Epigenetic deregulation of miR-29a and miR-1256 by isoflavone contributes to the inhibition of prostate cancer cell growth and invasion

The epigenetic regulation of genes has long been recognized as one of the causes of prostate cancer (PCa) development and progression. Recent studies have shown that a number of microRNAs (miRNAs) are also epigenetically regulated in different types of cancers including PCa. In this study, we found that the DNA sequence of the promoters of miR-29a and miR-1256 are partly methylated in PCa cells, which leads to their lower expression both in PCa cells and in human tumor tissues compared with normal epithelial cells and normal human prostate tissues. By real-time PCR, Western Blot analysis and miRNA mimic and 3′-UTR-Luc transfection, we found that TRIM68 is a direct target of miR-29a and miR-1256 and that the downregulation of miR-29a and miR-1256 in PCa cells leads to increased expression of TRIM68 and PGK-1 in PCa cells and in human tumor tissue specimens. Interestingly, we found that a natural agent, isoflavone, could demethylate the methylation sites in the promoter sequence of miR-29a and miR-1256, leading to the upregulation of miR-29a and miR-1256 expression. The increased levels of miR-29a and miR-1256 by isoflavone treatment resulted in decreased expression of TRIM68 and PGK-1, which is mechanistically linked with inhibition of PCa cell growth and invasion. The selective demethylation activity of isoflavone on miR-29a and miR-1256 leading to the suppression of TRIM68 and PGK-1 expression is an important biological effect of isoflavone, suggesting that isoflavone could be a useful non-toxic demethylating agent for the prevention of PCa development and progression.     

The clinical significance of circulating miR-21, miR-142, miR-143, and miR-146a in patients with prostate cancer

BackgroundProstate cancer (PCa) is the most common type of solid tissue cancer among men in western countries. In this study, we determined the levels of circulating miR-21, miR-142, miR-143, miR-146a, and RNU 44 levels as controls for early diagnosis of PCa.MethodsThe circulating miRNA levels in peripheral blood samples from 43 localized PCa patients, 12 metastatic PCa (MET) patients, and a control group of, 42 benign prostate hyperplasia (BPH) patients with a total of 97 volunteers were determined the by PCR method.ResultsNo differences in the DCT values were found among the groups. In PCa and PCaMet groups the expression of miR21 and miR142 were higher compared to the BHP group. No other differences were observed among the other groups. miR21 expression in the PCa group was 6.29 folds upregulated whereas in the PCaMet group 10.84 folds up-regulated. When the total expression of miR142 is evaluated, it showed a positive correlation with mir21 and mir 146 (both p<0.001). Also, the expression of miR146 shows a positive correlation with both miR21 and miR143 (both p<0.001). Expression of miRNAs was found to be an independent diagnostic factor in patients with Gleason score, PSA, and free PSA levels.ConclusionsOur study showed that co-expression of miR21, miR-142, miR-143, and miR-146a and the upregulation of miR-21 resulted in increased prostate carcinoma cell growth. In the PCaMet group, miR21 is the most upregulated of all miRNAs. These markers may provide a novel diagnostic tool to help diagnose PCa with aggressive behavior.     

Tumor-associated macrophages promote prostate cancer progression via exosome-mediated miR-95 transfer

Tumor-associated macrophages (TAMs) are vital constituents in mediating cell-to-cell communication within the tumor microenvironment. However, the molecular mechanisms underlying the interplay between TAMs and tumor cells that guide cell fate are largely undetermined. Extracellular vesicles, also known as exosomes, which are derived from TAMs, are the components exerting regulatory effects. Thus, understanding the underlying mechanism of “onco-vesicles” is of crucial importance for prostate cancer (PCa) therapy. In this study, we analyzed micro RNA sequences in exosomes released by THP-1 and M2 macrophages and found a significant increase in miR-95 levels in TAM-derived exosomes, demonstrating the direct uptake of miR-95 by recipient PCa cells. In vitro and in vivo loss-of-function assays suggested that miR-95 could function as a tumor promoter by directly binding to its downstream target gene, JunB, to promote PCa cell proliferation, invasion, and epithelial–mesenchymal transition. The clinical data analyses further revealed that higher miR-95 expression results in worse clinicopathological features. Collectively, our results demonstrated that TAM-mediated PCa progression is partially attributed to the aberrant expression of miR-95 in TAM-derived exosomes, and the miR-95/JunB axis provides the groundwork for research on TAMs to further develop more-personalized therapeutic approaches for patients with 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.     

Silencing LINC00491 inhibits prostate cancer development through the miR-384/TRIM44 axis

Accumulating evidence has demonstrated the key role of long noncoding (lnc)RNAs in tumorigenesis. Prostate cancer (PCa) is a cancer with high mortality that requires further exploration of the underlying molecular mechanisms. In the present study, we aimed to discover novel potential biomarkers for diagnosing PCa and targeting treatment. Overexpression of the lncRNA, LINC00491, was verified in PCa tumor tissues and cell lines using the real-time polymerase chain reaction. Cell proliferation and invasion were then analyzed via the Cell Counting Kit-8, colony formation, and transwell assays in vitro, and tumor growth in vivo. The interaction of miR-384 with LINC00491, as well as TRIM44, was investigated via bioinformatics analyses, subcellular fractionation, luciferase reporter gene assays, radioimmunoprecipitation, pull-down, and western blot analyses. LINC00491 was overexpressed in PCa tissues and cell lines. LINC00491 knockdown resulted in impaired cell proliferation and invasion in vitro and decreased tumor growth in vivo. Moreover, LINC00491 acted as a sponge for miR-384 and its downstream target, TRIM44. Additionally, miR-384 expression was downregulated in PCa tissues and cell lines, and its expression was negatively correlated with LINC00491. A miR-384 inhibitor restored the inhibitory effects of LINC00491 silencing on PCa cell proliferation and invasion. LINC00491 is a tumor promoter in PCa via enhancing TRIM44 expression by sponging miR-384 to facilitate the development of PCa. LINC00491 plays a significant role in PCa and could serve as both a biomarker for early diagnosis and a novel treatment target.     

Long noncoding RNA SNHG12 indicates the prognosis of prostate cancer and accelerates tumorigenesis via sponging miR-133b

Prostate cancer (PCa) is the second leading cause of death among American men. Increasing evidence has shown that long noncoding RNAs (lncRNAs) play important roles in tumorigenesis of PCa. In this study, we explored the biological functions of small nucleolar RNA host gene 12 (SNHG12) and investigated the interaction between miR-133b and SNHG12 in the progression of PCa. Data was downloaded from The Cancer Genome Atlas and Human Cancer Metastasis Database, and clinicopathological characteristics were analyzed with relapse-free survival rate. We detected SNHG12 expression level in PCa cells and tissues, and then analyzed its clinical significance, which revealed that SNHG12 has the potent to predict prognosis of PCa. Bioinformatic analysis revealed that SNHG12 was closely related to the progression of PCa and could target candidate microRNA (miR-133b). After transfecting SNHG12 silencing plasmid and miR-133b mimic/sponge, biological function assays were conducted and results illustrated that SNHG12 associated with miR-133b exerted biological effects on cancer cell growth, migration, and invasion. Direct interactions between miR-133b and SNHG12 have been found and SNHG12 acts as an oncogene to promote tumorigenesis of PCa by sponging tumor suppressor gene miR-133b.     

miR-888 is an expressed prostatic secretions-derived microRNA that promotes prostate cell growth and migration

microRNAs (miRNAs) are a growing class of small non-coding RNAs that exhibit widespread dysregulation in prostate cancer. We profiled miRNA expression in syngeneic human prostate cancer cell lines that differed in their metastatic potential in order to determine their role in aggressive prostate cancer. miR-888 was the most differentially expressed miRNA observed in human metastatic PC3-ML cells relative to non-invasive PC3-N cells, and its levels were higher in primary prostate tumors from cancer patients, particularly those with seminal vesicle invasion. We also examined a novel miRNA-based biomarker source called expressed prostatic secretions in urine (EPS urine) for miR-888 expression and found that its levels were preferentially elevated in prostate cancer patients with high-grade disease. These expression studies indicated a correlation for miR-888 in disease progression. We next tested how miR-888 regulated cancer-related pathways in vitro using human prostate cancer cell lines. Overexpression of miR-888 increased proliferation and migration, and conversely inhibition of miR-888 activity blocked these processes. miR-888 also increased colony formation in PC3-N and LNCaP cells, supporting an oncogenic role for this miRNA in the prostate. Our data indicates that miR-888 functions to promote prostate cancer progression and can suppress protein levels of the tumor suppressor genes RBL1 and SMAD4. This miRNA holds promise as a diagnostic tool using an innovative prostatic fluid source as well as a therapeutic target for aggressive prostate cancer.     

A transcriptional target of androgen receptor,miR-421 regulates proliferation and metabolism of prostate cancer cells

Prostate cancer is one of the most common malignancies, and microRNAs have been recognized to be involved in tumorigenesis of various kinds of cancer including prostate cancer (PCa). Androgen receptor (AR) plays a core role in prostate cancer progression and is responsible for regulation of numerous downstream targets including microRNAs. This study identified an AR-repressed microRNA, miR-421, in prostate cancer. Expression of miR-421 was significantly suppressed by androgen treatment, and correlated to AR expression in different prostate cancer cell lines. Furthermore, androgen-activated AR could directly bind to androgen responsive element (ARE) of miR-421, as predicted by bioinformatics resources and demonstrated by ChIP and luciferase reporter assays. In addition, over-expression of miR-421 markedly supressed cell viability, delayed cell cycle, reduced glycolysis and inhibited migration in prostate cancer cells. According to the result of miR-421 target genes searching, we focused on 4 genes NRAS, PRAME, CUL4B and PFKFB2 based on their involvement in cell proliferation, cell cycle progression and metabolism. The expression of these 4 downstream targets were significantly repressed by miR-421, and the binding sites were verified by luciferase assay. Additionally, we explored the expression of miR-421 and its target genes in human prostate cancer tissues, both in shared microarray data and in our own cohort. Significant differential expression and inverse correlation were found in PCa patients.     

Prognostic value of miR-21 for prostate cancer: a systematic review and meta-analysis

Elevated levels of miR-21 expression are associated with many cancers, suggesting it may be a promising clinical biomarker. In prostate cancer (PCa), however, there is still no consensus about the usefulness of miR-21 as an indicator of disease progression. This systematic review and meta-analysis was conducted to investigate the value of miR-21 expression as a prognostic measurement in PCa patients. Medline (Ovid), EMBASE, Web of Science, Scopus and Cochrane Library databases were systematically searched for relevant publications between 2010 to 2021. Studies exploring the relationship between miR-21 expression, PCa prognosis and clinicopathological factors were selected for review. Those reporting hazard ratio (HR) and 95% confidence intervals (CIs) were subject to meta-analyses. Fixed-effect models were employed to calculated pooled HRs and 95% CIs. Risk of bias in each study was assessed using QUIPS tool. Certainty of evidence in each meta-analysis was assessed using GRADE guidelines. A total of 64 studies were included in the systematic review. Of these, 11 were eligible for inclusion in meta-analysis. Meta-analyses revealed that high miR-21 expression was associated with poor prognosis: HR = 1.58 (95% CI = 1.19–2.09) for biochemical recurrence, MODERATE certainty; HR = 1.46 (95% CI = 1.06–2.01) for death, VERY LOW certainty; and HR = 1.26 (95% CI = 0.70–2.27) for disease progression, VERY LOW certainty. Qualitative summary revealed elevated miR-21 expression was significantly positively associated with PCa stage, Gleason score and risk groups. This systematic review and meta-analysis suggests that elevated levels of miR-21 are associated with poor prognosis in PCa patients. miR-21 expression may therefore be a useful prognostic biomarker in this disease.     

微RNA miR-29与人类疾病

人类微RNA miR-29包括miR-29a、miR-29b1、miR-29b2和miR-29c。miR-29在胚胎期组织不表达或低表达,而在生物体成熟组织细胞中广泛表达。实验证实的miR-29作用的靶点包括DNMT3A/3B、YY1、Mcl-1、BACE1以及HIV-1病毒等。miR-29与人类疾病密切相关,miR-29在多种肿瘤细胞、心肌梗塞与散发性阿尔茨海默病等病变组织细胞中表达受抑。本文就miR-29基因结构、基因表达与调控、生物学功能以及与疾病发生相关性作一综述。     

miR-195 Inhibits EMT by Targeting FGF2 in Prostate Cancer Cells

Prostate cancer (PCa) is one of the leading causes of deaths in America. The major cause of mortality can be attributed to metastasis. Cancer metastasis involves sequential and interrelated events. miRNAs and epithelial-mesenchymal transition (EMT) are implicated in this process. miR-195 is downregulated in many human cancers. However, the roles of miR-195 in PCa metastasis and EMT remain unclear. In this study, data from Memorial Sloan Kettering Cancer Center (MSKCC) prostate cancer database were re-analysed to detect miR-195 expression and its roles in PCa. miR-195 was then overexpressed in castration-resistant PCa cell lines, DU-145 and PC-3. The role of miR-195 in migration and invasion in vitro was also investigated, and common markers in EMT were evaluated through Western blot analysis. A luciferase reporter assay was conducted to confirm the target gene of miR-195; were validated in PCa cells. In MSKCC data re-analyses, miR-195 was poorly expressed in metastatic PCa; miR-195 could be used to diagnose metastatic PCa by measuring the corresponding expression. Area under the receiver operating characteristic curve (AUC-ROC) was 0.705 (P = 0.017). Low miR-195 expression was characterised with a shorter relapse-free survival (RFS) time. miR-195 overexpression suppressed cell migration, invasion and EMT. Fibroblast growth factor 2 (FGF2) was confirmed as a direct target of miR-195. FGF2 knockdown also suppressed migration, invasion and EMT; by contrast, increased FGF2 partially reversed the suppressive effect of miR-195. And data from ONCOMINE prostate cancer database showed that PCa patients with high FGF2 expression showed shorter RFS time (P = 0.046). Overall, this study demonstrated that miR-195 suppressed PCa cell metastasis by downregulating FGF2. miR-195 restoration may be considered as a new therapeutic method to treat metastatic PCa.     

Exosomal miR-375 promotes the activity of osteoblasts in prostate cancer

Studies have shown that exosomes influence tumour metastasis, diagnosis, and treatment. It has been found that exosomal miRNAs are closely linked to the metastatic microenvironment. However, the regulatory role of exosomes from prostate cancer (PCa) cells in bone metastasis remains poorly understood. Here, exosomes were isolated and purified by ultracentrifugation, total RNA from cells and total miRNA from exosomes were isolated, and the level of miR-375 was analyzed by RT-PCR. Exosome libraries from LNCaP cells and RWPE-1 cells were sequenced and filtered with an Illumina HiSeq^TM 2500 system. The activity of alkaline phosphatase, the extent of extracellular matrix mineralization, and the expression of osteoblast activity-related marker genes were measured to evaluate osteoblast activity. Morphological observation, particle size analysis, and molecular phenotyping confirmed that the isolated extracts contained exosomes. Differential expression analysis confirmed the high expression of miR-375 in LNCaP cell-derived exosomes. This study suggest that exosomes could enter osteoblasts and increase their miR-375 level. In addition, exosomal miR-375 could significantly promote the activity of osteoblasts.This study lays the foundation for further investigations on the function of exosomal miR-375 in the activation and differentiation of osteoblasts and the mechanism of bone metastasis in PCa.     

Targeting bone remodeling by isoflavone and 3,3'-diindolylmethane in the context of prostate cancer bone metastasis

Prostate cancer (PCa) bone metastases have long been believed to be osteoblastic because of bone remodeling leading to the formation of new bone. However, recent studies have shown increased osteolytic activity in the beginning stages of PCa bone metastases, suggesting that targeting both osteolytic and osteoblastic mediators would likely inhibit bone remodeling and PCa bone metastasis. In this study, we found that PCa cells could stimulate differentiation of osteoclasts and osteoblasts through the up-regulation of RANKL, RUNX2 and osteopontin, promoting bone remodeling. Interestingly, we found that formulated isoflavone and 3,3'-diindolylmethane (BR-DIM) were able to inhibit the differentiation of osteoclasts and osteoblasts through the inhibition of cell signal transduction in RANKL, osteoblastic, and PCa cell signaling. Moreover, we found that isoflavone and BR-DIM down-regulated the expression of miR-92a, which is known to be associated with RANKL signaling, EMT and cancer progression. By pathway and network analysis, we also observed the regulatory effects of isoflavone and BR-DIM on multiple signaling pathways such as AR/PSA, NKX3-1/Akt/p27, MITF, etc. Therefore, isoflavone and BR-DIM with their multi-targeted effects could be useful for the prevention of PCa progression, especially by attenuating bone metastasis mechanisms.     

Downregulation of miR-205 and miR-31 confers resistance to chemotherapy-induced apoptosis in prostate cancer cells

Advanced prostate cancers are known to acquire not only invasive capabilities but also significant resistance to chemotherapy-induced apoptosis. To understand how microRNAs (miRNAs) may contribute to prostate cancer resistance to apoptosis, we compared microRNA expression profiles of a benign prostate cancer cell line WPE1-NA22 and a highly malignant WPE1-NB26 cell line (derived from a common lineage). We found that miR-205 and miR-31 are significantly downregulated in WPE1-NB26 cells, as well as in other cell lines representing advanced-stage prostate cancers. Antiapoptotic genes BCL2L2 (encoding Bcl-w) and E2F6 are identified as the targets of miR-205 and miR-31, respectively. By downregulating Bcl-w and E2F6, miR-205 and miR-31 promote chemotherapeutic agents-induced apoptosis in prostate cancer cells. The promoter region of the miR-205 gene was cloned and was found to be hypermethylated in cell lines derived from advanced prostate cancers, contributing to the downregulation of the gene. Treatment with DNA methylation inhibitor 5-aza-2′-deoxycytidine induced miR-205 expression, downregulated Bcl-w, and sensitized prostate cancer cells to chemotherapy-induced apoptosis. Thus, downregulation of miR-205 and miR-31 has an important role in apoptosis resistance in advanced prostate cancer.     

miR-210 is induced by hypoxia and regulates neural cell adhesion molecule in prostate cells

Hypoxia in prostate tumours has been associated with disease progression and metastasis. MicroRNAs are short noncoding RNA molecules that are important in several cell processes, but their role in hypoxic signalling is still poorly understood. miR-210 has been linked with hypoxic mechanisms, but this relationship has been poorly characterised in prostate cancer. In this report, the link between hypoxia and miR-210 in prostate cancer cells is investigated. Polymerase chain reaction analysis demonstrates that miR-210 is induced by hypoxia in prostate cancer cells using in vitro cell models and an in vivo prostate tumour xenograft model. Analysis of The Cancer Genome Atlas prostate biopsy datasets shows that miR-210 is significantly correlated with Gleason grade and other clinical markers of prostate cancer progression. Neural cell adhesion molecule (NCAM) is identified as a target of miR-210, providing a biological mechanism whereby hypoxia-induced miR-210 expression can contribute to prostate cancer. This study provides evidence that miR-210 is an important regulator of cell response to hypoxic stress and proposes that its regulation of NCAM may play an important role in the pathogenesis of prostate cancer.     

Regulation and Methylation of Tumor Suppressor MiR-124 by Androgen Receptor in Prostate Cancer Cells

Prostate cancer (PCa) is the most frequently diagnosed cancer for men in the developed world. Androgen receptor signaling pathway plays an important role in prostate cancer progression. Recent studies show that microRNA miR-124 exerts a tumor suppressive function in prostate cancer. However, the relationship between AR and miR-124 is unclear. In the present study, we found a negative feedback loop between AR and miR-124 expression. On one hand, miR-124 was a positively regulated target gene of the AR, on the other hand, overexpression of miR-124 inhibited the expression of AR. In addition, we found that miR-124-2 and miR-124-3 promoters were hypermethylated in AR-negative PCa cells. Furthermore, overexpression of miR-124 inhibited proliferation rates and invasiveness capacity of PCa cells in vitro, and suppressed xenograft tumor growth in vivo. Taken together, our results support a negative feedback loop between AR and miR-124 expression. Methylation of miR-124-2 and miR-124-3 may serve as a biomarker for AR-negative PCa cells, and overexpression of miR-124 might be of potential therapeutic value for the treatment of PCa.     

LncRNA NEAT1 promotes docetaxel resistance in prostate cancer by regulating ACSL4 via sponging miR-34a-5p and miR-204-5p

Docetaxel resistance remains one of the main problems in clinical treatment of metastatic prostate cancer (PCa). Previous studies identified differently expressed lncRNAs in docetaxel-resistant PCa cell lines, while the potential mechanisms were still unknown. In the present study, we found NEAT1 was expressed at high levels in docetaxel-resistant PCa clinical samples and related cell lines. When knockdown NEAT1, cell proliferation and invasion in docetaxel-resistant PCa cells in vitro and in vivo were downregulated. Our further researches explained that NEAT1 exerts oncogenic function in PCa by competitively ‘sponging’ both miR-34a-5p and miR-204-5p. Inhibition of miR-34a-5p or miR-204-5p expression mimics the docetaxel-resistant activity of NEAT1, whereas ectopic expression of miR-34a-5p or miR-204-5p attenuates the anti-drug function of NEAT1 in PCa cells. Besides, we also found ACSL4 is a target of both miR-34a-5p and miR-204-5p, and ACSL4 was also inhibited by miR-34a-5p and miR-204-5p. Moreover, suppression of miR-34a-5p or/and miR-204-5p greatly restrained the expression of ACSL4 upon NEAT1 overexpression. Joint expression of miR-34a-5p and miR-204a-5p synergistically decreased the expression of ASCL4, indicating miR-34a-5p and miR-204a-5p collaboratively inhibit the expression of ACSL4. Innovatively, we concluded that NEAT1 contributes to the docetaxel resistance by increasing ACSL4 via sponging miR-34a-5p and miR-204-5p in PCa cells.