Generation and characterization of virus-free reprogrammed melanoma cells by the piggyBac transposon
Reprogramming of cancer cells to stem cell-like state provides a promising tool for the study of cancer pathogenesis and drug screening. However, most instances of direct reprogramming have been achieved by forced co-expression of defined transcription factors using viral vectors. Retroviral transduction as well as the ectopic expression of reprogramming factors may alter the differentiation potential of reprogrammed cancer cells or induce malignant transformation. Therefore, generation of reprogrammed cancer cells via virus-free reprogramming strategy needs to be studied.
Melanoma cells were reprogrammed by co-expression of doxycycline-inducible Oct4, Sox2, Klf4, and c-Myc using the piggyBac (PB) transposon system. The expression level of genes was analyzed through RT-PCR, Western blot, and immunofluorescence. Epigenetic modification of genes was detected by bisulfite genomic sequencing. Post reprogrammed melanoma cells were generated through differentiation of reprogrammed melanoma cells. Sensitivity to chemotherapeutic agents and metastasis potential were investigated in post reprogrammed melanoma cells.
The virus-free reprogrammed melanoma cells were positive for stem cell markers including Oct4, Nanog, and SSEA-1, and the promoters of Nanog and Oct4 were demethylated. Moreover, reprogrammed melanoma cells gained differentiation potential and higher sensitivity to differentiation-inducing drugs. Post reprogrammed melanoma cells showed lower proliferation rate and metastatic potential compared with the parental cells.
Our results indicate that PB transposon-based method is applicable to generate virus-free reprogrammed melanoma cells. These cells can differentiate into other lineages with loss of malignant phenotypes, which may provide a more suitable source for molding of cancer pathogenesis.