piggyBac Transposon-Based Immortalization of Human Deciduous Tooth Dental Pulp Cells with Multipotency and Non-Tumorigenic Potential

We aimed to immortalize primarily isolated human deciduous tooth-derived dental pulp cells (HDDPCs) by transfection with piggyBac (PB)-based transposon vectors carrying E7 from human papilloma virus 16 or complementary DNA (cDNA) encoding human telomerase reverse transcriptase (hTERT). HDDPCs were co-transfected with pTrans (conferring PB transposase expression) + pT-pac (conferring puromycin acetyltransferase expression) + pT-tdTomato (conferring tdTomato cDNA expression) and pT-E7 (conferring E7 expression) or pTrans + pT-pac + pT-EGFP (conferring enhanced green fluorescent protein cDNA expression) + pT-hTERT (conferring hTERT expression). After six days, these cells were selected in medium containing 5 μg/mL puromycin for one day, and then cultured in normal medium allowing cell survival. All resultant colonies were harvested and propagated as a pool. Stemness and tumorigenic properties of the established cell lines (“MT_E7” for E7 and “MT_hTERT” for hTERT) with untransfected parental cells (MT) were examined. Both lines exhibited proliferation similar to that of MT, with alkaline phosphatase activity and stemness-specific factor expression. They displayed differentiation potential into multi-lineage cells with no tumorigenic property. Overall, we successfully obtained HDDPC-derived immortalized cell lines using a PB-based transfection system. The resultant and parental cells were indistinguishable. Thus, E7 and hTERT could immortalize HDDPCs without causing cancer-associated changes or altering phenotypic properties.

Prognostic significance of TOP2A in non-small cell lung cancer revealed by bioinformatic analysis

Background Lung cancer has been a common malignant tumor with a leading cause of morbidity and mortality, current molecular targets are woefully lacking comparing to the highly progressive cancer. The study is designed to identify new prognostic predictors and potential gene targets based on bioinformatic analysis of Gene Expression Omnibus (GEO) database. Methods Four cDNA expression profiles GSE19188, GSE101929, GSE18842 and GSE33532 were chosen from GEO database to analyze the differently expressed genes (DEGs) between non-small cell lung cancer (NSCLC) and normal lung tissues. After the DEGs functions were analyzed, the protein–protein interaction network (PPI) of DEGs were constructed, and the core gene in the network which has high connectivity degree with other genes was identified. We analyzed the association of the gene with the development of NSCLC as well as its prognosis. Lastly we explored the conceivable signaling mechanism of the gene regulation during the development of NSCLC. Results A total of 92 up regulated and 214 down regulated DEGs were shared in four cDNA expression profiles. Based on their PPI network, TOP2A was connected with most of other genes and was selected for further analysis. Kaplan–Meier overall survival analysis (OS) revealed that TOP2A was associated with worse NSCLC patients survival. And both GEPIA analysis and immunohistochemistry experiment (IHC) confirmed that TOP2A was aberrant gain of expression in cancer comparing to normal tissues. The clinical significance of TOP2A and probable signaling pathways it involved in were further explored, and a positive correlation between TOP2A and TPX2 expression was found in lung cancer tissues. Conclusion Using bioinformatic analysis, we revealed that TOP2A could be adopted as a prognostic indicator of NSCLC and it potentially regulate cancer development through co-work with TPX2. However, more detailed experiments are needed to clarify its drug target role in clinical medical use.