FIG. 6.
Overexpression of long noncoding RNA H19 favors stemness gene expression and clonogenicity. RWPE-1 cells were transfected with either pcDNA-H19 or pcDNA-control and selected with G418 to obtain stable neor cell lines. (A) Overexpression of H19 in RWPE-1 cells increased expression of several stemness genes; expression was analyzed by qRT-PCR using total RNA from empty vector-expressing G418-resistant RWPE-1 cells (open bars) or from H19-expressing G418-resistant RWPE-1 cells (closed bars), and levels were normalized to those of RPLP0 internal control. Graphs show fold change over empty vector-expressing G418-resistant RWPE-1 cells for each gene. (B) Cells were cultivated at a low density in PrEGM culture medium in ultra-low attachment 24-well plates in liquid cultures, and RW-pcDNA-H19 formed more spheres than RW-pcDNA3 control cells; lower panels represent spheres formed after 15 days of culture. (C) RW-pcDNA-H19 cells grew faster than RW-pcDNA3 control cells; cell growth curves were obtained after plating 2×104 cells per well in six-well plates, and viable cell numbers were determined daily after trypsinization. (A, B, C) Data represent mean values±standard error of the mean of three independent experiments; P values were determined by Student's test ***P<0.001, **P<0.01.