Let-7b Expression Research Articles

HMGA2 As a Potential Molecular Target in MLL-AF4 Positive Infant Acute Lymphoblastic Leukemia

Leukemic cells of acute lymphoblastic leukemia (ALL) in infants are frequently characterised by chromosome translocations involving 11q23, resulting in the rearrangement of the mixed-lineage leukemia (MLL) gene and subsequent generation of MLL fusion gene. Among more than 50 genes which have been identified as the fusion partner of the MLL gene, fusion with AF4 is characteristically observed in infant ALL representing a hallmark of poor prognosis. Although recent progress of intensive chemotherapy with or without stem cell transplantation has improved its treatment outcome, the treatment is often accompanied by long-term side effects. Less toxic molecular targeting therapies are therefore necessary for infant ALL.We have previously reported that in infant ALL with MLL fusion gene, microRNA let-7b is significantly downregulated by DNA hypermethylation of its promoter region. The downregulation of let-7b is one of the consequences of oncogenic MLL fusion proteins contributing to leukemogenesis possibly through upregulation of let-7b-regulated target genes with oncogenic potential such as high mobility group AT-hook 2 (HMGA2). HMGA2 is a chromatin-remodelling factor, which alters chromatin architecture by binding to AT-rich regions in the DNA, either promoting or inhibiting the expression of its target genes. One of the targets of HMGA2 is CDKN2A gene which encodes 2 cell cycle regulators p16INK4A and p14ARF. This let-7b-HMGA2-CDKN2A axis regulates cellular growth and senescence of stem cells both in normal and pathological state such as cancer.We initially examined the expression of HMGA2 in leukemic cells obtained from 35 MLL-rearranged infant ALL patients (MLL-AF4, n = 26; MLL-AF9, n = 4; MLL-ENL, n = 5) using quantitative RT-PCR. As results, HMGA2 was highly expressed in most of the patients with MLL fusion gene, especially in MLL-AF4-positive cases, compared to those without the fusion. These results indicate that deregulation of let-7b-HMGA2 axis by MLL fusion may contribute to leukemogenesis and could be a possible target of molecular therapy against MLL-rearranged ALL.As let-7b is downregulated by promoter hypermethylation, demethylating agents such as 5-azacytidine could be applied to recover the expression of the gene in leukemic cells with MLL fusion gene. To test this possibility, leukemic cell lines with MLL-AF4 fusion gene were used. The administration of 5-azacytidine alone was able to restore the expression of suppressed let-7b as well as p16INK4A gene in the leukemic cells, but the effects was incomplete, showing persistent partial promoter methylation. In addition, the recovered expression disappeared when 5-azacytidine was removed. On the other hand, when HMGA2 inhibitor was combined with 5-azacytidine, the expression of let-7b was upregulated and sustained resulting in suppression of HMGA2 protein itself. This upregulation of let-7b and suppression of HMGA2 protein persisted even after the removal of 5-azacytidine, possibly through maintaining of the demethylating status by HMGA2 inhibitor. Inhibition of HMGA2 by either siRNA or HMGA2 inhibitor suppressed the growth of MLL-AF4-positive leukemic cells when analysed by MTT assay. The effects of HMGA2 inhibitor on cell growth inhibition became more prominent in combination with demethylating agent 5-azacytidine.Our results revealed the functional significance of let-7b and HMGA2 in controlling MLL-AF4-positive leukemic cell growth and the therapeutic potential of combining demethylating agent and the HMGA2 inhibitor in the treatment of MLL-AF4-positive ALL. DisclosuresNo relevant conflicts of interest to declare.

Repression of let-7 by transforming growth factor-β1-induced Lin28 upregulates collagen expression in glomerular mesangial cells under diabetic conditions.

Accumulation of mesangial extracellular matrix (ECM) proteins such as collagen type 1-α2 (Col1a2) and collagen type 4-α1 (Col4a1) is a key feature of diabetic nephropathy (DN). Transforming growth factor (TGF)-β1 plays important roles in ECM accumulation in DN, and evidence shows a mediatory role for microRNAs. In the present study, we found that microRNA let-7 family members (let-7b/c/d/g/i) were downregulated in TGF-β-treated mouse mesangial cells (MMCs) along with upregulation of Col1a2 and Col4a1. Ectopic expression of let-7b in TGF-β-treated MMCs attenuated Col1a2 and Col4a1 upregulation. Conversely, let-7b inhibitors increased Col1a2 and Col4a1 levels. Cotransfection of MMCs with mouse Col1a2 or Col4a1 3'-untranslated region luciferase constructs and let-7b inhibitors increased luciferase activity. However, constructs with let-7 target site mutations were unresponsive to TGF-β. TGF-β-induced 3'-untranslated region activity was attenuated by let-7b mimics, suggesting that Col1a2 and Col4a1 are direct targets of let-7b. In addition, Lin28b, a negative regulator of let-7 biogenesis, was upregulated in TGF-β-treated MMCs. Luciferase assays showed that the Lin28b promoter containing the Smad-binding element (SBE) responded to TGF-β, which was abolished in constructs without SBE. Chromatin immunoprecipitation assays showed TGF-β-induced enrichment of Smad2/3 at the Lin28b promoter, together suggesting that Lin28b is transcriptionally induced by TGF-β through SBE. Furthermore, let-7b levels were decreased, whereas Lin28b, Col1a2, and Col4a1 levels were increased, in glomeruli of diabetic mice compared with nondiabetic control mice, demonstrating the in vivo relevance of this Lin28/let-7/collagen axis. These results identify Lin28 as a new TGF-β target gene and suggest a novel role for the Lin28/let-7 pathway in controlling TGF-β-induced collagen accumulation in DN.

Mirna Expression in the Cervix Is Associated with Lead and Mercury Exposure during Pregnancy

Mirna Expression in the Cervix Is Associated with Lead and Mercury Exposure during PregnancyAbstract Number:2550 Alison Sanders*, Heather Burris, Allan Just, Katherine Svensson, Chitra Amarasiriwardena, Valeria Motta, Adriana Mercado-Garcia, Joel Schwartz, Martha Tellez-Rojo, Robert Wright, and Andrea Baccarelli Alison Sanders* Icahn School of Medicine at Mount Sinai, United States, E-mail Address: [email protected] Search for more papers by this author , Heather Burris Beth Isreal Deaconess Medical Center, Harvard Medical School, United States, E-mail Address: [email protected] Search for more papers by this author , Allan Just Harvard School of Public Health, United States, E-mail Address: [email protected] Search for more papers by this author , Katherine Svensson Icahn School of Medicine at Mount Sinai, United States, E-mail Address: [email protected] Search for more papers by this author , Chitra Amarasiriwardena Icahn School of Medicine at Mount Sinai, United States, E-mail Address: [email protected] Search for more papers by this author , Valeria Motta Harvard School of Public Health, United States, E-mail Address: [email protected] Search for more papers by this author , Adriana Mercado-Garcia National Institute of Public Health , Mexico, E-mail Address: [email protected] Search for more papers by this author , Joel Schwartz Harvard School of Public Health, United States, E-mail Address: [email protected] Search for more papers by this author , Martha Tellez-Rojo National Institute of Public Health , Mexico, E-mail Address: [email protected] Search for more papers by this author , Robert Wright Icahn School of Medicine at Mount Sinai, United States, E-mail Address: [email protected] Search for more papers by this author , and Andrea Baccarelli Harvard School of Public Health, United States, E-mail Address: [email protected] Search for more papers by this author AbstractToxic metals including lead (Pb) and mercury (Hg) are associated with adverse pregnancy outcomes. miRNAs are small non-coding RNAs that post-transcriptionally control gene expression. miRNAs are particularly susceptible to environmental exposures, and no previous studies have examined environmentally-associated alterations in cervix miRNA expression during pregnancy. This study aimed to analyze the association between miRNA expression in the cervix with Pb and Hg levels biomonitored during pregnancy. We obtained cervical swabs from 80 women between 18 to 20 weeks gestation in a cohort from Mexico City, Mexico. Cervical RNA was extracted and miRNA expression was quantified in a subset of 60 samples using the nCounter Analysis System from Nanostring. Women’s blood Pb was analyzed via ICP-MS and toenail Hg levels were analyzed with a direct Hg analyzer. We performed linear regression to examine the associations between expression of thirty-five miRNAs and metal exposures adjusting for women’s age, education, and environmental tobacco smoke. Expression of miR-188 was positively associated with maternal blood Pb levels (ß=0.3, p=0.04). Expression of let-7b (ß=-1.9, p=0.02), miR-205 (ß=-2.9, p=0.02), and miR-203 (ß=-2.3, p=0.03) were negatively associated with toenail Hg levels, whereas expression of miR-1283 (ß=1.4, p=0.01) and miR-888 (ß=1.0, p=0.03) were positively associated. Pathway analysis of the five Hg-associated miRNAs revealed enrichment for genes involved in cell cycle, cancer, and organismal injury and abnormalities (p=1x10-11). Notably, let-7, miR-205, and miR-203 are known to play a role in reproductive system diseases. We showed altered miRNA expression in the human cervix during pregnancy associated with biomonitored toxic metal levels. These findings highlight miRNAs as novel responders to maternal chemical exposure in specific tissues relevant to labor. The role of metals and cervical miRNA expression in pregnancy outcomes deserves further study.

Abstract A76: Ras pathway and HIF-1α contribute to the aggressiveness of Ewing sarcoma

Abstract Aim: Ewing sarcoma (ES) is the second most common and aggressive primary malignant bone tumor in children and adolescents. The hallmarks of the tumor are specific translocations; the most frequent involves the EWS gene on chromosome 22 and the FLI-1 gene on chromosome 11. Our aim was to identify microRNAs (miRs) and genes involved in the aggressiveness of ES. Out of the miRs located on these chromosomes, 3 belong to the let-7 family. Methods: We evaluated the expression levels of hsa-let-7a and hsa-let-7b by RQ-PCR in 57 primary ES tumors. We measured the protein levels of p-ERK, HIF-1a and EWS-FLI-1 following transfection with let-7a, let-7b or HIF-1a . The effect of a Ras inhibitor was evaluated in vitro and in vivo. Results: In the group of patients with localized disease, a significant correlation with outcome was observed. Progression free survival (PFS) at ten years for patients with high hsa-let-7b expression was 62% versus 29% for those with low hsa-let-7b expression level (p=0.029). Multivariate Cox regression analysis identified expression of let-7b as an independent prognostic factor in ES, with a 4.4 fold increased risk of relapse (p=0.021). hsa-let-7 is known to be a negative regulator of the RAS oncogene. Positive p-ERK immuno-staining was detected in 31 out of the 45 (69%) tumors analyzed, and a significant inverse correlation was identified between hsa-let-7a and p-ERK (p=0.037). One of the target genes of RAS is Hypoxia inducible factor 1 (HIF-1). We detected a high level of HIF-1α protein in ES cell lines, under normoxic conditions, which did not change following induced hypoxia. This implies for a mechanism that mimics hypoxia and probably contributes to the high frequency of metastasis and thereby aggressiveness of this tumor. Following transfection with hsa-let-7a and hsa-let-7b mimics (overexpression) the levels of RAS, p-ERK, HIF-1α and EWS-FLI-1 proteins were significantly reduced (60%, p=0.0019, 52%, p=0.003, 45%, p=0.0028 and 38%, p=0.006, respectively). Silencing of HIF-1α resulted in a decrease of 61% of EWS-FLI-1, suggesting that HIF-1α regulates EWS-FLI-1, and we were able to show that HIF-1α directly binds EWS promoter. This was accompanied by a reduction of 32% (p=0.009) in cell proliferation rate and by a significant decrease of 36% (p=0.0013) in cell migration ability. Using a RAS inhibitor, Salirasib, we observed a significant decrease of HIF-1α and EWS-FLI-1 protein levels in both in-vitro and in-vivo settings. In addition, a significant inhibition of tumor growth was demonstrated in the treated animals. Conclusions: These results imply that Ras and HIF-1α might contribute to the high frequency of metastasis and thereby aggressiveness of ES tumors. The reduction in tumor burden in a mouse model of ES following Salirasib treatment, demonstrates the therapeutic potential of this RAS inhibitor in ES. Citation Format: Michal Hameiri-Grossman, Adi Porat-Klein, Ian J. Cohen, Shifra Ash, Yoel Kloog, Ronit Haklai, Galit Elad-Sfadia, Avraham Weizman, Isaac Yaniv, Smadar Avigad. Ras pathway and HIF-1α contribute to the aggressiveness of Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A76.

Let-7b/g silencing activates AKT signaling to promote gastric carcinogenesis.

BackgroundAberrant AKT activation contributes to gastric cancer cell survival and chemotherapy resistance, however its regulation is poorly understood. microRNAs have been established to be important regulators in gastric carcinogenesis. Here, we showed the functional role and putative target of let-7b and let-7g (let-7b/g) in gastric carcinogenesis.MethodsThe expression of let-7b/g in gastric cancer cell lines and primary tumors were evaluated by miRNA qRT-PCR. The putative target gene of let-7b/g was explored by TargetScan followed by further validation. Functional analyses including MTT proliferation, monolayer colony formation, cell invasion assays and in vivo study were performed in both ectopic expression and knockdown approaches.Resultslet-7b/g was found down-regulated in gastric cancer and its downregulation was associated with poor survival and correlated with lymph node metastasis. let-7b/g inhibited AKT2 expression by directly binding to its 3’UTR, reduced p-AKT (S473) activation and suppressed expression of the downstream effector pS6. AKT2 mRNA expression showed negative correlation with the expression of let-7b/g in primary tumors. Short interfering RNA (siRNA) mediated knockdown of AKT2 phenocopied the tumor-suppressive effects of let-7b/g. Moreover, AKT2 re-expression partly abrogated the growth-inhibitory effect of let-7b/g.ConclusionIn conclusion, our findings reveal decreased let-7b/g contributes to aberrant AKT activation in gastric tumorigenesis and provide a potential therapeutic strategy for gastric cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0281-3) contains supplementary material, which is available to authorized users.

The KDM2B- let-7b -EZH2 axis in myelodysplastic syndromes as a target for combined epigenetic therapy.

Both DNA and histone methylation are dysregulated in the myelodysplastic syndromes (MDS). Based on preliminary data we hypothesized that dysregulated interactions of KDM2B, let-7b and EZH2 signals lead to an aberrant epigenetic landscape. Gene expression in CD34+ cells from MDS marrows was analyzed by NanoString miR array and validated by real-time polymerase chain reaction (PCR). The functions of KDM2B, let-7b and EZH2 were characterized in myeloid cell lines and in primary MDS cells. Let-7b levels were significantly higher, and KDM2B and EZH2 expression was lower in primary CD34+ MDS marrow cells (n = 44) than in healthy controls (n = 21; p<0.013, and p<0.0001, respectively). Overexpression of let-7b reduced EZH2 and KDM2B protein levels, and decreased cells in S-phase while increasing G0/G1 cells (p = 0.0005), accompanied by decreased H3K27me3 and cyclin D1. Silencing of KDM2B increased let-7b expression. Treatment with the cyclopentanyl analog of 3-deazaadenosine, DZNep, combined with the DNA hypomethylating agent 5-azacitidine, decreased levels of EZH2, suppressed methylation of di- and tri-methylated H3K27, and increased p16 expression, associated with cell proliferation. Thus, KDM2B, via let-7b/EZH2, promotes transcriptional repression. DZNep bypassed the inhibitory KDM2B/let-7b/EZH2 axis by preventing H3K27 methylation and reducing cell proliferation. DZNep might be able to enhance the therapeutic effects of DNA hypomethylating agents such as 5-azacitidine, currently considered standard therapy for patients with MDS.

IGF-1R, a target of let-7b, mediates crosstalk between IRS-2/Akt and MAPK pathways to promote proliferation of oral squamous cell carcinoma.

Insulin-like growth factor (IGF) signaling is involved in oral squamous cell carcinoma (OSCC), but IGF-1 receptor (IGF-1R)-mediated intricate regulatory networks among molecular interactions and signalling path ways in OSCC remain unclear. Here, we found that overexpression of IGF-1R and insulin receptor substrate-2 (IRS-2) was negatively associated with histological differentiation. IGF signaling stimulated OSCC cell growth. Conversely, overexpression of let-7b inhibited proliferation and colony formation and triggered S/G2 cell cycle arrest by targeting IGF-1R and IRS-2 through the Akt pathway. Also, the inverse relationship between expression of let-7b and IGF-1R/IRS-2 was confirmed in OSCC tumor xenografts and clinical specimens. Furthermore, by activating ERK1/2, IGF-1R transcriptionally upregulated IRS-2. Our results indicate that let-7b/IGF-1R-mediated crosstalk between IRS-2/Akt and MAPK is involved in OSCC and is a potential therapeutic target for therapy.

Differential expression of serum miR-126, miR-141 and miR-21 as novel biomarkers for early detection of liver metastasis in colorectal cancer.

MicroRNAs (miRNAs) have potential as diagnostic biomarkers in cancer. Evaluation of the association between miRNA expression patterns and early detection of liver metastasis in colorectal cancer (CRC) has not been reported. We investigated the expression of metastasis-associated miRs-31, 335, 206, 141, 126, 200b, 200c, 21, Let7a, Let7b and Let7c in localized, liver-metastatic and other organ-metastatic CRC (OM-CRC). Expressions of target miRNAs in serum were evaluated in 116 consecutive localized CRC (L-CRC), 72 synchronous liver-metastatic CRC (SLM-CRC) and 36 other OM-CRC by quantitative real-time PCR. Seven of 11 tested miRNAs could be detected from serum. Four miRNAs, miR-126, Let-7a, miR-141 and miR-21 were identified as metastasis-associated miRNAs. Compared with L-CRC, significant up-regulated expression was observed for miR-141 and miR-21 in SLM-CRC and OM-CRC, down-regulated expression was observed for miR-126 in SLM-CRC and OM-CRC, and up-regulated expression of Let-7a in OM-CRC. The receiver operating characteristic (ROC) curve showed serum miR-126 had a cut-off [log10 relative quantity (log10 (RQ))=-0.2005] with 77.78% sensitivity and 68.97% specificity with an area under curve (AUC) of 0.7564, miR-141 had a cut-off (log10 (RQ)=-0.2285) with 86.11% sensitivity and 76.11% specificity with an AUC of 0.8279, and miR-21 had a cut-off (log10 (RQ)=-0.1310) with 73.61% sensitivity and 66.38% specificity with an AUC of 0.7479. We identified liver metastasis-associated miRNAs, suggesting serum miR-126, miR-141 and miR-21 may be novel biomarkers for clinical diagnosis of early stage liver-metastatic CRC.

Significance of microRNA targeted estrogen receptor in male fertility.

Estrogen receptor-alpha (ERα) mediates estrogen action in regulation of different levels of the hypothalamic-pituitary-testis axis. It has a key role in spermatogenesis. Estrogen receptor alpha knock-out (ER koα) male mice were infertile and severe impairment in spermatogenesis and seminiferous tubules was observed. Recently, it has been reported that microRNA (miRNA) mir-100 and let-7b were predicted to target ERα gene. MiRNA are small, endogenous, single stranded RNA molecules that regulate gene expression and have been implicated in various disease states. It has been proved that some miRNAs expression is tissue- and disease-specific, giving potential for identifying miRNAs as a diagnostic tool. In this study, the change in the expression levels of mir-100, let-7b and ERα expression levels were evaluated in oligospermic infertile patients (n=43) compared to control fertile subjects (n=43). After washing and separating sperms, total RNA was isolated and then cDNA was synthesized. The expression levels of mir-100 and let-7b and ERα were evaluated by real time PCR. Mir-100, let-7b levels were significantly higher than those in control group (P=0.008 and P=0.009, respectively). We have found that, ERα level was significantly decreased in comparison with normal group (P< 0.0001). Changes in mir-100, let-7b and ERα expression levels in oligospermic patients may be associated with the susceptibility and progression of infertility. The results of this study indicate that miRNA can have a key role in spermatogenesis and might have a diagnostic and prognostic value in men infertility.

Transforming growth factor-β1-mediated renal fibrosis is dependent on the regulation of transforming growth factor receptor 1 expression by let-7b

Renal fibrosis results from excessive accumulation of extracellular matrix mainly driven by transforming growth factor-β1 (TGF-β1). Certain microRNAs have been implicated in this disease, and here we examine the role of let-7b. Rat proximal tubular epithelial cells (NRK52E) were treated with TGF-β1 for 3 days to assess the expression of markers of fibrosis and let-7b. These factors were also assessed in two mouse models representing early and more advanced diabetic nephropathy and in the non-diabetic adenine-induced renal fibrosis model. TGF-β1 downregulated the expression of let-7b and induced fibrogenesis in NRK52E cells. Ectopic expression of let-7b repressed TGF-β1 receptor 1 (TGFBR1) expression directly by targeting the two let-7b binding sites in the 3'-untranslated region of that gene, reduced expression of extracellular matrix proteins, decreased SMAD3 activity, and attenuated the profibrotic effects of TGF-β1. Knockdown of let-7b elevated TGFBR1 expression and mimicked some of the profibrotic effects of TGF-β1. Consistent with these observations, let-7b expression was also reduced in models of both diabetic and non-diabetic renal fibrosis with the upregulation of TGFBR1. Thus, let-7b microRNA represents a potential new target for the treatment of renal fibrosis in diabetic and non-diabetic nephropathy.

Vitamin D Receptor Genotype Modulates the Correlation between Vitamin D and Circulating Levels of let-7a/b and Vitamin D Intake in an Elderly Cohort

Background and Aims: Circulating microRNAs (miRNAs) are linked to disease and are potential biomarkers. Vitamin D may modulate miRNA profiles, and vitamin D status has been linked to risk of disease, including cardiovascular disease and cancers. We hypothesise that genotypic variance influences these relationships. We examined the correlations between vitamin D intake and circulating levels of the miRNAs let-7a/b, and the involvement of two common vitamin D receptor (VDR) polymorphisms, BsmI and ApaI. Methods: Two hundred participants completed food frequency and supplement questionnaires, and were assayed for circulating let-7b expression by qPCR. Polymorphisms were detected using restriction fragment length polymorphism-PCR. Results: let-7b expression negatively correlated with vitamin D intake (r_s = -0.20, p = 0.005). The magnitude and direction of correlation were maintained in the presence of the BsmI restriction site (r_s = -0.27, p = 0.0005). However, in the absence of BsmI restriction site, the direction of the correlation was reversed (r_s = +0.319, p = 0.0497). These correlations were significantly different (z-score = 2.64, p = 0.0085). The correlation between vitamin D intake and let-7a was only significant in those without the ApaI restriction site. Conclusions: The correlation between vitamin D intake and let-7a/b expression in this cohort varies with VDR genotype. This study highlights the importance of considering underlying genotypic variance in miRNA expression studies and in nutritional epigenetics generally.

Prognostic significance of let-7b expression in breast cancer and correlation to its target gene of BSG expression

Let-7 microRNAs (miRNAs) are found in a wide range of species, and alterations of let-7 miRNA family member expression levels in humans are associated with various types of cancer. However, few researchers have reported alterations in let-7b levels in breast cancer (BC). Specifically, the use of altered let-7 expression as a prognostic biomarker is of particular interest and significance. The aim of this study was to investigate whether let-7b could be used as a biomarker of tumor progression and patient prognosis in BC and to determine the target gene of let-7b. We retrospectively analyzed the clinical pathological characteristics of 80 BC. We utilized digoxigenin-labeled locked nucleic acid-miRNA probes to detect let-7b expression in 80 BC and 22 benign breast disease (BBD) histologic specimens by in situ hybridization, and also detect the expression of BSG-a potential target gene of let-7b-by immunohistochemistry. We observed that the levels of let-7b expression in BBD were higher than in BC specimens (P < 0.05), indicating that let-7b could inhibit growth and facilitate differentiation of BBD. Also, loss of let-7b expression on BC tissue specimens raised the possibility that let-7b could play a crucial role in the pathogenesis of BC. Furthermore, let-7b expression in breast cancer patients was inversely associated with tumor lymph node metastasis (P = 0.001), patient overall survival (P = 0.027), relapse-free survival (P = 0.016), and BSG protein expression (P = 0.001). Breast cancer patients with low let-7b expression had poor prognoses, indicating let-7b might act as cancer suppressor gene in BC development and progression by inhibiting the expression of BSG.

The Jumanjii Histone Demethylase KDM2B controls EZH2 Expression In Myelodysplastic Syndromes (MDS) Via Mir Let-7b (let7b), a Pathway That Is Bypassed By The Histone Methylation Inhibitor DZNep

Background and HypothesisDNA and histone methylation as well as microRNA (miR) expression are dysregulated in MDS. The histone demethylase KDM2B suppresses let7b, which post-transcriptionally controls EZH2, a histone-lysine N-methyl transferase that methylates lysine 27 on histone 3. We hypothesized that epigenetic dysregulation in MDS involves the KDM2B/ let7b/EZH2 axis (Figure). DZNep (cyclopentanyl analog of 3-deazaadenosine) is a novel compound, which results in loss of lysine27 methylation, re-expression of epigenetically silenced genes, and decreased cell proliferation.We propose that DZNep will bypass cellular dysregulation via the KDM2B/let7b/EZH2 axis and thereby enhance therapeutic efficacy when combined with azanucleosides. MethodsCD34+ MDS marrow cells were obtained by density separation and enriched by magnetic-activated cell sorting (MACS; Milteny Biotec, Auburn, CA); purity 95% to 98% by flow cytometry. For NanoString nCounter miR array total cell derived RNA or synthetic miR pools (IDT; 30 pmol per oligonucleotide) were used as input. Nano-string data were validated by real-time polymerase chain reaction (PCR). RNA was extracted using the RNeasy Mini kit (QIAGEN, Valencia, CA). Primers for let7b, U6, KDM2b and EZH2 were synthesized by Applied Biosystems. Total RNA was reverse transcribed with Taqman MiR Reverse Transcriptase kit (Applied Biosystems, Carlsbad, CA). Potential functional roles of KDM2B, let7b and EZH2 were determined in myeloid cell lines MDS-L, PL-21, KG1a,OCI-AML3 and ML1. Conditional knock-down of KDM2B was achieved with 2 short hairpins (open-biosystems). Expression of KDM2B was forced using pCMV-Myc-KDM2B (gift of Dr. Tzatsos) in cell lines with low/absent constitutive levels of KDM2B and high levels of let7b. miRZip plasmids (miRZip anti-sense) were used to knock-down let7b; pre-BAB-let7b (origene) served to overexpress let7b. Gene expression, protein levels and cell proliferation were analyzed by RQ-PCR, western blot and BrdU uptake. To determine the impact of modified let7b and KDM2B expression on treatment responses, MDS-L cells (high constitutive levels of EZH2) were treated with DZNep and methyltransferase inhibitor, 5-azacytidine, and subjected to the same analyses. ResultsLet7b levels were significantly higher in primary CD34+ MDS marrow cells (n=44) than in healthy controls (p= 0.0304), while KDM2B and EZH2 expression was reduced (n=21, p<0.0126, p<0.0001, respectively). Overexpression of let-7b in cells with low constitutive levels (PL21, ML1 and KG1a) reduced EZH2 and KDM2B protein levels, and decreased S-phase while increasing G0/G1 cells (p=0.0005); this was associated with a decrease of H3K27me3 and cyclin D1. Silencing of KDM2B in cells with high (MDS-L) and low (KG1a) constitutive levels of let-7b resulted in increased levels of let7b. This increase in let-7b was associated with reduced levels of EZH2 protein and H3K27me3. These findings were accompanied by reduced proliferation (fewer cells in S-phase, more cells in G0/G1). Addition of DZNep to the myeloid cell lines under study resulted in a decrease of let-7b expression, increased levels of EZH2, but reduced cell proliferation. When DZNep (1 uM/ml) was combined with 5-azacytidine (AZA, 5 uM/ml), levels of EZH2 increased in parallel with reduction of H3K27me3 and cell proliferation. KG1a cells overexpressing let-7b (to mimic conditions in primary MDS cells) showed further increase of EZH2 in response to DZNep and AZA. Summary and ConclusionThe KDM2B/ let7b/EZH2 axis is involved in epigenetic regulation in MDS. KDM2B, via let-7b/EZH2, promotes transcriptional repression in myeloid cell lines and primary MDS cells; KDM2B controls epigenetic modifications and cell proliferation. DZNep bypasses the “inhibitory” KDM2B/let7b/EZH2 axis by preventing H3K27 methylation and reducing cell proliferation, independent of EZH2 expression. Thus, the use of DZNep would add histone demethylation to the effect of azanucleosides on DNA methylation, thereby enhancing the therapeutic success in MDS. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Meta‐analysis of transcriptome reveals let‐7b as an unfavorable prognostic biomarker and predicts molecular and clinical subclasses in high‐grade serous ovarian carcinoma

High-grade serous ovarian carcinoma (HG-SOC) is a heterogeneous, poorly classified, lethal disease that frequently exhibits altered expressions of microRNAs. Let-7 family members are often reported as tumor suppressors; nonetheless, clinicopathological functions and prognostic values of individual let-7 family members have not been addressed in HG-SOC. In our work, we performed an integrative study to investigate the potential roles, clinicopathological functions and prognostic values of let-7 miRNA family in HG-SOC. Using microarray and clinical data of 1,170 HG-SOC patients, we developed novel survival prediction and system biology methods to analyze prognostic values and functional associations of let-7 miRNAs with global transcriptome and clinicopathological factors. We demonstrated that individual let-7 members exhibit diverse evolutionary history and distinct regulatory characteristics. Statistical tests and network analysis suggest that let-7b could act as a global synergistic interactor and master regulator controlling hundreds of protein-coding genes. The elevated expression of let-7b is associated with poor survival rates, which suggests an unfavorable role of let-7b in treatment response for HG-SOC patients. A novel let-7b-defined 36-gene prognostic survival signature outperforms many clinicopathological parameters, and stratifies HG-SOC patients into three high-confidence, reproducible, clinical subclasses: low-, intermediate- and high-risk, with 5-year overall survival rates of 56-71%, 12-29% and 0-10%, respectively. Furthermore, the high-risk and low-risk subclasses exhibit strong mesenchymal and proliferative tumor phenotypes concordant with resistance and sensitivity to primary chemotherapy. Our results have led to identification of promising prognostic markers of HG-SOC, which could provide a rationale for genetic-based stratification of patients and optimization of treatment regimes.

Molecule of the month: miRNA and Human Prion brain disease.

Human prion brain disease has been studied intensely since 1920. Examples of such diseases include Creutzfeldt-Jakob disease (CJD), fatal familial insomnia, Gerstmann-Straussler- Scheinker disease, and Kuru. Central in the epidemiology and pathogenesis of prion diseases is the prion protein itself and the gene for this protein resides on chromosome 12, at locus 20pterp12. The prion protein is termed PRNP, has a monomer molecular weight of 22-36 Kd and is linked to glycosylphosphatidyl- inositol (GPI) on cell surfaces. Various mutants of PRNP have roles in disease pathogenesis. Interestingly, the functions of the normal prion protein include synaptic interactions, cell adhesion, and transmembrane signaling [1]. Within the human genome, an RNA universe includes noncoding RNA, microRNA (miRNA), which regulates many genes and pathways. Processes influenced and regulated by prion gene-associated miRNAs include cell death, neuronal differentiation, development, synapse function, neurogenesis, as well as neurodegeneration and neuropathogenesis. Studies in mouse brains indicated that prion disease results in deregulation of miRNAs as follows. MiR-342-3p, miR-146a, miR-139-5p, miR-320, miR-128, miR-328, and let-7b miRNAs were up regulated; miR-337-3p and miR-338-3p miRNAs were down regulated. Gene promoters that were cognate to several of these miRNAs included E2F-1 (cell cycle re-entry and neurodegeneration), MAZ (inflammatory response transcription factor), PAX6 (neurogenesis), KROX (transcription factor), and Early Growth Response 1 (EGR1), a Nerve Growth Factor-Induced Protein. Additional work indicated effects on Nmethyl- D-aspartic acid (NMDA) receptor glutamate receptor (ionotropic, N-methyl D-aspartate 2A, GRIN2A) as well [2, 3]. MiRNAs also participate in neuronal development, dendrite, spine, and synapse, as well as neuron sub-type specificity and they are involved in CNS ischemia, recovery from ischemia, spinal cord injury, and traumatic brain injury [4]. Exosomes are released from cells and are 50-130 nm size vesicles. Exosomes from normal and prion-diseased humans and animals contain molecular mixtures including cellular prion protein, PrP(C), and the abnormal infectious form, PrP (Sc). Exosomes released from prion-infected neuronal cells contained miRNAs, other non-coding RNA, messenger RNA fragments, retroviral RNA repeat regions, transfer RNA fragments, small cytoplasmic RNA, small nuclear RNA, and small nucleolar RNA. Compared to uninfected exosomes, infected exosomes contain increased expression of miRNAs miR-424, miR-29b, miR-342-3p, miR-128a, miR-21, let-7b, let-7i, and miR-222 as well as miR-146 at decreased levels [5]. In addition to prion diseases, miRNAs are also involved in Parkinson's and Huntington's diseases [4]. In (Figures 1A, 1B,1C & 1D), it illustrates the gene expression and protein networks involving the proteins that interact with miRNAs in prion-related disease [3, 4, 5]. The study of miRNAs is a new field and much has been accomplished. In this article, we summarize just a few of the protein interactions of proteins whose expression levels are perturbed in conjunction with miRNAs in prion disease. The identification of the miRNAs involved in the disease process and the subsequent interacting protein networks provide several pivotal junctures that could be attacked in developing treatments and possibly cures for prion-related diseases. It is left as a puzzle for the interested reader to identify the various genes and their functions in the figures [6, 7, 8]. Figure 1 A) Network of input prion - protein, PRNP with immediate input neighbors. In this figure, line-colors and various interactions with other genes are red Down-regulation, green Up-regulation, beige Regulation, purple Co-expression, brown Physical Interaction, ...

Let-7b expression determines response to chemotherapy through the regulation of Cyclin D1 in Glioblastoma

BackgroundGlioblastoma is the most common type of primary brain tumors. Cisplatin is a commonly used chemotherapeutic agent for Glioblastoma patients. Despite a consistent rate of initial responses, cisplatin treatment often develops chemoresistance, leading to therapeutic failure. Cellular resistance to cisplatin is of great concern and understanding the molecular mechanisms is an utter need.MethodsGlioblastoma cell line U251 cells were exposed to increasing doses of cisplatin for 6 months to establish cisplatin-resistant cell line U251R. The differential miRNA expression profiles in U251 and U251R cell lines were identified by microarray analysis and confirmed by Q-PCR. MiRNA mimics were transfected into U251R cells, and cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis.ResultsU251R cells showed 3.1-fold increase in cisplatin resistance compared to its parental U251 cells. Microarray analysis identified Let-7b and other miRNAs significantly down-regulated in U251R cells compared to U251 cells. Transfection of Let-7b mimics greatly re-sensitized U251R cells to cisplatin, while transfection of other miRNAs has no effect or slightly effect. Cyclin D1 is predicted as a target of Let-7b through bioinformatics analysis. Over-expression of Let-7b mimics suppressed cyclin D1 protein expression and inhibited cyclin D1-3’-UTR luciferase activity. Knockdown of cyclin D1 expression significantly increased cisplatin-induced G1 arrest and apoptosis.ConclusionsCollectively, our results indicated that cisplatin treatment leads to Let-7b suppression, which in turn up-regulates cyclin D1 expression. Let-7b may serve as a marker of cisplatin resistance, and can enhance the therapeutic benefit of cisplatin in glioblastoma cells.

The Heterochronic microRNA let-7 Inhibits Cell Motility by Regulating the Genes in the Actin Cytoskeleton Pathway in Breast Cancer

The heterochronic gene let-7 serves as a tumor suppressor microRNA by targeting various oncogenic pathways in cancer cells. Considerable evidence indicates that reduced expression of let-7 might be associated with poor clinical outcome in patients with cancer. Here, we report that the expression levels of three let-7 family members, let-7a, let-7b, and let-7g, were significantly decreased in the patients with breast cancer with lymph node metastasis compared with those without lymph node metastasis. Enforced expression of let-7b significantly inhibits breast cancer cell motility and affects actin dynamics. Using bioinformatic and experimental approaches, four genes in the actin cytoskeleton pathway, including PAK1, DIAPH2, RDX, and ITGB8, were identified as let-7 direct targets. Blocking the expression of PAK1, DIAPH2, and RDX significantly inhibits breast cancer cell migration induced by let-7b repression. Our results indicate that reconstitution of let-7 expression in tumor cells could provide a novel therapeutic strategy for the treatment of metastatic disease.

Let-7b Is Involved in the Inflammation and Immune Responses Associated with Helicobacter pylori Infection by Targeting Toll-Like Receptor 4

ObjectivesToll-like receptors (TLRs) are important initiators in native immune responses to microbial infections. TLR4 is up-regulated in response to H.pylori infection in gastric epithelial cells. However, the regulatory mechanisms for the expression of TLR4 in H.pylori infection have not been clearly defined. The aims of this study are to present the evidence that microRNA let-7b directly regulates TLR4 expression in human gastric epithelial cells, and subsequently influences the activation of NF-κB and the expression of the downstream genes in H.pylori infection.MethodsThe expression of let-7b was determined in gastric mucosa specimens and in two gastric epithelial cell lines using quantitative RT-PCR. The expression of TLR4 was determined by immunohistochemistry staining and RT-PCR. The potential target of let-7b was identified by luciferase reporter assay and Western blot. Let-7b mimics and inhibitors were used to examine the effects of let-7b on NF-κB activity. The expression of the downstream genes of NF-κB was also determined in cells infected with H.pylori 26695.ResultsLet-7b was significantly decreased in gastric mucosa specimens and in gastric epithelial cell lines (AGS, GES-1) infected with H.pylori 26695 (cagA+). Let-7b was complementary to the 3′-UTR of TLR4 mRNA and regulated TLR4 expression via post-transcriptional suppression in gastric epithelium. Infection of H.pylori induced the expression of TLR4 and activated NF-κB in AGS and GES-1 cells. Overexpression of let-7b by mimics downregulated TLR4, and subsequently attenuated NF-κB, MyD88, NF-κB1/p50, RelA/p65. The expression of IL-8, COX-2 and CyclinD1 was inhibited in H.pylori infected cells with let-7b overexpression. Both TAK-242 (TLR4 inhibitor) and SN50 (NF-κB inhibitor) significantly inhibited the H.pylori induced downregulation of let-7b.ConclusionsLet-7b targets at TLR4 mRNA, and regulates the activation of NF-κB and the expression of the downstream genes related to the inflammation and immune responses in H.pylori infection.

MicroRNA Profile to Predict Gemcitabine Resistance in Bladder Carcinoma Cell Lines

MicroRNAs (miRNA) are small, noncoding RNAs with important regulatory roles in development, differentiation, cell proliferation, and death as well as the complex process of acquired drug resistance. The goal of this study was to identify specific miRNAs and their potential protein targets that confer acquired resistance to gemcitabine in urothelial carcinoma of the bladder (UCB) cell lines. Gemcitabine-resistant cells were established from 6 cell lines following exposure to escalating concentrations of the drug and by passaging cells in the presence of the drug over a 2- to 3-month period. Differential miRNA expression was identified in a microarray format comparing untreated controls with resistant cell lines, representing the maximum tolerated concentration, and results were validated via qRT-PCR. The involvement of specific miRNAs in chemoresistance was confirmed with transfection experiments, followed by clonogenic assays and Western blot analysis. Gemcitabine resistance was generated in 6 UCB cell lines. Microarray analysis comparing miRNA expression between gemcitabine-resistant and parental cells identified the differential expression of 66 miRNAs. Confirmation of differential expression was recorded via qRT-PCR in a subset of these miRNAs. Within this group, let-7b and let-7i exhibited decreased expression, while miR-1290 and miR-138 displayed increased expression levels in gemcitabine-resistant cells. Transfection of pre-miR-138 and pre-miR-1290 into parental cells attenuated cell death after exposure to gemcitabine, while transfection of pre-miR-let-7b and pre-miR-let-7i into the resistant cells augmented cell death. Mucin-4 was up-regulated in gemcitabine-resistant cells. Ectopic expression of let-7i and let-7b in the resistant cells resulted in the down-regulation of mucin-4. These results suggest a role for miRNAs 1290, 138, let-7i, and let-7b in imparting resistance to gemcitabine in UCB cell lines in part through the modulation of mucin-4. Alterations in these miRNAs and/or mucin-4 may constitute a potential therapeutic strategy for improving the efficacy of gemcitabine in UCB.

Validation of Expression Patterns for Nine miRNAs in 204 Lymph-Node Negative Breast Cancers

IntroductionAlthough lymph node negative (LN-) breast cancer patients have a good 10-years survival (∼85%), most of them still receive adjuvant therapy, while only some benefit from this. More accurate prognostication of LN- breast cancer patient may reduce over- and under-treatment. Until now proliferation is the strongest prognostic factor for LN- breast cancer patients. The small molecule microRNA (miRNA) has opened a new window for prognostic markers, therapeutic targets and/or therapeutic components. Previously it has been shown that miR-18a/b, miR-25, miR-29c and miR-106b correlate to high proliferation.MethodsThe current study validates nine miRNAs (miR-18a/b miR-25, miR-29c, miR-106b, miR375, miR-424, miR-505 and let-7b) significantly correlated with established prognostic breast cancer biomarkers. Total RNA was isolated from 204 formaldehyde-fixed paraffin embedded (FFPE) LN- breast cancers and analyzed with quantitative real-time Polymerase Chain Reaction (qPCR). Independent T-test was used to detect significant correlation between miRNA expression level and the different clinicopathological features for breast cancer.ResultsStrong and significant associations were observed for high expression of miR-18a/b, miR-106b, miR-25 and miR-505 to high proliferation, oestrogen receptor negativity and cytokeratin 5/6 positivity. High expression of let-7b, miR-29c and miR-375 was detected in more differentiated tumours. Kaplan-Meier survival analysis showed that patients with high miR-106b expression had an 81% survival rate vs. 95% (P = 0.004) for patients with low expression.ConclusionHigh expression of miR-18a/b are strongly associated with basal-like breast cancer features, while miR-106b can identify a group with higher risk for developing distant metastases in the subgroup of Her2 negatives. Furthermore miR-106b can identify a group of patients with 100% survival within the otherwise considered high risk group of patients with high proliferation. Using miR-106b as a biomarker in conjunction to mitotic activity index could thereby possibly save 18% of the patients with high proliferation from overtreatment.