miR-9 Acts as an OncomiR in Prostate Cancer through Multiple Pathways That Drive Tumour Progression and Metastasis

Identification of dysregulated microRNAs (miRNAs) in prostate cancer is critical not only for diagnosis, but also differentiation between the aggressive and indolent forms of the disease. miR-9 was identified as an oncomiR through both miRNA panel RT-qPCR as well as high-throughput sequencing analysis of the human P69 prostate cell line as compared to its highly tumorigenic and metastatic subline M12, and found to be consistently upregulated in other prostate cell lines including DU-145 and PC3. While miR-9 has been characterized as dysregulated either as an oncomiR or tumour suppressor in a variety of other cancers including breast, ovarian, and nasopharyngeal carcinomas, it has not been previously evaluated and proven as an oncomiR in prostate cancer. miR-9 was confirmed an oncomiR when found to be overexpressed in tumour tissue as compared to adjacent benign glandular epithelium through laser-capture microdissection of radical prostatectomy biopsies. Inhibition of miR-9 resulted in reduced migratory and invasive potential of the M12 cell line, and reduced tumour growth and metastases in male athymic nude mice. Analysis showed that miR-9 targets e-cadherin and suppressor of cytokine signalling 5 (SOCS5), but not NF-ĸB mRNA. Expression of these proteins was shown to be affected by modulation in expression of miR-9

While protein levels of SOCS5 are increased upon miR-9 inhibition, messenger RNA levels are not significantly different.

<p>SOCS5 expression in the P69, M12, and M12 stably transformed with vector expressing miR-9 inhibitor. A: messenger RNA relative quantitation shows that mRNA levels are not significantly impacted by miR-9. mRNA is normalized to GAPDH and reported as relative to the M12 cell line; results are compiled data from three independent lysates, each performed in triplicate. Regardless of similar SOCS5 mRNA levels, (B) Western blot analysis and (C) quantitation show that miR-9 inhibition results in increased levels of SOCS5. Blot is representative of 6 independent experiments, Quantitation the average of 3 independent experiments normalized to β-Actin and reported as relative to the M12 cell line (p<0.05). (D) The proven SOCS5 mRNA:miR-9 hybrid [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159601#pone.0159601.ref021" target="_blank">21</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159601#pone.0159601.ref022" target="_blank">22</a>]. miR-9 is the top sequence, and hybridized below is the complementary region of the 3’-UTR of SOCS5. (E) SOCS5 expression is suppressed by miR-9. M12 cells were transiently transfected with a dual luciferase reporter construct containing a portion of the SOCS5 3’-UTR, with the wild type or mutated miR-9 binding seed region. Firefly luciferase expression is reported as normalized to renilla luciferase activity and relative to mutated seed region expression. Results are the mean of 2 independent experiments, each performed in triplicate. (p = .016).</p

Tumour growth and metastasis is reduced upon miR-9 inhibition.

<p>A: Subcutaneous injections into the flank of the respective cell lines into nude athymic mice. Tumour reduction was significantly reduced in mice injected with M12 cells transformed with miR-9 inhibitor (p <.0001). Results are reported as the average ratio of tumour volume to the final average M12 tumour volume. N = 5 mice for each treatment. B: Intraprostatic (IP) injection into nude athymic mice resulted in 0 metastatic lesions when miR-9 was inhibited, as compared to 7 lesions in the mouse injected with M12 cells alone.</p

Inhibition of miR-9 significantly reduces migratory and invasive potential, but has no effect on cell proliferation.

<p>M12 cells were stably transformed with scrambled control or miR-9 inhibitor, plated on a ThinCert transwell membrane (with basement membrane added for invasion assay) and assessed for (A) migratory (p < 0.0001), (B) invasive potential (p < 0.0001), and (C) proliferation. Data is the mean of 3 independent experiments, each performed in triplicate (A&B) or one representative of three independent experiments (C).</p

messenger RNA and protein levels of CDH1 are increased upon miR-9 inhibition.

<p>A: RT-qPCR analysis shows that mRNA levels are impacted by miR-9, and miR-9 inhibition relieves mRNA and protein levels (p<0.03 for P69 and miR-9 inhibited lines vs. M12). mRNA is normalized to GAPDH and reported as relative to the M12 cell line using the comparative C_T method. Results are compiled data from two biological replicates, each performed in triplicate. B: Proven binding site for miR-9 in e-cadherin. Adapted from RNAHybrid [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159601#pone.0159601.ref021" target="_blank">21</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159601#pone.0159601.ref022" target="_blank">22</a>] analysis of CDH1 mRNA (NM_004360.3). miR-9 is the left sequence, and hybridized on the right is the complementary region of the 3’-UTR of CDH1. C: Western blot analysis and D: quantitation. Blot is representative of 5 independent experiments, Quantitation the average of 3 independent experiments normalized to β-Actin and reported as relative to the M12 cell line (p<0.05). (E) e-cadherin expression is suppressed by miR-9. M12 cells were transiently transfected with a firefly luciferase reporter construct containing a portion of the CDH1 3’-UTR, with the wild type or mutated miR-9 binding seed region along with a renilla luciferase plasmid. Firefly luciferase expression is reported as normalized to renilla luciferase activity and relative to mutated seed region expression. Results are the mean of 2 independent experiments, each performed in triplicate. (p <0.01). Western blot analysis shows that NF-kB levels do not change significantly between M12 or M12 cells stably transfected with miR-9 inhibitor. Blot (F) is representative of 3 independent experiments and quantitation (G) is from one representative experiment.</p

miR-9 levels are upregulated in all prostate cancer cell line models.

<p>A: miR-9 levels were significantly upregulated in M12 cells as relative to the parental P69 line (p = .002). Data is the mean of 3 independent experiments, each performed in duplicate (F6) or triplicate (P69, M2182, M12). B: miR-9 levels in DU-145 and PC3 cells are upregulated as relative to P69 cells (p <.001). Data was normalized to RNU48 (dC_T) and expressed relative to P69 using the comparative C_T method. Data is the mean of three technical replicates. C: miR-9 expression is upregulated in 75% of positive patient tumours. miR-9 levels were measured in tumour and benign tissue separated from prostate biopsies using laser-captured microdissection (LCM). Data was normalized to RNU48 and expressed relative to the benign tissue using the comparative C_T method, and is the average of 3 technical replicates. miR-9 was undetected in both benign and tumour of the fifth patient, and thus was not included in the analysis.</p