Long non-coding RNA SIX1-1 promotes proliferation of cervical cancer cells via negative transcriptional regulation of RASD1.

Cervical cancer poses a significant health burden for women globally, and the rapid proliferation of cervical cancer cells greatly worsens patient prognosis. Long non-coding RNAs (lncRNAs) play a crucial role in regulating tumor cell proliferation. However, the involvement of lncRNAs in cervical cancer cell proliferation remains unclear. In this study, we investigated the lncRNA SIX1-1, which was found to be upregulated in cervical cancer tissues and cell lines. Functional assays revealed that knockdown of SIX1-1 inhibited cell proliferation in vitro and reduced tumor growth in vivo. Mechanistically, SIX1-1 was predominantly localized in the nucleus and could bind with DNMT1 protein. The expression of SIX1-1 enhanced the interaction of DNMT1 with RASD1 promoter, leading to the methylation of the promoter and decreased mRNA transcription. Then RASD1 downregulation activated the cAMP/PKA/CREB signaling pathway, promoting cell proliferation. Rescue experiments showed that knockdown of RASD1 restored the inhibited cell proliferation caused by decreased expression of SIX1-1, indicating that RASD1 acted as the functional mediator of SIX1-1. In conclusion, SIX1-1 promoted cervical cancer cell proliferation by modulating RASD1 expression. This suggests that targeting the SIX1-1/RASD1 axis could be a potential antitumor strategy for cervical cancer.

KIAA1429 promotes gastric cancer progression by destabilizing RASD1 mRNA in an m6A-YTHDF2-dependent manner.

BACKGROUND: KIAA1429, a regulatory subunit of the N6-methyladenosine (m6A) methyltransferase complex, has been implicated in the progression of various cancers. However, the role of KIAA1429 in gastric cancer (GC) and its underlying mechanisms remain elusive. This study aimed to investigate the role of KIAA1429 in GC and to elucidate the underlying mechanisms. METHODS: The expression patterns and clinical relevance of KIAA1429 in GC were assessed using quantitative real-time PCR (qRT-PCR), Western blotting, immunohistochemistry (IHC), and bioinformatic analysis. In vitro and in vivo loss- and gain-of-function assays, m6A dot blot assays, methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA-seq, MeRIP-qPCR, dual luciferase reporter assays, RNA stability assays, RNA immunoprecipitation (RIP) assays, and RNA pull-down assays were performed to investigate the biological functions and underlying molecular mechanisms of KIAA1429 in GC. RESULTS: Both the mRNA and protein expression of KIAA1429 were greater in GC tissues than in normal gastric tissues. High KIAA1429 expression correlated positively with poor prognosis in GC patients. KIAA1429 not only promoted GC cell proliferation, colony formation, G2/M cell cycle transition, migration, and invasion in vitro but also enhanced GC tumor growth and metastasis in vivo. Mechanistically, KIAA1429 increased the m6A level of RASD1 mRNA and enhanced its stability in an m6A-YTHDF2-dependent manner, thereby upregulating its expression. RASD1 knockdown partially rescued the KIAA1429 knockdown-induced impairment of pro­oncogenic ability in GC cells. The expression levels of KIAA1429 and RASD1 were negatively correlated in GC tissues. CONCLUSIONS: KIAA1429 plays a pro­oncogenic role in GC by downregulating RASD1 expression through destabilizing RASD1 mRNA in an m6A-YTHDF2-dependent manner. KIAA1429 may serve as a prognostic biomarker and therapeutic target for GC.

Rasd1 is involved in white matter injury through neuron-oligodendrocyte communication after subarachnoid hemorrhage.

AIMS: Rasd1 has been reported to be correlated with neurotoxicity, metabolism, and rhythm, but its effect in case of subarachnoid hemorrhage (SAH) remained unclear. White matter injury (WMI) and ferroptosis participate in the early brain injury (EBI) after SAH. In this work, we have investigated whether Rasd1 can cause ferroptosis and contribute to SAH-induced WMI. METHODS: Lentivirus for Rasd1 knockdown/overexpression was administrated by intracerebroventricular (i.c.v) injection at 7 days before SAH induction. SAH grade, brain water content, short- and long-term neurobehavior, Western blot, real-time PCR, ELISA, biochemical estimation, immunofluorescence, diffusion tensor imaging (DTI), and transmission electron microscopy (TEM) were systematically performed. Additionally, genipin, a selective uncoupling protein 2(UCP2) inhibitor, was used in primary neuron and oligodendrocyte co-cultures for further in vitro mechanistic studies. RESULTS: Rasd1 knockdown has improved the neurobehavior, glia polarization, oxidative stress, neuroinflammation, ferroptosis, and demyelination. Conversely, Rasd1 overexpression aggravated these changes by elevating the levels of reactive oxygen species (ROS), inflammatory cytokines, MDA, free iron, and NCOA4, as well as contributing to the decrease of the levels of UCP2, GPX4, ferritin, and GSH mechanistically. According to the in vitro study, Rasd1 can induce oligodendrocyte ferroptosis through inhibiting UCP2, increasing reactive oxygen species (ROS), and activating NCOA4-mediated ferritinophagy. CONCLUSIONS: It can be concluded that Rasd1 exerts a modulated role in oligodendrocytes ferroptosis in WMI following SAH.

Sevoflurane protects against ischemia-reperfusion injury in mice after total knee arthroplasty via facilitating RASD1-mediated protein kinase A pathway activation.

This study aimed to explore effects of Sevoflurane on ischemia-reperfusion (I/R) injury after total knee arthroplasty (TKA). To explore potential molecular mechanism, Ras related dexamethasone induced 1 (RASD1), a Protein kinase A (PKA) activator, frequently associated with various models of I/R injury, was also investigated. In vivo mouse models with I/R injury after TKA and in vitro cell models with I/R injury were induced. Contents of creatinine kinase (CK), lactic dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA), serum levels of inflammatory factors, expression of PKA pathway-related genes and cell proliferation and apoptosis were measured. RASD1 was altered and PKA pathway was inhibited in mice and cells to elucidate the involvement of RASD1 and PKA pathway in Sevoflurane treatment on I/R injury. RASD1 was upregulated in I/R injury after TKA. Sevoflurane treatment or silencing RASD1 reduced RASD1 expression, CK, LDH and MDA contents, inflammation, apoptosis, but increased proliferation, SOD content, cAMP expression, and extents of PKA and cAMP responsive element binding protein (CREB) phosphorylation in skeletal muscle cells of I/R injury. Additionally, PKA pathway activation potentiated the therapeutic effect of Sevoflurane on I/R injury after TKA. Altogether, Sevoflurane treatment confines I/R injury after TKA via RASD1-mediated PKA pathway activation.

Mettl14-mediated m6A modification modulates neuron apoptosis during the repair of spinal cord injury by regulating the transformation from pri-mir-375 to miR-375.

BACKGROUND: Spinal cord injury (SCI) is a disabling disorder, resulting in neurological impairments. This study investigated the mechanism of methyltransferase-like 14 (Mettl14) on apoptosis of spinal cord neurons during SCI repair by mediating pri-microRNA (miR) dependent N6-methyladenosine (m6A) methylation. METHODS: The m6A content in total RNA and Mettl14 levels in spinal cord tissues of SCI rats were detected. Mettl14 expression was intervened in SCI rats to examine motor function, neuron apoptosis, and recovery of neurites. The cell model of SCI was established and intervened with Mettl14. miR-375, related to SCI and positively related to Mettl14, was screened out. The expression of miR-375 and pri-miR-375 after Mettl14 intervention was detected. The expression of pri-miR-375 combined with DiGeorge critical region 8 (DGCR8) and that modified by m6A was detected. Furthermore, the possible downstream gene and pathway of miR-375 were analysed. SCI cell model with Mettl14 intervention was combined with Ras-related dexamethasone-induced 1 (RASD1)/miR-375 intervention to observe the apoptosis. RESULTS: Mettl14 level and m6A content in spinal cord tissue were significantly increased. After Mettl14 knockdown, the injured motor function was restored and neuron apoptosis was reduced. In vitro, Mettl14 silencing reduced the apoptosis of SCI cells; miR-375 was reduced and pri-miR-375 was increased; miR-375 targeted RASD1. Silencing Mettl14 inactivated the mTOR pathway. The apoptosis in cells treated with silencing Mettl14 + RASD1/miR-375 was inhibited. CONCLUSIONS: Mettl14-mediated m6A modification inhibited RASD1 and induced the apoptosis of spinal cord neurons in SCI by promoting the transformation of pri-miR-375 to mature miR-375.

Prognostic value of RASD1 transcript levels in adult Philadelphia-negative B-cell acute lymphoblastic leukemia.

OBJECTIVES: Ras-related dexamethasone-induced 1 (RASD1) is abnormally expressed in many solid cancers. However, its potential role in adults with B-cell acute lymphoblastic leukemia (B-ALL) is unclear. Therefore, we aim to clarify the abnormal expression of the tumor-associated biomarker, RASD1, as a potential target for diagnosis and prognosis in adult Philadelphia-negative B-ALL. METHODS: The expression of RASD1 was detected with RT-qPCR in 92 adults with de novo Ph-negative B-ALL and 40 healthy controls. The correlation between RASD1 transcript levels and relapse was assessed. RESULTS: RASD1 transcript levels in patients with Ph-negative B-ALL (median 81.76%, range 0.22%-1824.52%) were significantly higher than those in healthy controls (7.59%, 0.46%-38.66%; P<0.0001). Patients with low RASD1 transcript levels had a lower 5-year relapse-free survival (RFS, 47.5% [32.9%, 62.1%] vs. 63.1% [49.0%, 77.2%]; P = 0.012) and a higher 5-year cumulative incidence of relapse (CIR, 52.0% [37.4%, 66.6%] vs. 36.2% [22.2%, 50.2%]; P = 0.013) especially in patients receiving chemotherapy only. Multivariate analysis showed that a low RASD1 transcript level was an independent risk factor for RFS (HR = 2.938 [1.427, 6.047], P = 0.003) and CIR (HR = 3.367 [1.668, 6.796], P = 0.001) in patients with Ph-negative B-ALL. CONCLUSIONS: RASD1 transcript levels were significantly higher in patients with Ph-negative B-ALL and a low RASD1 transcript level was independently correlated with increased relapse risk.

Hsa-miR-375/RASD1 Signaling May Predict Local Control in Early Breast Cancer.

BACKGROUND: In order to characterize the various subtypes of breast cancer more precisely and improve patients selection for breast conserving therapy (BCT), molecular profiling has gained importance over the past two decades. MicroRNAs, which are small non-coding RNAs, can potentially regulate numerous downstream target molecules and thereby interfere in carcinogenesis and treatment response via multiple pathways. The aim of the current two-phase study was to investigate whether hsa-miR-375-signaling through RASD1 could predict local control (LC) in early breast cancer. RESULTS: The patient and treatment characteristics of 81 individuals were similarly distributed between relapse (n = 27) and control groups (n = 54). In the pilot phase, the primary tumors of 28 patients were analyzed with microarray technology. Of the more than 70,000 genes on the chip, 104 potential hsa-miR-375 target molecules were found to have a lower expression level in relapse patients compared to controls (p-value < 0.2). For RASD1, a hsa-miR-375 binding site was predicted by an in silico search in five mRNA-miRNA databases and mechanistically proven in previous pre-clinical studies. Its expression levels were markedly lower in relapse patients than in controls (p-value of 0.058). In a second phase, this finding could be validated in an independent set of 53 patients using ddPCR. Patients with enhanced levels of hsa-miR-375 compared to RASD1 had a higher probability of local relapse than those with the inverse expression pattern of the two markers (log-rank test, p-value = 0.069). CONCLUSION: This two-phase study demonstrates that hsa-miR-375/RASD1 signaling is able to predict local control in early breast cancer patients, which-to our knowledge-is the first clinical report on a miR combined with one of its downstream target proteins predicting LC in breast cancer.

Prenatal exposure to lipopolysaccharide induces changes in the circadian clock in the SCN and AA-NAT activity in the pineal gland.

The mammalian circadian pacemaker in the suprachiasmatic nucleus (SCN) regulates behavioral and physiological processes in a 24-h cycle. During its development, the SCN can be sensitive to external stimuli which may change the circadian phenotypes in adulthood. Here, we investigated the effects of prenatal exposure to endotoxin lipopolysaccharide (LPS) on the developing rhythms in expression of Per1, Per2, Nr1d1 and Rasd1 along the rostrocaudal axis of the SCN, and on the rhythm of the rate-limiting enzyme in melatonin synthesis, pineal alkylamine N-acetyltransferase (AA-NAT). The prenatal LPS treatment induced anxiety-like behavior in adulthood as shown before and affected the rhythmicity of clock genes in the SCN. The major effect was observed for Nr1d1 expression; the least affected gene was Per2. The Nr1d1 in the LPS-treated group was arrhythmic at postnatal day 3, but showed significantly higher amplitude at postnatal day 20 at all SCN parts, similarly to the AA-NAT activity in pineal glands, thus suggesting adaptive flexibility of the developing SCN to immune challenges in early development.

High RASD1 transcript levels at diagnosis predicted poor survival in adult B-cell acute lymphoblastic leukemia patients.

B-cell acute lymphoblastic leukemia (B-ALL) in adults remains a highly challenging disease. Identifying new prognostic biomarkers is necessary to help select the best therapeutic schedules and to improve prognosis. We performed bioinformatics analyses of transcriptomic data to identify aberrantly-expressed mRNA transcripts in B-ALL and focused on RASD1 (Ras-related dexamethasone-induced 1). To date, no information is available on the prognostic value of RASD1 in B-ALL. Fifty-three consecutive adults with de novo B-ALL were enrolled in this study. Our data suggested that RASD1 was abnormally overexpressed in B-ALL. High RASD1 transcript levels at diagnosis were associated with lower survival probabilities (44% [20%-61%] vs. 79% [60%-97%]; P = 0.037) and were also an independent prognostic factor in adult B-ALL (HR = 4.9 [1.5-15.9]; P = 0.008). Functional in vitro analyses and bioinformatic analyses indicated that RASD1 promoted cell proliferation, cell cycle progression and chemotherapy resistance and inhibited cell apoptosis. These data demonstrated that RASD1 might serve as a novel prognostic biomarker for adult B-ALL and as a potential therapeutic target in adult B-ALL patients.

Differential ability of formononetin to stimulate proliferation of endothelial cells and breast cancer cells via a feedback loop involving MicroRNA-375, RASD1, and ERα.

For postmenopausal cardiovascular disease, long-term estrogen therapy may increase the risk of breast cancer. To reduce this risk, estrogen may be replaced with the phytoestrogen formononetin, but how formononetin acts on vascular endothelial cells (ECs) and breast cancer cells is unclear. Here, we show that low concentrations of formononetin induced proliferation and inhibited apoptosis more strongly in cultured human umbilical vein endothelial cells (HUVECs) than in breast cancer cells expressing estrogen receptor α (ERα) (MCF-7, BT474) or not (MDA-MB-231), and that this differential stimulation was associated with miR-375 up-regulation in HUVECs. For the first time, we demonstrate the presence of a feedback loop involving miR-375, ras dexamethasone-induced 1 (RASD1), and ERα in normal HUVECs, and we show that formononetin stimulated this feedback loop in HUVECs but not in MCF-7 or BT474 cells. In all three cell lines, formononetin increased Akt phosphorylation and Bcl-2 expression. Inhibiting miR-375 blocked these changes and increased proliferation in HUVECs, but not in MCF-7 or BT474 cells. In ovariectomized rats, formononetin increased uterine weight and caused similar changes in levels of miR-375, RASD1, ERα, and Bcl-2 in aortic ECs as in cultured HUVECs. In mice bearing MCF-7 xenografts, tumor growth was stimulated by 17ß-estradiol but not by formononetin. These results suggest selective action of formononetin in ECs (proliferation stimulation and apoptosis inhibition) relative to breast cancer cells, possibly via a feedback loop involving miR-375, RASD1, and ERα. This differential effect may explain why formononetin may not increase the risk of postmenopausal breast cancer.

MiR-301b-3p/3584-5p enhances low-dose mono-n-butyl phthalate (MBP)-induced proliferation by targeting Rasd1 in Sertoli cells.

To investigate the possible molecular mechanism of low concentration plasticizer mono-n-butyl phthalate (MBP) -induced juvenile Sertoli cells (SCs) proliferation, we evaluated global alterations of miRNA and mRNA expression in rat SCs treated with 0.1mM MBP. Microarray analysis revealed that miR-3584-5p and miR-301b-3p were up-regulated and their common target gene Dexamethasone-induced Ras-related protein 1 (Rasd1) was down-regulated. Further work suggested that SCs proliferation induced by low concentration MBP in vitro might be mediated by Rasd1 regulating ERK1/2 signaling pathway. The present study is first to investigate the effect of low-dose MBP on SCs proliferation and may enhance our understanding on the modes of action of low concentration MBP on male reproductive system. We hope the results will contribute to explain the causes of precocious puberty and testicular tumors induced by exogenous chemicals.

Effects of In Utero Exposure to Di-n-Butyl Phthalate on Testicular Development in Rat.

Humans are inevitably exposed to ubiquitous phthalate esters (PAEs). In utero exposure to di-n-butyl phthalate (DBP) induces abnormal development of the testis and reproductive tract in male offspring, which correspond closely with the human condition of testicular dysgenesis syndrome (TDS)-like syndrome. However, the underlying mechanisms have not been elucidated in detail. In this study, pregnant rats were orally exposed to either corn oil (controls) or DBP at three different doses by gavage during Gestational Days 12.5-21.5. Pathological examinations were performed for toxicity evaluation. Proliferation and apoptosis related proteins (ras related dexamethasone induced 1 (Rasd1), mitogen-activated protein kinase kinases1/2 (MEK1/2), Bcl-2, and Bax) were measured for mechanisms exploration. The results showed that different doses of DBP caused male developmental and reproductive toxicity in rats, including the decrease of anogenital distance (AGD), the histological damage of testis, and apoptosis of seminiferous tubule cells. Our data suggested that DBP played chronic and continuous toxic roles on male reproductive system by disrupting expression of Rasd1 and MEK1/2 as well as Bcl-2/Bax ratio. Further research is warranted.

Rasd1 is an estrogen-responsive immediate early gene and modulates expression of late genes in rat anterior pituitary cells.

Dexamethasone-induced Ras-related protein 1 (Rasd1) is a member of the Ras superfamily of monomeric G proteins that have a regulatory function in signal transduction. Here we investigated the role of Rasd1 in regulating estrogen-induced gene expression in primary cultures of rat anterior pituitary cells. Rasd1 mRNA expression in anterior pituitary cells decreased after treatment with forskolin or serum and increased after treatment with 17ß-estradiol (E2). Increases in Rasd1 mRNA expression occurred as early as 0.5 h after E2 treatment, peaked at 1 h and were sustained for as long as 96 h. This rapid and profound increase in Rasd1 mRNA expression induced by E2 was also seen in GH4C1 cells, an estrogen receptor-positive somatolactotroph cell line. Among pituitary estrogen-responsive late genes studied, basal mRNA expression of Pim3 and Igf1 genes was decreased by RNA interference-mediated knockdown of Rasd1 expression, whereas basal expression of the Giot1 gene was increased. Moreover, Rasd1 knockdown enhanced stimulation of Pim3 mRNA expression and attenuated inhibition of Fosl1 mRNA expression 24 h after E2 treatment. These changes in mRNA expression were accompanied by enhanced activity of promoters containing CRE, AP-1 and SRE binding sequences. These results suggest that Rasd1 is an estrogen-responsive immediate early gene and modulates E2 induction of at least several late genes in anterior pituitary cells.

Rapid expression of RASD1 is regulated by estrogen receptor-dependent intracellular signaling pathway in the mouse uterus.

Dexamethasone-induced RAS-related protein 1 (RASD1) is a signaling protein that is involved in various cellular processes. In a previous study, we found that RASD1 expression was down-regulated in the uterine endometrium of repeated implantation failure patients. The study aim was to determine whether RASD1 is expressed in the endometrium of mouse uterus and how it is regulated by steroid hormones during the estrous cycle. In this study, we investigated RASD1 expression and regulation in an ovariectomized female mouse model. Rasd1 mRNA was highly expressed in mouse reproductive tissues, including the uterus. Rasd1 expression was detected exclusively in the endometrial epithelium at the proestrus stage of the estrous cycle. Rasd1 expression in uteri increased with administration of estradiol, but not progesterone. Its expression was rapidly induced within 2 h after E2 treatment. Pretreatment with ICI 182,780, an estrogen receptor antagonist, reduced RASD1 protein expression. In addition, we identified that rapid expression of Rasd1 was mediated by the estrogen intracellular signaling including both p38-mitogen-activated protein kinase and the extracellular signal-regulated kinase. These findings suggest that RASD1 acts as a novel signaling molecule and plays an important role in regulating dynamic uterine remodeling during the estrous cycle in the uterus.

Dexamethasone activates transient receptor potential canonical 4 (TRPC4) channels via Rasd1 small GTPase pathway.

Canonical transient receptor potential 4 (TRPC4) channels are calcium-permeable, nonselective cation channels that are widely distributed in mammalian cells. It is generally speculated that TRPC4 channels are activated by Gq/11-PLC pathway or directly activated by Gi/o proteins. Although many mechanistic studies regarding TRPC4 have dealt with heterotrimeric G proteins, here, we first report the functional relationship between TRPC4 and small GTPase, Rasd1. Rasd1 selectively activated TRPC4 channels, and it was the only Ras protein among Ras protein family that can activate TRPC4 channels. For this to occur, it was found that certain population of functional Gαi1 and Gαi3 proteins are essential. Meanwhile, dexamethasone, a synthetic glucocorticoid and anti-inflammatory drug was known to increase messenger RNA (mRNA) level of Rasd1 in pancreatic ß-cells. We have found that dexamethasone triggers TRPC4-like cationic current in INS-1 cells via increasing protein expression level of Rasd1. This relationship among dexamethasone, Rasd1, and TRPC4 could suggest a new therapeutic agent for hospitalized diabetes mellitus (DM) patients with prolonged dexamethasone prescription.

Downregulated RASD1 and upregulated miR-375 are involved in protective effects of calycosin on cerebral ischemia/reperfusion rats.

Isoflavone calycosin is a typical phytoestrogen extracted from Chinese medical herb Radix Astragali. It has been reported that estrogens could provide neuroprotective effects, and dietary intake of phytoestrogens could reduce stroke injury in cerebral ischemia/reperfusion (I/R) animal models. In the present study, we investigate the molecular mechanisms underlying the neuroprotective effects of calycosin on middle cerebral artery occlusion (MCAO) rats. Focal cerebral ischemia was induced in male rats by MCAO, neurological deficits and brain edema was evaluated after 24h of reperfusion. The results shown calycosin significantly reduced the infarcted volume and the brain water content, and improved the neurological deficit. To provide insight into the functions of estrogen receptor (ER)-mediated signaling pathway in neuroprotection by calycosin, the expression of miR-375, ER-α, RASD1 (Dexamethasone-induced Ras-related protein 1) and Bcl-2 was determined by RT-PCR or western blot assay. Calycosin exhibited a downregulation of RASD1, and an upregulation of ER-α, miR-375 and Bcl-2. Our finding illustrated that calycosin had been shown neuroprotective effects in cerebral ischemia/reperfusion rats, and the molecular mechanisms may correlate with the positive feedback between ER-α and miR-375, along with the regulation of downstream targets.

MiR-375, a microRNA related to diabetes.

MiR-375 is an important small non-coding RNA that is specifically expressed in islet cells of the pancreas. miR-375 is required for normal pancreatic genesis and influences not only ß-cell mass but also α-cell mass. miR-375 is also important to glucose-regulated insulin secretion through the regulation of the expression of Mtpn and Pdk1 genes. When human embryonic stem cells (hESCs) differentiate into endodermal lineages, miR-375 is highly expressed in the definitive endoderm, which suggests that miR-375 may have a distinct role in early development. miR-375 plays an important role in the complex regulatory network of pancreatic development, which could be regulated by pancreatic genes, such as NeuroD1, Ngn3, Pdx1 and Hnf6; additionally, miR-375 regulates genes related to pancreas development, cell growth and proliferation and insulin secretion genes to exert its function. Because of the special role of miR-375, it may be a potential target to treat diabetes. Antagonising miR-375 may enhance the effects of exendin-4 in patients, and controlling the expression of miR-375 could assist mature hESCs-derived ß-cells.

MicroRNA 'signature' during estrogen-mediated mammary carcinogenesis and its reversal by ellagic acid intervention.

Dysregulated miRNA expression has been associated with the development and progression of cancers, including breast cancer. The role of estrogen (E2) in regulation of cell proliferation and breast carcinogenesis is well-known. Recent reports have associated several miRNAs with estrogen receptors in breast cancers. Investigation of the regulatory role of miRNAs is critical for understanding the effect of E2 in human breast cancer, as well as developing strategies for cancer chemoprevention. In the present study we used the well-established ACI rat model that develops mammary tumors upon E2 exposure and identified a 'signature' of 33 significantly modulated miRNAs during the process of mammary tumorigenesis. Several of these miRNAs were altered as early as 3 weeks after initial E2 treatment and their modulation persisted throughout the mammary carcinogenesis process, suggesting that these molecular changes are early events. Furthermore, ellagic acid, which inhibited E2-induced mammary tumorigenesis in our previous study, reversed the dysregulation of miR-375, miR-206, miR-182, miR-122, miR-127 and miR-183 detected with E2 treatment and modulated their target proteins (ERα, cyclin D1, RASD1, FoxO3a, FoxO1, cyclin G1, Bcl-w and Bcl-2). This is the first systematic study examining the changes in miRNA expression associated with E2 treatment in ACI rats as early as 3 week until tumor time point. The effect of a chemopreventive agent, ellagic acid in reversing miRNAs modulated during E2-mediated mammary tumorigenesis is also established. These observations provide mechanistic insights into the new molecular events behind the chemopreventive action of ellagic acid and treatment of breast cancer.