Figure 4

Expression of telomerase-related genes in RNAi methods. (a) Inhibitory effect of full-length RGM249 dsRNA (100 nM dsRNA) on the transcriptional expression of telomerase-related genes in a hepatocellular carcinoma cell line (HMc-Li7) as determined by RT-PCR. RGM249, hTERT, and RPL22 were significantly suppressed. The lower blotting demonstrates gene expression using a transfection reagent without dsRNA. (b) RT-PCR showing the inhibitory effects of RGM249-specific siRNA on RGM249 mRNA, hTERT mRNA, and RPL22 mRNA in HLF cells, which express RGM249 mRNA in 4 hepatoma cell lines in the strongest manner. siRNA ① and siRNA ② correspond to those in Table 2. (c) Quantitative evaluation of the suppressive effect of siRNA in which the measurement, in the case without siRNA, was regarded as 100% for standardization (N = 5), suggesting that siRNA ① has a functional sequence. RGM249 siRNA transcriptionally suppressed more than 80% of hTERT expression and approximately 50% of RPL22 expression. RGM249 mRNA, ▯ hTERT mRNA, RPL22 mRNA. d) Quantitative evaluation of the inhibitory effect of RGM249 shRNA on RGM249 mRNA, hTERT mRNA, and RPL22 mRNA. From left, transfection using only transfection reagents, LacZ shRNA as the gene control, wtRGM249 shRNA, and mt-1RGM249 or mt-2RGM249 shRNA with a different mutation (a nucleotide T or C) in the functional sequence. A total of 5 transfections were performed and data were statistically analyzed using the Mann-Whitney test. Compared with LacZ shRNA, RGM249 and mt-1RGM249 shRNA significantly suppressed hTERT mRNA (P = 0.002 and P = 0.034, respectively). mt-1RGM249 and mt-2RGM249 correspond to those in Table 2. ▯ RGM249 mRNA, hTERT mRNA, RPL22 mRNA, *: P < 0.05; **: P < 0.01. Quantitative evaluation of the suppressive effect of shRNA in which the measurement, in the case without siRNA, was regarded as 100% for standardization (N = 5). (e) Telomerase activity was quantitatively compared using image analyzer between transfectants with LacZ shRNA (in proliferative state) and RGM249 shRNA (in senesced state) and RGM249 shRNA, and was compared with the parental cells (112 and 54, respectively, which had an intensity of 100). Although the results depended on the timing of cell harvests after transfection, telomerase activity in transfectants with RGM249 shRNA was reduced to approximately half compared with that in parental cells, and we observed a periodicity of 6 bp due to reduced telomerase in the shRNA lane. NC: telomerase negative control.