RESUMO
We established a semi-high-throughput in vivo screening platform using hyperactive piggyBac (hyPB) transposons (designated as PB-miR) to identify microRNAs (miRs) that inhibit hepatocellular carcinoma (HCC) development in vivo, following miR overexpression in hepatocytes. PB-miRs encoding six different miRs from the miR-17-92 cluster and nine miRs from outside this cluster were transfected into mouse livers that were chemically induced to develop HCC. In this slow-onset HCC model, miR-20a significantly inhibited HCC. Next, we developed a more aggressive HCC model by overexpression of oncogenic Harvey rat sarcoma viral oncogene homolog (HRASG12V) and c-MYC oncogenes that accelerated HCC development after only 6 weeks. The tumor suppressor effect of miR-20a could be demonstrated even in this rapid-onset HRASG12V/c-MYC HCC model, consistent with significantly prolonged survival and decreased HCC tumor burden. Comprehensive RNA expression profiling of 95 selected genes typically associated with HCC development revealed differentially expressed genes and functional pathways that were associated with miR-20a-mediated HCC suppression. To our knowledge, this is the first study establishing a direct causal relationship between miR-20a overexpression and liver cancer inhibition in vivo. Moreover, these results demonstrate that hepatocyte-specific hyPB transposons are an efficient platform to screen and identify miRs that affect overall survival and HCC tumor regression.
RESUMO
Antiparasite responses are associated with the recruitment of monocytes that differentiate to macrophages and dendritic cells at the site of infection. Although classically activated monocytic cells are assumed to be the major source of TNF and NO during Trypanosoma brucei brucei infection, their cellular origin remains unclear. In this study, we show that bone marrow-derived monocytes accumulate and differentiate to TNF/inducible NO synthase-producing dendritic cells (TIP-DCs) in the spleen, liver, and lymph nodes of T. brucei brucei-infected mice. Although TIP-DCs have been shown to play a beneficial role in the elimination of several intracellular pathogens, we report that TIP-DCs, as a major source of TNF and NO in inflamed organs, could contribute actively to tissue damage during the chronic stage of T. brucei brucei infection. In addition, the absence of IL-10 leads to enhanced differentiation of monocytes to TIP-DCs, resulting in exacerbated pathogenicity and early death of the host. Finally, we demonstrate that sustained production of IL-10 following IL-10 gene delivery treatment with an adeno-associated viral vector to chronically infected mice limits the differentiation of monocytes to TIP-DCs and protects the host from tissue damage.
