miR-154 inhibits EMT by targeting HMGA2 in prostate cancer cells

Epithelial–mesenchymal transition (EMT) is a crucial process that plays an important role in the invasion and metastasis of human cancers. High-mobility group AT-hook 2 (HMGA2) has been found to be involved in the EMT program, with its aberrant expression having been observed in a variety of malignant tumors. However, the mechanisms regulating HMGA2 expression remain incompletely understood. The objective of this study was to investigate whether mir-154 plays a critical role in EMT by regulating HMGA2. The expression levels of HMGA2 were examined in four samples of prostate cancer (PCa) tissue and adjacent non-tumorous tissue by Western blot analysis. The effects of forced expression of miR-154 or HMGA2 knockdown on PCa cells were evaluated by cell migration and invasion assays and Western blot analysis. HMGA2 was upregulated in the PCa tissue samples compared with the adjacent normal ones. Forced expression of miR-154 or HMGA2 knockdown significantly reduced the migratory and invasive capabilities of PCa cells in vitro and inhibited EMT gene expression, increased the levels of E-cadherin, an epithelial marker, and decreased the levels of vimentin, a mesenchymal marker. HMGA2 is a direct target gene of miR-154 by dual-luciferase reporter assay. Our findings suggest that miR-154 plays a role in regulating EMT by targeting HMGA2. Understanding the targets and regulating pathways of miR-154 may provide new insights into the underlying pathogenesis of PCa.     

HEF1 promotes epithelial mesenchymal transition and bone invasion in prostate cancer under the regulation of microRNA‐145

The principal problem arising from prostate cancer (PCa) is its propensity to metastasize to bones, and it's crucial to understand the mechanism of tumor progression to metastasis in order to develop therapies that may reduce the morbidity and mortality of PCa patients. Although we had identified that microRNA(miR)‐145 could repress bone metastasis of PCa via regulating epithelial–mesenchymal transition (EMT) in previous study, it is still unknown how miR‐145 regulated EMT. In the present study, we constructed a luciferase reporter system and identified HEF1 as a direct target of miR‐145. More importantly, HEF1 was shown to promote migration, invasion and EMT of PC‐3 cells, a human PCa cell line originated from a bone metastatic PCa specimen. And HEF1 was also shown to partially mediate miR‐145 suppression of EMT and invasion. Furthermore, inhibition of HEF1 repressed bone invasion of PC‐3 cells in vivo. Expression of HEF1 was negatively correlated with miR‐145 in primary PCa and bone metastatic specimens, but HEF1 was higher in samples which were more likely to commit to bone metastasis or those with higher free prostate‐specific antigen (fPSA) levels and Gleason scores. Taken together, these findings indicate that HEF1 promotes EMT and bone invasion in prostate cancer by directly targeted by miR‐145, and miR‐145 suppresses EMT and invasion, at least in part, through repressing HEF1. J. Cell. Biochem. 114: 1606–1615, 2013. © 2013 Wiley Periodicals, Inc.     

Long noncoding RNA SNHG12 promotes cell proliferation and activates Wnt/β-catenin signaling in prostate cancer through sponging microRNA-195

Long noncoding RNAs (lncRNAs) serve critical roles in multiple human malignant tumors, including prostate cancer (PCa). Currently, the biological role of oncogenic lncRNA SNHG12 in PCa remains largely unclear. In the present study, we found that SNHG12 was highly expressed in human PCa tissues and cell lines. In addition, gain-of-function and loss-of-function studies showed that overexpression of SNHG12 promoted, while downregulation suppressed the proliferation, invasion, and migration of PCa cells in vitro. Knockdown of SNHG12 also repressed PCa xenograft tumor growth in vivo. Further in-depth mechanistic studies showed that SNHG12 might serve as a competing endogenous RNA for miR-195 in PCa cells, and miR-195 expression level was negatively associated with the expression of SNHG12 in PCa tissues. Finally, we found that the activity of Wnt/β-catenin signaling is enhanced by SNHG12 overexpression and rescued by co-transfection with miR-195 mimics in PCa cells. Collectively, the present study indicated the oncogenic function of SNHG12 in PCa and our findings might provide a new target in the treatment of PCa.     

Stromal-induced downregulation of miR-1247 promotes prostate cancer malignancy

Cancer progression is strictly dependent on the relationship between tumor cells and the surrounding stroma, which supports cancer malignancy promoting several crucial steps of tumor progression, including the execution of the epithelial to mesenchymal transition (EMT) associated with enhancement in cell invasion, resistance to both anoikis and chemotherapeutic treatments. Recently it has been highlighted the central role of microRNAs (miRNAs) as regulators of tumor progression. Notably, in several tumors a strong deregulation of miRNAs is observed, supporting proliferation, invasion, and metabolic reprogramming of tumor cells. Here we demonstrated that cancer-associated fibroblasts induce a downregulation of miR-1247 in prostate cancer (PCa) cells. We proved that miR-1247 repression is functional for the achievement of EMT and increased cell invasion as well as stemness traits. These phenomena contribute to promote the metastatic potential of PCa cells as demonstrated by increased lung colonization in in vivo experiments. Moreover, as a consequence of miR-1247 downregulation, we observed a correlated increased expression level of neuropilin-1, a miR-1247 target involved as a coreceptor in the epidermal growth factor receptor signaling. Taken together, our data highlight miR-1247 as a potential target for molecular therapies aimed to block the progression and diffusion of PCa.     

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.     

MicroRNA-7 Inhibits Tumor Metastasis and Reverses Epithelial-Mesenchymal Transition through AKT/ERK1/2 Inactivation by Targeting EGFR in Epithelial Ovarian Cancer

Epidermal growth factor receptor (EGFR) overexpression and activation result in increased proliferation and migration of solid tumors including ovarian cancer. In recent years, mounting evidence indicates that EGFR is a direct and functional target of miR-7. In this study, we found that miR-7 expression was significantly downregulated in highly metastatic epithelial ovarian cancer (EOC) cell lines and metastatic tissues, whereas the expression of, EGFR correlated positively with metastasis in both EOC patients and cell lines. Overexpression of miR-7 markedly suppressed the capacities of cell invasion and migration and resulted in morphological changes from a mesenchymal phenotype to an epithelial-like phenotype in EOC. In addition, overexpression of miR-7 upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, accompanied with EGFR inhibition and AKT/ERK1/2 inactivation. Similar to miR-7 transfection, silencing of EGFR with this siRNA in EOC cells also upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, and decreased phosphorylation of both Akt and ERK1/2, confirming that EGFR is a target of miR-7 in reversing EMT. The pharmacological inhibition of PI3K-AKT and ERK1/2 both significantly enhanced CK-18 and β-catenin expression and suppressed vimentin expression, indicating that AKT and ERK1/2 pathways are required for miR-7 mediating EMT. Finally, the expression of miR-7 and EGFR in primary EOC with matched metastasis tissues was explored. It was showed that miR-7 is inversely correlated with EGFR. Taken together, our results suggested that miR-7 inhibited tumor metastasis and reversed EMT through AKT and ERK1/2 pathway inactivation by reducing EGFR expression in EOC cell lines. Thus, miR-7 might be a potential prognostic marker and therapeutic target for ovarian cancer metastasis intervention.     

MUC15 loss facilitates epithelial-mesenchymal transition and cancer stemness for prostate cancer metastasis through GSK3β/β-catenin signaling

Patients with prostate cancer (PCa) have a high incidence of relapse and metastasis. Unfortunately, the molecular mechanisms underlying these processes have not been fully elucidated. In our study, we demonstrate that MUC15, a member of the mucin family, is a novel tumor suppressor in PCa that modulates epithelial-mesenchymal transition (EMT) and cancer stemness, contributing to PCa metastasis. First, MUC15 expression was found to be decreased in PCa tissues compared with para-carcinoma tissues. Moreover, we observed that MUC15 suppressed cell migration and invasion, both in vitro and in vivo, but had no effect on cell proliferation. Mechanistically, knockdown of MUC15 increased GSK3β phosphorylation and promoted β-catenin nuclear translocation. Therefore, the β-catenin-specific inhibitors XAV939 and PRI-724 rescued EMT in MUC15-deficient cell lines. Taken together, these results indicate that MUC15 is downregulated in PCa tissues and serves as a potential target to prevent PCa metastasis, which can inhibit EMT and cancer stemness via the GSK3β/β-catenin signaling pathway.     

Epithelial to Mesenchymal Transition Is Mechanistically Linked with Stem Cell Signatures in Prostate Cancer Cells

Background

Current management of patients diagnosed with prostate cancer (PCa) is very effective; however, tumor recurrence with Castrate Resistant Prostate Cancer (CRPC) and subsequent metastasis lead to poor survival outcome, suggesting that there is a dire need for novel mechanistic understanding of tumor recurrence, which would be critical for designing novel therapies. The recurrence and the metastasis of PCa are tightly linked with the biology of prostate cancer stem cells or cancer-initiating cells that is reminiscent of the acquisition of Epithelial to Mesenchymal Transition (EMT) phenotype. Increasing evidence suggests that EMT-type cells share many biological characteristics with cancer stem-like cells.

Methodology/Principal Findings

In this study, we found that PCa cells with EMT phenotype displayed stem-like cell features characterized by increased expression of Sox2, Nanog, Oct4, Lin28B and/or Notch1, consistent with enhanced clonogenic and sphere (prostasphere)-forming ability and tumorigenecity in mice, which was associated with decreased expression of miR-200 and/or let-7 family. Reversal of EMT by re-expression of miR-200 inhibited prostasphere-forming ability of EMT-type cells and reduced the expression of Notch1 and Lin28B. Down-regulation of Lin28B increased let-7 expression, which was consistent with repressed self-renewal capability.

Conclusions/Significance

These results suggest that miR-200 played a pivotal role in linking the characteristics of cancer stem-like cells with EMT-like cell signatures in PCa. Selective elimination of cancer stem-like cells by reversing the EMT phenotype to Mesenchymal-Epithelial Transition (MET) phenotype using novel agents would be useful for the prevention of tumor recurrence especially by eliminating those cells that are the “Root Cause” of tumor development and recurrence.     

Long noncoding RNA LEF1-AS1 silencing suppresses the initiation and development of prostate cancer by acting as a molecular sponge of miR-330-5p via LEF1 repression

Prostate cancer (PCa) is one of the major cancers affecting males with high mortality around the world. Recent studies have found that some long noncoding RNAs play a critical part in the cellular processes of PCa. In our study, aberrant expressed lymphoid enhancer-binding factor-1 antisense RNA 1 (LEF1-AS1), microRNA-330-5p (miR-330-5p), and lymphoid enhancer-binding factor-1 (LEF1) were screened out from a microarray database, the role of the novel noncoding RNA regulatory circuitry in the initiation and development of PCa was investigated. LEF1-AS1 and LEF1 were highly expressed while miR-330-5p was poorly expressed in PCa. Following that, the PCa PC-3 cell line was adopted for subsequently experiments, in which the expression of LEF1-AS1 and miR-330-5p was subsequently altered by means of exogenous transfection. After that, the effects of up- or downregulation of LEF1-AS1 and miR-330-5p on epithelial–mesenchymal transition (EMT) and the cell ability for proliferation, invasion, migration in vitro, and tumorigenesis and lymph node metastasis (LNM) in vivo were evaluated. RNA crosstalk revealed that LEF1-AS1 bound to miR-330-5p and LEF1 was the target gene of miR-330-5p. Silenced LEF1-AS1 or elevated miR-330-5p exhibited inhibited EMT processes, reduced ability of proliferation, invasion and migration, coupling with decreased tumorigenesis and LNM in nude mice. The key findings of this study collectively propose downregulation of LEF1-AS1 competing with miR-330-5p to inhibit EMT, invasion and migration of PCa by LEF1 repression.     

miR-19a-3p在前列腺癌细胞侵袭和迁移中的作用

miR-19a-3p通过多种机制调节癌细胞的增殖和转移,然而,miR-19a-3p在前列腺癌转移中的生物学作用和机制尚不清楚。本研究旨在考察mir-19a-3p在前列腺癌中的表达情况,及其对细胞侵袭和迁移的影响。本研究发现,miR-19a-3p在骨转移性前列腺癌组织和前列腺癌细胞中明显下调。上调miR-19a-3p可显著抑制前列腺癌细胞的侵袭和迁移。然而,下调miR-19a-3p则会逆转上述变化。此外,本研究还发现miR-19a-3p通过靶向TGF-β信号传导的下游效应物SMAD2来抑制前列腺癌细胞中TGF-β信号传导的活性,从而抑制前列腺癌细胞的侵袭和迁移。因此,本研究表明mir-19a-3p与前列腺癌的发生发展密切相关,mir-19a-3p有望成为前列腺癌的新治疗靶点及生物标志物。     

FOXO3a inhibits the EMT and metastasis of breast cancer by regulating TWIST-1 mediated miR-10b/CADM2 axis

BackgroundBreast cancer is the most common malignancy and has been considered as a leading cause of cancer death in women. Exploring the mechanism of breast cancer metastasis is extremely important for seeking novel therapeutic strategies and improving prognosis.MethodsClinical specimens and pathological characteristics were collected for evaluating the expression of forkhead box class O 3a (FOXO3a) and twist-related protein 1 (TWIST-1) in breast cancer tissues. CCK-8 assay was used to analyze cell proliferation. Cell invasion and migration were assessed by transwell assays. The expression of FOXO3a, TWIST-1, miR-10b, CADM2, FAK, phosphor-AKT and the epithelial-mesenchymal transition (EMT)-related protein (N-cadherin, E-cadherin and vimentin) were analyzed by RT-qPCR, immunohistochemical staining, immunofluorescence assay or western blot, respectively. Xenograft mouse models were used to analyze the role of the FOXO3a in breast cancer.ResultsFOXO3a was down-regulated and TWIST-1 was up-regulated in breast cancer tissues. Overexpression of FOXO3a or knockdown of TWIST-1 suppressed the proliferation, invasion, migration and EMT of breast cancer cells. Overexpression of TWIST-1 could reverse the effect of FOXO3a on the proliferation, invasion, migration and EMT of breast cancer. Moreover, FOXO3a suppressed the growth and metastasis of breast cancer by targeting TWIST1 in vivo.ConclusionFOXO3a inhibited the EMT and metastasis of breast cancer via TWIST-1/miR-10b/CADM2 axis.     

MicroRNA-197 induces epithelial–mesenchymal transition and invasion through the downregulation of HIPK2 in lung adenocarcinoma

The major cause of cancer-related deaths in patients with lung adenocarcinoma (LAD) is due to distant metastasis. Many reports have indicated that miRNA plays a key role in tumour metastasis. The expression of miR-197 is correlated with LAD progression, however, the mechanism of miR-197 is still unknown in the processing of LAD. A Boyden chamber migration/invasion assay was used for the metastatic function study in vitro. Real-time PCR and Western blot assays were employed to analyse the EMT hallmark changes in both the mRNA and protein levels. \(3^{\prime }\)-UTR reporter luciferase assay was used to show that HIPK2 is a direct target of miR-197. miR-197 enhances LAD cell migration and invasion miR-197. The downregulation of miR-197 suppresses the EMT and migration ability. HIPK2 is a direct functional target of miR-197 in LAD metastasis. In summary, miR-197 controls EMT and metastasis by directly silencing HIPK2. The findings suggest that interfering with the miR-197-dependent regulation of HIPK2 could be a useful approach for the treatment of patients with late stage metastatic LAD.     

Exosomal long noncoding RNA HOXD-AS1 promotes prostate cancer metastasis via miR-361-5p/FOXM1 axis

Development of distant metastasis is the main cause of deaths in prostate cancer (PCa) patients. Understanding the mechanism of PCa metastasis is of utmost importance to improve its prognosis. The role of exosomal long noncoding RNA (lncRNA) has been reported not yet fully understood in the metastasis of PCa. Here, we discovered an exosomal lncRNA HOXD-AS1 is upregulated in castration resistant prostate cancer (CRPC) cell line derived exosomes and serum exosomes from metastatic PCa patients, which correlated with its tissue expression. Further investigation confirmed exosomal HOXD-AS1 promotes prostate cancer cell metastasis in vitro and in vivo by inducing metastasis associated phenotype. Mechanistically exosomal HOXD-AS1 was internalized directly by PCa cells, acting as competing endogenous RNA (ceRNA) to modulate the miR-361-5p/FOXM1 axis, therefore promoting PCa metastasis. In addition, we found that serum exosomal HOXD-AS1 was upregulated in metastatic PCa patients, especially those with high volume disease. And it is correlated closely with Gleason Score, distant and nodal metastasis, Prostatic specific antigen (PSA) recurrence free survival, and progression free survival (PFS). This sheds a new insight into the regulation of PCa distant metastasis by exosomal HOXD-AS1 mediated miR-361-5p/FOXM1 axis, and provided a promising liquid biopsy biomarker to guide the detection and treatment of metastatic PCa.Subject terms: Bone metastases, Prostate cancer     

Tumour-suppressive microRNA-224 inhibits cancer cell migration and invasion via targeting oncogenic TPD52 in prostate cancer

Our recent study of the microRNA expression signature of prostate cancer (PCa) revealed that microRNA-224 (miR-224) is significantly downregulated in PCa tissues. Here, we found that restoration of miR-224 significantly inhibits PCa cell migration and invasion. Additionally, we found that oncogenic TPD52 is a direct target of miR-224 regulation. Silencing of the TPD52 gene significantly inhibits cancer cell migration and invasion. Moreover, TPD52 expression is upregulated in cancer tissues and negatively correlates with miR-224 expression. We conclude that loss of tumour-suppressive miR-224 enhances cancer cell migration and invasion in PCa through direct regulation of oncogenic TPD52.     

The microRNA -23b/-27b Cluster Suppresses the Metastatic Phenotype of Castration-Resistant Prostate Cancer Cells

MicroRNAs (miRs) are small, endogenous, non-coding RNAs that regulate the stability and/or translation of complementary mRNA targets. MiRs have emerged not only as critical modulators of normal physiologic processes, but their deregulation may significantly impact prostate and other cancers. The expression of miR-23b and miR-27b, which are encoded by the same miR cluster (miR-23b/-27b), are downregulated in metastatic, castration-resistant tumors compared to primary prostate cancer and benign tissue; however, their possible role in prostate cancer progression is unknown. We found that ectopic expression of miR-23b/-27b in two independent castration-resistant prostate cancer cell lines resulted in suppression of invasion and migration, as well as reduced survival in soft agar (a measure of anoikis). However, there was no effect of miR-23b/-27b on cell proliferation suggesting that these miRs function as metastasis (but not growth) suppressors in prostate cancer. Conversely, inhibition of miR-23b/-27b in the less aggressive androgen-dependent LNCaP prostate cancer cell line resulted in enhanced invasion and migration also without affecting proliferation. Mechanistically, we found that introduction of miR-23b/-27b in metastatic, castration-resistant prostate cancer cell lines resulted in a significant attenuation of Rac1 activity without affecting total Rac1 levels and caused increased levels of the tumor suppressor E-cadherin. Inhibition of these miRs had the opposite effect in androgen-dependent LNCaP cells. These results suggest that miR-23b/-27b are metastasis suppressors that might serve as novel biomarkers and therapeutic agents for castration-resistant disease.     

PAX3 silencing inhibits prostate cancer progression through the suppression of the TGF‐β/Smad signaling axis

Multiple studies have confirmed the pro‐oncogenic effects of PAX3 in an array of cancers, but its role in prostate cancer (PCa) remains largely undefined. The aim of this study is to investigate the role of PAX3 in PCa. PAX3 expression was compared between PCa tumor tissue and nontumor tissues and PCa cell lines and normal prostate epithelial cells (PNT2) by western blot analysis and immunohistochemistry staining. MTT and immunofluorescence assays were used to detect PCa cell proliferation. Flow cytometry was used to evaluate cell apoptosis in PCa. Transwell assays were used for the determination of cell migration and PCa cell invasion. PAX3 expression was higher in PCa tissues and human PCa cell lines. Moreover, PAX3 silencing inhibited the proliferation, metastasis, and epithelial–mesenchymal transition (EMT) of PCa cells, and increased the rates of apoptosis. PAX3 silencing inhibited transforming growth factor‐β (TGF‐β)/Smad signaling in PCa cells. The effects of si‐PAX3 on the proliferation, apoptosis, metastasis, and EMT of PCa cells were alleviated by TGF‐β1 treatment. PAX3 silencing inhibits PCa progression through the inhibition of TGF‐β/Smad signaling. This reveals PAX3 as a novel biomarker and therapeutic target for future PCa treatments.     

miR-219-5p targets TBXT and inhibits breast cancer cell EMT and cell migration and invasion

miR-219-5p has been reported to act as either a tumor suppressor or a tumor promoter in different cancers by targeting different genes. In the present study, we demonstrated that miR-219-5p negatively regulated the expression of TBXT, a known epithelial–mesenchymal transition (EMT) inducer, by directly binding to TBXT 3′-untranslated region. As a result of its inhibition on TBXT expression, miR-219-5p suppressed EMT and cell migration and invasion in breast cancer cells. The re-introduction of TBXT in miR-219-5p overexpressing cells decreased the inhibitory effects of miR-219 on EMT and cell migration and invasion. Moreover, miR-219-5p decreased breast cancer stem cell (CSC) marker genes expression and reduced the mammosphere forming capability of cells. Overall, our study highlighted that TBXT is a novel target of miR-219-5p. By suppressing TBXT, miR-219-5p plays an important role in EMT and cell migration and invasion of breast cancer cells.