The online version of this article (doi:10.1186/s12943-017-0688-6) contains supplementary material, which is available to authorized users.
The primary issue arising from prostate cancer (PCa) is its high prevalence to metastasize to bone, which severely affects the quality of life and survival time of PCa patients. miR-210-3p is a well-documented oncogenic miRNA implicated in various aspects of cancer development, progression and metastasis. However, the clinical significance and biological roles of miR-210-3p in PCa bone metastasis remain obscure.
miR-210-3p expression was evaluated by real-time PCR in 68 bone metastatic and 81 non-bone metastatic PCa tissues. The biological roles of miR-210-3p in the bone metastasis of PCa were investigated both in vitro by EMT and Transwell assays, and in vivo using a mouse model of left cardiac ventricle inoculation. Bioinformatics analysis, real-time PCR, western blot and luciferase reporter analysis were applied to discern and examine the relationship between miR-210-3p and its potential targets. RT-PCR was performed to identify the underlying mechanism of miR-210-3p overexpression in bone metastasis of PCa. Clinical correlation of miR-210-3p with its targets was examined in human PCa and metastatic bone tissues.
miR-210-3p expression is elevated in bone metastatic PCa tissues compared with non-bone metastatic PCa tissues. Overexpression of miR-210-3p positively correlates with serum PSA levels, Gleason grade and bone metastasis status in PCa patients. Upregulating miR-210-3p enhances, while silencing miR-210-3p represses the EMT, invasion and migration of PCa cells in vitro. Importantly, silencing miR-210-3p significantly inhibits bone metastasis of PC-3 cells in vivo. Our results further demonstrate that miR-210-3p maintains the sustained activation of NF-κB signaling via targeting negative regulators of NF-κB signaling (TNF-α Induced Protein 3 Interacting Protein 1) TNIP1 and (Suppressor Of Cytokine Signaling 1) SOCS1, resulting in EMT, invasion, migration and bone metastasis of PCa cells. Moreover, our results further indicate that recurrent gains (amplification) contribute to miR-210-3p overexpression in a small number of PCa patients. The clinical correlation of miR-210-3p with SOCS1, TNIP1 and NF-κB signaling activity is verified in PCa tissues.
Our findings unravel a novel mechanism for constitutive activation of NF-κB signaling pathway in the bone metastasis of PCa, supporting a functional and clinical significance of epigenetic events in bone metastasis of PCa.
Additional file 2: Table S2. A list of primers used in the reactions for real-time RT-PCR. (PDF 9 kb)
Additional file 3: Table S3. The clinicopathological characteristics in 149 patients with prostate cancer. (PDF 54 kb)
Additional file 4: Figure S1. miR-210-3p expression is upregulated in bone metastatic PCa tissues and cells. (a) Percentages and number of samples showed high or low miR-210-3p expression in our PCa patients with different bone metastasis. P < 0.001. (b) miR-210-3p expression was elevated in PCa cells compared with that in stromal cells in GSE17321 dataset. *P < 0.05. (PDF 62 kb)
Additional file 5: Table S4. The relationship between miR-210-3p and clinicopathological characteristics in 149 patients with prostate cancer. (PDF 58 kb)
Additional file 6: Figure S2. Silencing miR-210-3p repressed EMT, invasion and migration in PC-3 cells in vitro. Real-time PCR analysis of miR-210-3p expression in PC-3 cells transduced with antagomiR-210-3p compared to controls. Transcript levels were normalized by U6 expression. Error bars represent the mean ± s.d. of three independent experiments. *P < 0.05.
Additional file 7: Figure S3. Silencing miR-210-3p inhibits NF-κB signaling activity in PC-3 cells. (a) Gene set enrichment analysis (GSEA) revealed that miR-210-3p expression significantly and positively correlated with the NF-κB signaling. (b) NF-κB transcriptional activity was repressed by silencing miR-210-3p in the indicated PC-3 cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. (c) Western blotting of nuclear NF-κB/p65 expression. The nuclear protein p84 was used as the nuclear protein marker. (d) Real-time PCR analysis of TWIST1, MMP13 and IL11 in the indicated cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. (e and f) NF-κB signaling inhibitors LY2409881 and JSH-23 inhibited the NF-κB transcriptional activity in a dose-dependent manner in the indicated cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. (PDF 128 kb)
Additional file 8: Figure S4. miR-210-3p targets multiple negative regulators of NF-κB signaling. (a) Predicted miR-210-3p targeting sequence and mutant sequences in 3’UTR s of SOCS1 and TNIP1. (b) Real-time PCR analysis of TNIP1, SOCS1, PIAS4 and PDLIM7 expression in the indicated cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. (c) Western blotting of TNIP1, SOCS1, PIAS4 and PDLIM7 expression in the indicated cells. α-Tubulin served as the loading control. (d) Luciferase assay of cells transfected with pmirGLO-3’UTR reporter of TNIP1 and SOCS1 in the miR-210-3p silencing PC-3 cells. *P < 0.05. (e and f) Individual silencing of TNIP1 and SOCS1 rescued the NF-κB activity (e) and invasion (f) abilities repressed by miR-210-3p silencing in PCa cells. *P < 0.05 and **P < 0.01. (PDF 185 kb)
Additional file 9: Figure S5. miR-210-3p expression levels was markedly elevated in metastatic bone tissues compared with that in primary PCa tissues with bone metastasis (BM, n = 6; Bone, n = 7). *P < 0.05. (PDF 28 kb)
Additional file 10: Figure S6. Clinical correaltion of miR-210-3p with SOCS1, TNIP1 and nuclear p65 in human PCa and bone tissues. (a-c) Correlation between miR-210-3p levels and SOCS1, TNIP1 and nuclear p65 expression in PCa and bone tissues.The expression levels of SOCS1, TNIP1 and nuclear p65 were quantified by densitometry using Quantity One Software, and normalized to the levels of α-tubulin and p84, respectively. The sample 1 was used as a standard. The relative expressions of miR-210-3p and these proteins were used to perform the correlation analysis. (PDF 88 kb)
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- Oncogenic miR-210-3p promotes prostate cancer cell EMT and bone metastasis via NF-κB signaling pathway
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