Yubo Tang

Identification of miRs-143 and -145 that is associated with bone metastasis of prostate cancer and involved in the regulation of EMT.

The principal problem arising from prostate cancer (PCa) is its propensity to metastasize to bone. MicroRNAs (miRNAs) play a crucial role in many tumor metastases. The importance of miRNAs in bone metastasis of PCa has not been elucidated to date. We investigated whether the expression of certain miRNAs was associated with bone metastasis of PCa. We examined the miRNA expression profiles of 6 primary and 7 bone metastatic PCa samples by miRNA microarray analysis. The expression of 5 miRNAs significantly decreased in bone metastasis compared with primary PCa, including miRs-508-5p, -145, -143, -33a and -100. We further examined other samples of 16 primary PCa and 13 bone metastases using real-time PCR analysis. The expressions of miRs-143 and -145 were verified to down-regulate significantly in metastasis samples. By investigating relationship of the levels of miRs-143 and -145 with clinicopathological features of PCa patients, we found down-regulations of miRs-143 and -145 were negatively correlated to bone metastasis, the Gleason score and level of free PSA in primary PCa. Over-expression miR-143 and -145 by retrovirus transfection reduced the ability of migration and invasion in vitro, and tumor development and bone invasion in vivo of PC-3 cells, a human PCa cell line originated from a bone metastatic PCa specimen. Their upregulation also increased E-cadherin expression and reduced fibronectin expression of PC-3 cells which revealed a less invasive morphologic phenotype. These findings indicate that miRs-143 and -145 are associated with bone metastasis of PCa and suggest that they may play important roles in the bone metastasis and be involved in the regulation of EMT Both of them may also be clinically used as novel biomarkers in discriminating different stages of human PCa and predicting bone metastasis.

Pristimerin Inhibits Prostate Cancer Bone Metastasis by Targeting PC-3 Stem Cell Characteristics and VEGF-Induced Vasculogenesis of BM-EPCs

Background/Aims: Prostate cancer (PCa) is one of the most common malignant cancers and a major leading cause of cancer deaths in men. Cancer stem-like cells are shown to be highly tumorigenic, pro-angiogenic and can significantly contribute to tumor new vessel formation and bone marrow derived-EPCs (BM-EPCs) are shown to recruit to the angiogenic switch in tumor growth and metastatic progression, suggesting the importance of targeting cancer stem cells (CSCs) and EPCs for novel tumor therapies. Pristimerin, an active component isolated from Celastraceae and Hippocrateaceae, has shown anti-tumor effects in some cell lines in previous studies. However, the effect and mechanism of Pristimerin on CSCs and EPCs in PCa bone metastasis are not well studied. Methods: The effect of Pristimerin on PC-3 stem cell characteristics and metastasis were detected by spheroid formation, CD133 and CD44 protein expression, matrix-gel invasive assay and colony-formation assay in vitro, VEGF and pro-inflammatory cytokines expression by ELISA assay, and tumor tumorigenicity by X-ray and MR in NOD-SCID mice model in vivo. In addition, we also detected the effect of Pristimerin on VEGF-induced vasculogenesis and protein expression of BM-EPCs. Results: Pristimerin could significantly inhibit spheroid formation and protein expression of CD133 and CD44, reduce VEGF and pro-inflammation cytokines expression of PC-3 cell, and prevent the xenografted PC-3 tumor growth in the bone of nude mice. The present data also showed that Pristimerin significantly inhibited VEGF-induced vasculogenesis of BM-EPCs by suppressing the EPCs functions including proliferation, adhesion, migration, tube formation and inactivation the phosphorylation of VEGFR-2, Akt and eNOS. Conclusion: These data provide evidence that Pristimerin has strong potential for development as a novel agent against prostate bone metastasis by suppressing PC-3 stem cell characteristics and VEGF-induced vasculogenesis of BM-EPCs.

Inhibitory action of pristimerin on hypoxia‑mediated metastasis involves stem cell characteristics and EMT in PC-3 prostate cancer cells

The aim of the present study was to investigate whether pristimerin affects the bone metastasis, stem cell characteristics and epithelial-mesenchymal transition (EMT) of prostate cancer (PCa) PC-3 cells subjected to hypoxia. The PC-3 cells were cultured under hypoxia or normoxia for 48 h and were then treated with increasing concentrations of pristimerin from 0 to 0.8 µmol/l, under normoxia. Hypoxia‑inducible factor-1α (HIF-1α) was detected by western blotting. Proliferation was assessed with the CCK-8 assay. Transwell invasion assay was used to analyze the potency of invasion. Stem cell characteristics were detected by sphere formation, colony formation assay and western blotting, including CD44, KLF4, OCT4 and AGO2, which are stem cell characteristic-related markers. EMT was confirmed by the expression changes of EMT-related markers, including N-cadherin, fibronectin, vimentin and ZEB1, which were evaluated by western blotting. The addition of pristimerin to the medium reduced the hypoxia-induced PC-3 cell proliferation in a dose-dependent manner. Pristimerin effectively inhibited hypoxia‑induced invasion of the PCa cells in vitro. Moreover, the treatment of cells with pristimerin induced the reversal of hypoxia-induced stem cell characteristics and EMT, which was confirmed by sphere formation, colony formation assay and the expression changes of CSC- and EMT-related markers. The reversal of hypoxia‑induced stem cell characteristics and EMT in the PCa cells by low-dose pristimerin was dose‑dependent. These results showed that treatment with pristimerin may be a potential strategy for the suppression of hypoxia-induced metastasis through the reversal of hypoxia-induced stem cell characteristics and EMT in cancer cells, which justifies the potential use of pristimerin as a practical chemopreventive approach for patients with PCa.

Downregulation of miR-141-3p promotes bone metastasis via activating NF-κB signaling in prostate cancer

BackgroundClinically, prostate cancer (PCa) exhibits a high avidity to metastasize to bone. miR-141-3p is an extensively studied miRNA in cancers and downregulation of miR-141-3p has been widely reported to be involved in the progression and metastasis of several human cancer types. However, the clinical significance and biological roles of miR-141-3p in bone metastasis of PCa are still unclear.MethodsmiR-141-3p expression was examined in 89 non-bone metastatic and 52 bone metastatic PCa tissues by real-time PCR. Statistical analysis was performed to investigate the clinical correlation between miR-141-3p expression levels and clinicopathological characteristics in PCa patients. The biological roles of miR-141-3p in bone metastasis of PCa were evaluated both in vitro and a mouse intracardial model in vivo. Bioinformatics analysis, Western blot, luciferase reporter and miRNA immunoprecipitation assays were performed to explore and examine the relationship between miR-141-3p and its potential targets. Clinical correlation of miR-141-3p with its targets was examined in clinical PCa tissues.ResultsmiR-141-3p expression is reduced in bone metastatic PCa tissues compared with non-bone metastatic PCa tissues. Low expression of miR-141-3p positively correlates with serum PSA levels, Gleason grade and bone metastasis status in PCa patients. Furthermore, upregulating miR-141-3p suppresses the EMT, invasion and migration of PCa cells in vitro. Conversely, silencing miR-141-3p yields an opposite effect. Importantly, upregulating miR-141-3p dramatically reduces bone metastasis of PC-3 cells in vivo. Our results further show that miR-141-3p inhibits the activation of NF-κB signaling via directly targeting tumor necrosis factor receptor-associated factor 5(TRAF5) and 6 (TRAF6), which further suppresses invasion, migration and bone metastasis of PCa cells. The clinical negative correlation of miR-141-3p expression with TRAF5, TRAF6 and NF-κB signaling activity is demonstrated in PCa tissues.ConclusionOur findings unravel a novel mechanism underlying the bone metastasis of PCa, suggesting that miR-141-3p mimics might represent a potential therapeutic avenue for the treatment of PCa bone metastasis.

Downregulation of miR‑505‑3p predicts poor bone metastasis‑free survival in prostate cancer

The principal issue deriving from prostate cancer (PCa) is its propensity to metastasize to bone. To date, bone metastasis remains incurable, and therapeutic strategies are limited. Therefore, it is of paramount importance to explore predictive markers for bone metastasis of PCa. In the present study, we reported that miR-505-3p was significantly downregulated in bone metastatic PCa tissues compared with that in non-bone metastatic PCa tissues, but there was no significant difference in miR-505-3p expression between PCa and adjacent normal tissues. miR-505-3p expression was inversely associated with serum PSA levels, Gleason grade, N and M classification, and short bone metastasis-free survival in PCa patients, but had no effect on overall survival in PCa patients. Furthermore, upregulation of miR-505-3p suppressed the activity of TGF-β signaling by directly targeting downstream effectors of TGF-β signaling, SMAD2 and SMAD3, further inhibiting the invasion and migration abilities of PCa cells. Therefore, our findings unraveled a novel mechanism by which miR-505-3p inhibits bone metastasis of PCa, supporting the notion that miR-505-3p may serve as a predictive marker for bone metastasis of PCa.

Downregulation of miR-133a-3p promotes prostate cancer bone metastasis via activating PI3K/AKT signaling

BackgroundBone metastasis is a leading cause of morbidity and mortality in advanced prostate cancer (PCa). Downexpression of miR-133a-3p has been found to contribute to the progression, recurrence and distant metastasis in PCa. However, clinical significance of miR-133a-3p in bone metastasis of PCa, and the biological role of miR-133a-3p and its molecular mechanisms underlying bone metastasis of PCa remain unclear.MethodsmiR-133a-3p expression was evaluated in 245 clinical PCa tissues by real-time PCR. Statistical analysis was performed to evaluate the clinical correlation between miR-133a-3p expression and clinicopathological features, and overall and bone metastasis-free survival in PCa patients. The biological roles of miR-133a-3p in the bone metastasis of PCa were investigated both in vitro and in vivo. Bioinformatics analysis, real-time PCR, western blot and luciferase reporter analysis were applied to demonstrate the relationship between miR-133a-3p and its potential targets. Western blotting and luciferase assays were examined to identify the underlying pathway involved in the anti-tumor role of miR-133a-3p. Clinical correlation of miR-133a-3p with its targets was verified in human PCa tissues.ResultsmiR-133a-3p expression is reduced in PCa tissues compared with the adjacent normal tissues and benign prostate lesion tissues, particularly in bone metastatic PCa tissues. Low expression of miR-133a-3p is significantly correlated with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients by statistical analysis. Moreover, upregulating miR-133a-3p inhibits cancer stem cell-like phenotypes in vitro and in vivo, as well as attenuates anoikis resistance in vitro in PCa cells. Importantly, administration of agomir-133a-3p greatly suppresses the incidence of PCa bone metastasis in vivo. Our results further demonstrate that miR-133a-3p suppresses bone metastasis of PCa via inhibiting PI3K/AKT signaling by directly targeting multiple cytokine receptors, including EGFR, FGFR1, IGF1R and MET. The negative clinical correlation of miR-133a-3p with EGFR, FGFR1, IGF1R, MET and PI3K/AKT signaling activity is determined in clinical PCa tissues.ConclusionOur results unveil a novel mechanism by which miR-133a-3p inhibits bone metastasis of PCa, providing the evidence that miR-133a-3p may serve as a potential bone metastasis marker in PCa, and delivery of agomir-133a-3p may be an effective anti-bone metastasis therapeutic strategy in PCa.

Transcriptional downregulation of miR-133b by REST promotes prostate cancer metastasis to bone via activating TGF-β signaling

High avidity of bone metastasis is an important characteristic in prostate cancer (PCa). Downexpression of miR-133b has been reported to be implicated in the development, progression and recurrence in PCa. However, clinical significance and biological roles of miR-133b in bone metastasis of PCa remain unclear. Here we report that miR-133b is downregulated in PCa tissues and further decreased in bone metastatic PCa tissues. Downexpression of miR-133b positively correlates with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients. Upregulating miR-133b inhibits invasion, migration in vitro and bone metastasis in vivo in PCa cells. Mechanistically, we find that miR-133b suppresses activity of TGF-β signaling via directly targeting TGF-β receptor I and II, which further inhibits bone metastasis of PCa cells. Our results further reveal that overexpression of REST contributes to miR-133b downexpression via transcriptional repression in PCa tissues. Importantly, silencing miR-133b enhances invasion and migration abilities in vitro and bone metastasis ability in vivo in REST-silenced PCa cells. The clinical correlation of miR-133b with TGFBRI, TGFBRII, REST and TGF-β signaling activity is verified in PCa tissues. Therefore, our results uncover a novel mechanism of miR-133b downexpression that REST transcriptionally inhibits miR-133b expression in PCa cells, and meanwhile support the notion that administration of miR-133b may serve as a rational regimen in the treatment of PCa bone metastasis.

Acidic stress induces apoptosis and inhibits angiogenesis in human bone marrow‑derived endothelial progenitor cells

Bone marrow-derived endothelial progenitor cells (BM-EPCs) are exposed to acidotic environments in a variety of physiological and pathological conditions, including in tumors. However, the effect of acidosis on the function of BM-EPCs is still not fully understood. In the present study, BM-EPCs were isolated and cultured at an extracellular pH (pHe) of 6.5 or pHe 7.4 in vitro prior to various experiments being performed. Cellular proliferation, migration and tube formation ability were detected by DNA content quantification, Transwell assay and Matrigel-based angiogenesis assay. ELISA and western blot analysis measured protein secretion and expression, respectively. The results demonstrated that BM-EPCs cultured at pHe 6.5 compared with at pHe 7.4 demonstrated: Induced apoptosis; inhibited cellular proliferation, migration and adhesion; markedly reduced vascular endothelial growth factor (VEGF) expression; and the capacity to incorporate into vascular networks. Acidic pHe 6.5 induced ratio expression of B-cell lymphoma 2 (Bcl2)/Bcl2 associated X-protein (Bax), which in turn induced apoptosis, and inhibited cellular proliferation and other functional activities, with involvement of activation of VEGF receptor 2, protein kinase B and p38 mitogen activated protein kinase. These observations raise the possibility that the acidic extracellular environment may perform an important role in the vasculogenesis of BM-EPCs in tumor microenvironments. Therefore, culturing cancer cells at a lower pH that simulates endogenous tumor conditions may improve retention of the cellular heterogeneity identified in tumors.