We previously identified saRNAs that activated that expression of E-cadherin, p21, and VEGF in human cells 
. Sequence alignment utilizing public genome databases revealed that the intended targeted sites for each human saRNA in primates (e.g.
Chimpanzee, Orangutan, and Rhesus monkey) were all highly-conserved with human (data not shown). Conservation was confirmed by sequencing DNA amplified from non-human primate COS1 (African green monkey, AGM) and WES (chimpanzee) cell lines (, GenBank accession numbers for the resulted sequences are listed in Materials and Methods
). Our results also revealed subtle species-specific variations in sequence between human, AGM, and chimpanzee excluding the possibility of potential contamination of non-human primate cell lines with human cells (). In order to replicate the phenomenon of RNAa in non-human primate cells, we transfected both cell lines with human saRNA targeting the E-cadherin promoter at position −215 (dsEcad-215) or p21 promoter at position −322 (dsP21–322) relative to the transcription start sites (TSS). Compared to control treatments, dsEcad-215 and dsP21–322 caused a ~1.6- (p
<0.05) and ~3.7-fold (p
<0.05) increase in E-cadherin and p21 expression levels, respectively, in both cell lines (). Similar to what we observed in human cells 
, activation of either E-cadherin or p21 in COS1 and WES cells significantly inhibited cell proliferation and survival reflecting their roles as tumor suppressor genes (Fig. S1
). We also tested previously identified human VEGF saRNA dsVEGF-706 and newly designed dsVEGF-359 in both primate cell lines. In WES cells, dsVEGF-359 and dsVEGF-706 induced VEGF mRNA expression by ~3.0- (p
0.2) and ~5.3-fold (p
<0.05), respectively (). In COS1 cells, dsVEGF-706 significantly induced VEGF mRNA expression by ~2-fold (p
<0.01, ). ELISA confirmed that the secreted form of VEGF (VEGF165
) was induced following saRNA treatment (Fig. S2
Primate saRNA sequences and target alignments.
RNA activation of different genes in non-human primate cells.
To expand the list of known RNAa-responsive genes in primate cells, we used a set of design rules (Materials and Methods
) to select saRNA targets for the following clinically relevant genes: p53, PAR4, WT1, RB1, p27, NKX3-1, VDR, IL2, and pS2. Genome sequence alignment revealed that these saRNA target sites are well conserved between human and primates ( and data not shown). Of the nine genes, p53, PAR4, WT1 and NKX3-1 were induced ≥1.5-fold by their corresponding saRNA in at least one of the two non-human primate cell lines ().
To activate NKX3-1 expression, two saRNAs were designed that targeted the human NKX3-1 promoter at positions −360 (dsNKX3-1-360) and −381 (dsNKX3-1-381) relative to the TSS. The human dsNKX3-1-381 target sequence perfectly matched those in the NKX3-1 promoter of chimpanzee and AGM, while the dsNKX3-1-360 target site shared 94.7% and 84.2% sequence identity with its cognates in chimpanzee and AGM, respectively (). Compared to controls, dsNKX3-1-381 robustly induced NKX3-1 mRNA expression in both WES (~3.5-fold, p
<0.05) and COS1 (~5.7-fold, p
<0.01) cells (), as well as displayed altered morphology and decreased cell density characteristic of the predicted tumor suppressor function of NKX3-1 
). Interestingly, despite having non-perfect homology, dsNKX3-1-360 was still capable of inducing NKX3-1 mRNA expression by ~2-fold in both cell lines (, p
<0.05). To evaluate the specificity of the saRNA (i.e.
whether improved sequence complementarity would enhance RNAa activity), we designed species-specific NKX3-1 saRNAs that perfectly matched their respective chimpanzee (dsNKX3-1-360-PT) and AGM (dsNKX3-1-360-CA) targeted sequences. Both species-specific saRNAs were found to have stronger RNAa activity compared to human dsNKX3-1-360; NKX3-1 mRNA expression was increased by ~4.8- and ~3.3-fold in WES and COS1 cells, respectively (p
<0.01, ). These results suggest that NKX3-1 activation is sequence-dependent and RNAa in non-human primates can tolerate mismatches in a manner analogous to microRNA target recognition.
The p53 saRNA targeted sequence position −285 (dsP53–285) relative to the TSS in the human p53 promoter. The −285 site is comprised of a repetitive region unique to the p53 promoter that is almost identical amongst human, chimpanzee, and AGM (). In WES cells, activation of p53 by dsP53–285 resulted in PARP cleavage indicative of caspase-dependent apoptosis; a consequence of p53 overexpression 
(). To evaluate cell cycle distribution, DNA content was analyzed by flow cytometry in cells stained with propidium iodide. Transfection of dsP53–285 caused a significant increase in G1/G0 and G2/M populations with a concurrent decline in S populations as compared to control treatments (). Cell cycle arrest is associated with p53 induction and activation of downstream cell cycle inhibitory protein p21 
. Consistent with this observation, p21 was also induced by over 10-fold in dsP53-285-transfected cells ().
Activation of p53 by saRNA causes cell cycle arrest and induction of p21 in WES cells.
We further extended our analysis of RNAa into mouse cells by selecting Cyclin B1 (Ccnb1) as a candidate gene. Using the same design rules, five saRNA targets were chosen on the mouse Ccnb1 promoter between −1200 to −270 bp relative to the TSS (). We transfected the corresponding saRNAs into NIH/3T3 and transgenic adenocarcinoma mouse prostate (TRAMP) C1 cells and identified two saRNAs (dsCcnb1–313 and dsCcnb1–597) that elevated Ccnb1 mRNA expression levels. In NIH/3T3 cells, dsCcnb1–313 and dsCcnb1–597 induced Ccnb1 levels by ~5.5- (p
<0.01) and ~3.3-fold (p
<0.05), respectively; whereas dsCcnb1–597 induced Ccnb1 levels by ~3.2-fold (p
<0.05) in TRAMP C1 cells (). Because Ccnb1 promotes entry into mitosis, induction of Ccnb1 by dsCcnb1–313 or dsCcnb1–597 also increased the phosphorylation of histone H3 at serine 10 (p-H3S10) correlating with chromosome condensation during mitosis 
Ccnb1 activation in mouse cells by promoter-targeting saRNAs.
To explore RNAa in rat cells, we designed five saRNAs targeting sequences between −500 and −250 bp relative to the TSS in the promoter of rat chemokine receptor CXCR4 (). Primary rat adipose-derived stem cells (rADSCs) were transfected with each duplex and CXCR4 expression levels were evaluated by RT-PCR. Remarkably, two saRNAs (dsCXCR4–359 and dsCXCR4–438) were identified that activated CXCR4 expression (). Compared to control treatments, dsCXCR4–359 and dsCXCR4–438 both induced CXCR4 levels by ~2.5-fold in rADSCs ().
RNAa-mediated induction of CXCR4 in primary rat stem cells.