Figure 6.

Expression of miRNA199a in Tumorspheres of Hepa1-6 and Its Implication for Targeted Gene Delivery

(A) Photomicrographs of tumorspheres of Hepa1-6 after 5 days in culture in serum-free, stem-cell-enriching media (scale bars, 50 μM on the left and 200 μm on the right). Real-time qPCR was performed on cDNA of the Hepa1-6 and Hepa1-6 tumorspheres to compare the markers of stemness, (B) CD44, (C) CD133, and (D) Oct4, as well as (E) miRNA199a. Comparison of miRNA199a levels in Hepa1-6 and Hepa1-6 tumorspheres: (F) Hepa1-6 and Hepa1-6 tumorspheres were transfected with CMV-GLuc-miR199a*3, and secreted GLuc was reported as percentage of CMV-GLuc. (G) Hepa1-6 and Hepa1-6 tumorspheres were transduced with pscAAV8-GLuc and pscAAV8-GLuc-miR199a*3, and relative GLuc expression was reported. (H) Similarly, Hepa1-6 and Hepa1-6 tumorspheres were transfected with CMV-CD and CMV-CD-miR199a*3, and percent proliferation was calculated following incubation with 5-FC. Percent proliferation for CMV-CDmiR199a*3 was normalized with CMV-CD. A two-tailed t test was performed to analyze the difference between groups (data are reported as mean ± SD, n > 3; *p < 0.05; **p < 0.01; ***p < 0.001).