Fig. 2.
Effect of increased expression of RLN2 in LNCaP prostate cancer cells. a LNCaP sublines LNCaP-RLN2/C1 and LNCaP-RLN2/C2, which express elevated levels of RLN2, were generated by stable transfection of RLN2 in LNCaP cells. Quantitative RT-PCR (qRT-PCR) analysis for RLN2 mRNA levels in these sublines compared with vector-transfected LNCaP cells and LNCaP-R273H, a subline previously shown to express extremely high levels of this peptide hormone, demonstrated that the two RLN2 LNCaP sublines expressed RLN2 at significantly higher levels compared with LNCaP but not as high as in p53R273H-transfected cells. Triplicate samples were run for each experimental group, and the resulting Ct values for each group were within 0.5 Ct of each other. b Immunocytochemical analysis of the cell lines confirmed this trend. c Next-generation sequencing (NGS) followed by gene ontology (GO) analysis revealed 12.7% of genes that are differentially expressed between the RLN2 LNCaP and LNCaP-vector sublines are linked to proliferation. Other key processes associated with increased RLN2 expression were transcription (18.6%), metabolism (16.4%), signal transduction (11.7%), and proteolysis (6.2%). d The RLN2 LNCaP sublines express high levels of NSE and low levels of NEP, suggesting a neuroendocrine-like phenotype. While lower levels of AR were observed in the RLN2 LNCaP sublines, assessment of PSA levels indicates that the AR pathway is much more active. e MTT proliferation assay determined that the RLN2 LNCaP sublines are able to grow in the absence of androgen, as was the LNCaP-R273H subline, and that the difference in proliferation at the day 5 time point was statistically significant when comparing the LNCaP-vector and all three LNCaP sublines (asterisk signifies p < 0.05)