407040

MIR34A

Snail1 is a zinc finger transcriptional repressor whose pathological expression has been linked to cancer cell epithelial-mesenchymal transition (EMT) programs and the induction of tissue-invasive activity, but pro-oncogenic events capable of regulating Snail1 activity remain largely uncharacterized. Herein, we demonstrate that p53 loss-of-function or mutation promotes cancer cell EMT by de-repressing Snail1 protein expression and activity. In the absence of wild-type p53 function, Snail1-dependent EMT is activated in colon, breast, and lung carcinoma cells as a consequence of a decrease in miRNA-34 levels, which suppress Snail1 activity by binding to highly conserved 3' untranslated regions in Snail1 itself as well as those of key Snail1 regulatory molecules, including beta-catenin, LEF1, and Axin2. Although p53 activity can impact cell cycle regulation, apoptosis, and DNA repair pathways, the EMT and invasion programs initiated by p53 loss of function or mutation are completely dependent on Snail1 expression. These results identify a new link between p53, miR-34, and Snail1 in the regulation of cancer cell EMT programs.

Recently, the inhibition of epithelial-mesenchymal-transition (EMT) by p53 has been described as a new mode of tumor suppression which presumably prevents metastasis. Here we report that activation of p53 down-regulates the EMT-inducing transcription factor SNAIL via induction of the miR-34a/b/c genes. Suppression of miR-34a/b/c caused up-regulation of SNAIL and cells displayed EMT markers and related features, as enhanced migration and invasion. Ectopic miR-34a induced mesenchymal-epithelial-transition (MET) and down-regulation of SNAIL, which was mediated by a conserved miR-34a/b/c seed-matching sequence in the SNAIL 3'-UTR. miR-34a also down-regulated SLUG and ZEB1, as well as the stemness factors BMI1, CD44, CD133, OLFM4 and c-MYC. Conversely, the transcription factors SNAIL and ZEB1 bound to E-boxes in the miR-34a/b/c promoters, thereby repressing miR-34a and miR-34b/c expression. Since ectopic miR-34a prevented TGF-beta-induced EMT, the repression of miR-34 genes by SNAIL and related factors is part of the EMT program. In conclusion, the frequent inactivation of p53 and/or miR-34a/b/c found in cancer may shift the equilibrium of these reciprocal regulations towards the mesenchymal state and thereby lock cells in a metastatic state.

BACKGROUND: Hypoxia-induced renal tubular cell epithelial-mesenchymal transition (EMT) is an important event leading to renal fibrosis. MicroRNAs (miRNAs) are small non-coding RNA molecules that bind to their mRNA targets, thereby leading to translational repression. The role of miRNA in hypoxia-induced EMT is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: miRNA profiling was performed for the identification of differentially expressed miRNAs in HK-2 cells under normal and low oxygen, and the results were then verified by quantitative real time RT-PCR (qRT-PCR). The function of miRNAs in hypoxia-induced renal tubular cell EMT was assessed by the transfection of specific miRNA inhibitors and mimics. Luciferase reporter gene assays and western blot analysis were performed to validate the target genes of miR-34a. siRNA against Jagged1 was designed to investigate the role of the miR-34a-Notch pathway in hypoxia induced renal tubular cell EMT. miRNA-34a was identified as being downregulated in hypoxic renal tubular epithelial cells. Inhibition of miR-34a expression in HK-2 cells, which highly express endogenous miR-34a, promoted a mesenchymal phenotype accompanied by reduced expression of the epithelial marker Z0-1, E-cadherin and increased expression of the mesenchymal markers alpha-SMA and vimentin. Conversely, miR-34a mimics effectively prevented hypoxia-induced EMT. Transfection of miRNA-34a in HK-2 cells under hypoxia abolished hypoxia-induced expression of Notch1 and Jagged1 as well as Notch downstream signals, such as snail. Western blot analysis and luciferase reporter gene assays showed direct evidence for miR-34a targeting Notch1 and Jagged1. siRNAs against Jagged1 or Notch1 effectively prevented miR-34a inhibitor-induced tubular epithelial cell EMT. CONCLUSIONS/SIGNIFICANCE: Our study provides evidence that the hypoxia-induced decrease of miR-34a expression could promote EMT in renal tubular epithelial cells by directly targeting Notch1 and Jagged1, and subsequently, Notch downstream signaling.

The interactions between kidney and thyroid functions have been known for many years, but how the thyroid affects the kidney function is largely unknown. Here we analyzed the role of T3 on the tubular epithelial-to-mesenchymal transition (EMT), which is recognized to play pivotal roles in the process of renal fibrosis. T3 was found to significantly inhibit the TGFbeta1 induced EMT in human proximal tubular epithelial cell line HK-2. Meanwhile, T3 induced the expression of miR34a. Molecularly, the T3 receptor could directly bind the T3R recognition motif at the -1505 to -1526bp and -604 to -609bp regions in the miR34a promoter and transcriptionally activate the expression of miR34a upon T3 treatment. Inhibition of the miR34a by miR34a knockdown nearly blocked the effects of T3 on EMT. Taken together, our study here revealed that thyroid hormone T3 could inhibit TGFbeta1 induced renal tubular epithelial to mesenchymal transition by increasing miR34a expression.CI - Copyright (c) 2013 Elsevier Inc. All rights reserved.

UNLABELLED: The microRNA-34a (miR-34a), a tumor-suppressive microRNA (miRNA), is implicated in epithelial-mesenchymal transition (EMT) and cancer stem cells. Lymphoid enhancer-binding factor-1 (LEF1) is a key transcription factor in the Wnt signaling pathway, and has been suggested to be involved in regulation of cell proliferation and invasion. Here, the molecular mechanism of miR-34a and LEF1 in cooperatively regulating prostate cancer cell invasion is described. Molecular profiling analysis of miRNA levels in prostate cancer cells revealed a negative correlation between miR-34a and LEF1 expression, and the downregulation of LEF1 by miR-34a was confirmed by luciferase assays. Furthermore, miR-34a specifically repressed LEF1 expression through direct binding to its 3'-untranslated regions (3'-UTR). miR-34a modulated the levels of LEF1 to regulate EMT in prostate cancer cells. Functionally, miR-34a negatively correlated with the migration and invasion of prostate cancer cells through LEF1. An analysis of miR-34a expression levels in matched human tumor and benign tissues demonstrated consistent and statistically significant downregulation of miR-34a in primary prostate cancer specimens. These data strongly suggest that miR-34a/LEF1 regulation of EMT plays an important role in prostate cancer migration and invasion. IMPLICATIONS: The miR-34a-LEF1 axis represents a potential molecular target for novel therapeutic strategies in prostate cancer.CI - (c)2015 American Association for cancer Research.

MicroRNAs (miRs) are short non-coding single stranded RNAs regulating the translation of target mRNAs in normal and cancer cells in which they are frequently dysregulated promoting tumor progression. cancer stem cells (CSCs) of head and neck squamous cell carcinoma (HNSCC), identified by aldehyde-dehydrogenase expression (ALDH), are a cell subset within the tumor cell population that takes part in the genesis and progression of cancer. The relevance of epithelial-mesenchymal transition (EMT) has recently been recognized for tumor development and metastasis. Several studies have illustrated that miRs regulate EMT of CSC. CSC from 8 HNSCC lines, 4 of which are human papillomavirus (HPV)positive, were enriched by spheroid culture (spheroid-derived cells, SDC) and compared to their parental monolayer-derived cells (MDC) to analyze expression patterns of miR34a, CSC-related transcription factors (CSC-TFs: Sox2, Nanog, Oct3/4) and EMT-related TFs (EMT-TFs: Twist, Snail1, Snail2) by RT-qPCR. Flow cytometry was used to quantify and enrich ALDH+ CSCs. Transfection of miR34a mimics was used to evaluate its regulatory potential for CSC marker profiles as well as CSC- and EMT-TFs expression in HNSCC-SDC. Invasive, colony-forming and clonogenic capability of the miR34a mimics transfected SDC after sorting for ALDH+ and ALDH- cells was assessed by Matrigel invasion, clonogenicity and spheroid formation assay, respectively. miR34a expression levels were significantly downregulated in the majority of SDC derived from HNSCC-lines as compared to parental MDC (-1.6-16.4-fold). For EMT- and CSC-related TF expression, all HNSCC-derived SDC showed a significantly increased level compared to parental MDC (expression of ALDH was found in SDC (2-3-fold). Compared to the HPV+, the HPV- group showed a significantly higher mean expression level of EMT-TFs, CSCs-TFs and ALDH (30.3 v.s. 12.8%). Transfection of miR34a mimics significantly reduced the EMT- and CSC-related TF expression level in UM-SCC9 (HPV-) and UM-SCC47 (HPV+) SDC. Simultaneously, the ALDH expression was reduced significantly (1.5-2-fold) and the invasive capacity (EMT formation of CSC-phenotype and functionally reduced clonogenic and invasive capacity in HNSCC cell lines. Therapeutic modulation of miR34a in HNSCC and CSCs may reduce the rate of metastasis and recurrence of tumors after therapy.

BACKGROUND: In ovarian cancer (OC) cells, Snail was reported to induce the epithelial-to-mesenchymal transition (EMT), which is a critical step in OC metastasis. At present little is known about controlling Snail expression in OC cells by using specific microRNAs (miRNAs). METHODS: We first used a computational target prediction analysis to identify 6 candidate miRNAs that bind to the 3'-untranslated region (3'-UTR) region of the Snail mRNA. Among these miRNAs, two miRNAs (miR-137 and miR-34a) with a potential to regulate Snail were validated by quantitative real-time PCR, Western blot analysis, and Snail 3'-UTR reporter assays. We assessed the effects of miR-137 and miR-34a on EMT, invasion and sphere formation in OC cells. We also evaluated the expression of miR-137 and miR-34a in OC tissues and adjacent normal tissues and analyzed the relationship between their expression and patient survival. RESULTS: We report that OC tissues possess significantly decreased levels of miR-137 and miR-34a and increased expression of Snail when compared to their adjacent normal tissues, and lower miR-137 and miR-34a expression correlates with worse patient survival. Using luciferase constructs containing the 3'-UTR region of Snail mRNA combined with miRNA overexpression and mutagenesis, we identified miR-137 and miR-34a as direct suppressors of Snail in OC cells. The introduction of miR-137 and miR-34a resulted in the suppression of Snail at both the transcript and protein levels, and effectively suppressed the EMT phenotype and sphere formation of OC cells. However, the inhibition of miR-137 and miR-34a with antisense oligonucleotides promoted EMT and OC cell invasion. Moreover, ectopic expression of Snail significantly reversed the inhibitory effects of miR-137 and miR-34a on OC cell invasion and sphere formation. CONCLUSIONS: These findings suggest that both miR-137 and miR-34a act as Snail suppressors to negatively regulate EMT, invasive and sphere-forming properties of OC cells.

AIM: This study intented to clarify the intracellular effect of PAI-1 on Non-small cell lung cancer (NSCLC) metastasis and the precise mechanism involved. METHODS: The metastatic properties of NSCLC cells were determined by transwell assays and wound-healing assay in vitro. The mRNA and protein expressions of genes were analyzed by Real-time qPCR and western blot, respectively. Pulmonary metastasis model of NSCLC cells was established to evaluate the pro-metastasis effect of PAI-1 and anti-metastatic effect of miR-34a in vivo. The gene targets of miR-34a were confirmed by luciferase reporter assays. Chromatin immunoprecipitation assay was employed to detect the transcriptional regulation of miR-34a. Co-immunoprecipitation assay was performed to observe the interaction of proteins. RESULTS: PAI-1, which was elevated in NSCLC patients with recurrence and metastasis, augmented NSCLC metastasis and was negatively related to the prognosis of NSCLC. miR-34a, which was decreased in NSCLC patients with metastasis, attenuated NSCLC metastasis and was positively correlated with the prognosis of NSCLC. Moreover, PAI-1 was identified as the target gene of miR-34a and activated the Stat3 signaling pathway to promote epithelial-mesenchymal transition (EMT) in NSCLC cells. PAI-1 interacted with PIAS3 to regulate Stat3-dependent gene expression and miR-34a was transcriptionally suppressed by Stat3 to form a positive regulatory loop through Stat3 signaling. CONCLUSION: Our findings suggest that PAI-1 and miR-34a, which can be clinically utilized as biomarkers for the clinical prognosis or diagnosis of NSCLC, are potential targets for the treatment of NSCLC.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

Docetaxel resistance is a primary clinical obstacle in the therapy of advanced prostate cancer (PCa). Aberrant expression of miR-27b and miR-34a has been revealed to be implicated in drug resistance of different tumors. Nevertheless, the roles of miR-27b and miR-34a in docetaxel resistance of PCa and their molecular mechanisms are far from being elucidated. In this study, we found that miR-27b and miR-34a were significantly downregulated in docetaxel-resistant PCa cells. Gain-of-function experiments showed that overexpression of miR-27b or miR-34a enhanced docetaxel sensitivity and inhibited EMT in docetaxel-resistant PCa cells. Moreover, miR-27b and miR-34a was demonstrated to directly target ZEB1 and suppress ZEB1 expression. Loss-of-function analysis disclosed that ZEB1 knockdown enhanced docetaxel sensitivity and suppressed EMT in docetaxel-resistant PCa cells. Rescue experiments presented that ZEB1 overexpression abolished the effects of miR-27b or miR-34a overexpression on docetaxel sensitivity and EMT in docetaxel-resistant PCa cells. Finally, tumor xenograft assay confirmed the contribution of miR-27b and miR-34a in improving docetaxel sensitivity in PCa in vivo. In summary, miR-27b and miR-34a overexpression enhanced docetaxel sensitivity of PCa partly through inhibiting EMT by targeting ZEB1, providing new insights into the molecular mechanism of miR-27b and miR-34a in modulating docetaxel resistance and potential therapy targets in advanced PCa.CI - Copyright (c) 2017 Elsevier Masson SAS. All rights reserved.

epithelial-mesenchymal transition (EMT) is considered a prerequisite for tumor invasion and metastasis in many cancers. However, the mechanisms underlying EMT in nasopharyngeal carcinoma (NPC) is largely unknown. In this study, we found that transforming growth factor-beta (TGF-beta), which reportedly promotes EMT in multiple cancers, can trigger EMT and increase the invasive and migratory capacities of NPC cells. Conversely, the downregulation of SMAD4, a vital member of the canonical TGF-beta pathway, reversed the TGF-beta-induced EMT, invasion, and migration. Further experiments revealed that SMAD4 was the target of miRNA-34a, which was downregulated in NPC tissues and suppressed NPC cell metastasis in vivo. miRNA-34a overexpression also antagonized the TGF-beta-induced EMT progression, invasion, and migration through SMAD4 inhibition. However, the restoration of SMAD4 expression rescued the inhibitory effects of miRNA-34a on tumorigenesis. All these results confirmed that miRNA-34a suppressed the TGF-beta-induced EMT, invasion, and migration of NPC cells by directly targeting SMAD4, which indicated the potential of miR-34a as a therapeutic target against NPC.CI - Copyright (c) 2018 Elsevier Masson SAS. All rights reserved.

The long noncoding RNA (LncRNA) SNHG7 (small nuclear RNA host gene 7) is a novel LncRNA and functions as an oncogene to promote tumor proliferation and inhibits apoptosis in many cancers. However, the role of SNHG7 in osteosarcoma (OS) remains to be further investigated. In this study, OS tissues and corresponding relative normal tissues (n = 30) were collected to determine the expression of SNHG7 in OS. The results indicated that high SNHG7 level in OS correlated with high Enneking stage, distant metastasis, and short overall survival time of OS patients. Moreover, miRNA-34a (miR-34a) is a classic tumor suppressor. Bioinformatic analysis predicted that SNHG7 harbored miR-34a binding sites, and the authors found that SNHG7 negatively correlated with miR-34a in OS tissues and the SNHG7 inhibition induced the restoration of miR-34a in OS cell lines MG63 and SaOS2, leading to the reactivation of miR-34a-mediated tumor suppression. Knockdown of SNHG7 in tumor cells significantly impaired the cell vitality, migration and invasion or TGF-beta-induced epithelial-mesenchymal transition (EMT), induced apoptosis, and G1/S arrest via miR-34a. Mechanistically, the targets of miR-34a could be upregulated by SNHG7, including proliferation-related Notch1, apoptosis-related BCL-2, cell cycle-related CDK6, and EMT-related SMAD4. The oncogene role of SNHG7 in vivo was also confirmed and found that knockdown of SNHG7 delayed the tumor growth with increased miR-34a level and Ki-67 level in OS tissues. These findings demonstrated that the LncRNA SNHG7 is upregulated during the development of OS via inhibition of tumor suppressor miR-34s signals.