Figure 2. Antisense-mediated regulation of sense mRNA and protein.
(A) Knockdown of brain derived neurotrophic factor (BDNF) natural antisense transcript, BDNF-AS, in HEK293T cells (n=12 per treatment) with each of three unique siRNAs (10 nM) targeting the non-overlapping region of BDNF-AS transcript, caused 2–6 fold upregulation of BDNF (sense) mRNA (n=6 for each data point/treatment ***= P < 0.001, **= P < 0.01). Similar results were obtained from experiments using Human cortical neuron (HCN), glioblastoma (MK059) cells, mouse N2a cells and neurospheres “data not shown”. Scrambled sequences, mock transfection and control siRNAs were used as controls. Control siRNA for this and other experiments is an inert siRNA (CCUCUCCACGCGCAGUACATT) that does not target any known sequence in the mammalian genome. All measurements were normalized to the 18S rRNA and graphed as a percentage of each mRNA to the negative siRNA control sample.
(B) We assessed changes in BDNF and BDNF-AS transcripts over a period of time, following BDNF-AS knockdown (n=6 for each data point/treatment). siRNA knockdown of human BDNF-AS resulted in efficient and consistent downregulation of BDNF-AS, starting at 6 h and continuing on to 72 h. BDNF mRNA levels rose at 18 h, remaining high for more than 72 h, reversing to pre-treatment levels at 96 h. Note that the peak at 48 h is consistent and reproducible. Although BDNF-AS knockdown begins after 6 h, upregulation of BDNF started 18 h post-treatment. This time lag between the depletion of BDNF-AS and the increase of BDNF mRNA shows the sequential order of events indicating that the cells require time to adapt to the removal of the antisense transcript before upregulating BDNF.
(C) siRNA-mediated knockdown of BDNF-AS transcript caused an increase in BDNF protein levels measured by ELISA. Cells were transfected with 10 nM of two active siRNAs for BDNF-AS, scrambled siRNAs or a control siRNA for 48 hours. The supernatants of these cells were concentrated and analyzed for BDNF protein by ELISA, using a commercially available kit. BDNF protein was significantly increased (n=6 per treatment, ***=P < 0.0001, **= P<0.001) with siRNA targeting BDNF-AS transcript.
(D) Western blots confirmed that knockdown of the non-protein-coding BDNF-AS, with BDNF-AS siRNA1, but not control non-targeting siRNA transcript increased BDNF protein levels without changing the levels of beta-actin. Collectively, these data suggest that there is a discordant relationship between the sense and antisense BDNF transcripts in which BDNF-AS suppresses the expression of BDNF mRNA and protein. Removal of this negative regulatory effect, by BDNF-AS knockdown, causes upregulation of BDNF mRNA and protein levels.
(E) Dose-dependent increases in Bdnf following Bdnf-AS depletion: We performed dose-response experiments using 11 different concentrations (1:3 serial dilutions ranging from 300nM to 5pM) of mBdnf-AntagoNAT9 (n=6 per data point/treatment) and we observed a dose-dependent increase in Bdnf mRNA levels at 1–300 nM concentration with an EC50 of 6.6 nM.
(F) Selective knockdown of GDNF-AS increases GDNF mRNA: We treated cells with various AntagoNATs targeting a low abundance noncoding antisense RNA, GDNF-AS. We observed that GDNF-AntagoNAT5 and GDNF-AntagoNAT6 increase the GDNF mRNA by 3–4 fold (n=6 per treatment *= P < 0.05, **= P < 0.01).