Figure 4. siRNA targeting the region near rs36115365 reduces expression of TERT.

(a) Eight double-stranded siRNAs were designed to target the potential gene-regulatory region encompassing rs36115365. (b) Three of eight double-stranded siRNAs targeting the rs36115365 locus can inhibit the gene expression of TERT but not CLPTM1L, ACTB, and GAPDH when transfected into PANC-1 (pancreatic cancer, upper left), A549 (lung cancer, upper right), NTERA-2 (testicular cancer, lower left) and UACC903 (melanoma, lower right). Expression values were normalized to a scrambled control siRNA. Experiments were performed in triplicate and repeated at least three times; error bars represent standard error of the mean (s.e.m.). (c) Allele-specific TERT expression after siRNA targeting (by siRNA3) of the regulatory region encompassing rs36115365. TERT expression was assayed using a quantitative allelic-discrimination TaqMan assay for a synonymous mRNA coding SNP in the TERT gene (rs2736098) that is genetically linked to rs36115365 (r²=0.14, D′=1.0). Two pancreatic cancer cell lines (Panc 05.04, left; and IMIM-PC-1, right) and one lung cancer line (A549, center) heterozygous for both SNPs were assayed. Expression of the allele of rs2736098 that is linked to the C-allele of rs36115365 (labelled ‘C allele') was reduced to a greater extent than the allele linked to rs36115365-G. The degree of knockdown of each allele was normalized to that from a scrambled siRNA control. Experiments were performed in triplicate and repeated three times. Mean measures for three independent experiments are plotted; error bars represent s.e.m.