSCs exhibited typical NL cell morphologies. Subsequently, NL cells were characterized by examining the expression of neuronal markers and by analysis of cell functions. The findings demonstrated that the NL cells produced by neuritin treatment expressed the neuronal markers neuron-specific enolase and microtubule associate protein 2, and secreted the neurotransmitter 5-hydroxytryptamine. Furthermore, the NL cells exhibited certain partial neural-electrophysiological functions. In conclusion, neuritin treatment may be an effective method for inducing the differentiation of BM-MSCs towards NL cells. This may provide an alternative, potentially complementary tool for disease modeling and the development of cell-based therapies.

ABCA1 Is Coordinated with ABCB1 in the Arsenic-Resistance of Human Cells

Arsenic is one of the most widespread global environmental toxicants associated with endemic poisoning. ATP-binding cassette (ABC) proteins are transmembrane channels that transport and dispose of lipids and metabolic products across the plasma membrane. The majority of ABC family members (including ABCB1 and ABCC1) are reported to play a role in the development of arsenic and drug resistance in mammals. Previously, we established a human arsenic-resistant ECV-304 (AsRE) cell line and identified ABCA1 as a novel arsenic resistance gene. In the current study, we further investigated the potential contribution of ABCA1, ABCB1, and ABCC1 to arsenic resistance through measurement of survival rates and arsenic accumulation in AsRE cells with RNA interference. The arsenic resistance capacity of ABCC1 was the strongest among the three genes, while those of ABCA1 and ABCB1 were similar. Double or triple gene knockdown of ABCA1, ABCB1, and ABCC1 via RNA interference led to a decrease significant in arsenic resistance when ABCA1/ABCB1 or ABCB1/ABCC1 were simultaneously silenced. Interestingly, no differences were evident between cells with ABCA1/ABCC1 and ABCC1 only knockdown. Our findings suggest that ABCA1 and ABCB1 proteins display similar arsenic resistance capabilities and possibly coordinate to promote arsenic resistance in AsRE cells.