Dicer Expression Research Articles

Integrative Analysis of lncRNAs in Kidney Cancer to Discover A New lncRNA (LINC00847) as A Therapeutic Target for Staphylococcal Enterotoxin tst Gene.

Objective Bacterial toxin can cause cell death through induction of apoptosis in cancer cell lines as well as changes in the expression patterns of long non-coding RNAs (lncRNAs) and genes. In the present study, the effect of tst gene on ACHN cell lines was reported along with proposing a novel pathway of apoptosis in kidney cancer. Materials and Methods In this experimental study, effective lncRNAs and genes were predicted from different criteria for renal cell carcinoma (RCC) by bioinformatics methods and lncRNA-miRNA-mRNA interaction was constructed; then the effect of Staphylococcus aureus tst gene on induction of apoptosis pathways on ACHN and HDF cell lines was investigated. Results After creation of lncRNA-miRNA-mRNA interaction, changes in expression levels of lncRNA LINC00847 (P=0.0024) and PTEN gene (P=0.0027) were identified, as potential apoptosis biomarkers for kidney cancer, after treating ACHN cell line by pCDNA3.1 (+)-tst compared to the empty vector. In contrast, there was no statistically significant difference in DICER1 expression levels in ACHN-tst cell (P≥0.05). In addition, transfection by pcDNA3.1 (+)-tst could increase ACHN cell apoptosis level (P<0.0001) compared to the pcDNA3.1 (+) group; but no significant effect was observed on normal cells. ConclusionIt is suggested that lncRNA LINC00847, discovered in this study, could provide a new landscape for researches aimed to determine relationship between functional lncRNA and RCC pathways. pcDNA3.1 (+)-tst was found to increase apoptosis in the transfected cells.

Reactivation of Epstein–Barr virus by a dual-responsive fluorescent EBNA1-targeting agent with Zn2+-chelating function

Epstein-Barr nuclear antigen 1 (EBNA1) plays a vital role in the maintenance of the viral genome and is the only viral protein expressed in nearly all forms of Epstein-Barr virus (EBV) latency and EBV-associated diseases, including numerous cancer types. To our knowledge, no specific agent against EBV genes or proteins has been established to target EBV lytic reactivation. Here we report an EBNA1- and Zn2+-responsive probe (ZRL5P4) which alone could reactivate the EBV lytic cycle through specific disruption of EBNA1. We have utilized the Zn2+ chelator to further interfere with the higher order of EBNA1 self-association. The bioprobe ZRL5P4 can respond independently to its interactions with Zn2+ and EBNA1 with different fluorescence changes. It can selectively enter the nuclei of EBV-positive cells and disrupt the oligomerization and oriP-enhanced transactivation of EBNA1. ZRL5P4 can also specifically enhance Dicer1 and PML expression, molecular events which had been reported to occur after the depletion of EBNA1 expression. Importantly, we found that treatment with ZRL5P4 alone could reactivate EBV lytic induction by expressing the early and late EBV lytic genes/proteins. Lytic induction is likely mediated by disruption of EBNA1 oligomerization and the subsequent change of Dicer1 expression. Our probe ZRL5P4 is an EBV protein-specific agent that potently reactivates EBV from latency, leading to the shrinkage of EBV-positive tumors, and our study also suggests the association of EBNA1 oligomerization with the maintenance of EBV latency.

Exosomes-mediated synthetic Dicer substrates delivery for intracellular Dicer imaging detection

Ribonuclease Dicer initiates gene-silencing process by cleaving exogenously long RNA duplexes into small interfering RNA (siRNA) or endogenous precursor microRNAs (pre-miRNAs) into mature miRNAs. It holds great promise in cancer diagnosis and therapeutics due to its molecular ruler role. However, the intracellular Dicer detection remains a key challenge and Dicer related gene therapy has never been explored. In this study, we design a fluorescent labeling Dicer substrate and effectively deliver it into cell by exosomes derived from the target parent cells for intracellular Dicer expression level monitor and gene therapy. Using pre-miRNA let-7a as a model, the Dicer substrates with two terminals labeled with fluorescent and quencher group respectively was obtained by T4 RNA mediated ligase reaction from two short RNA sequences. Then, the substrate was packaged into exosomes by electroporation and delivered to target cells for intracellular dicer imaging detection. After packaging substrates into exosomes with little immunogenicity and good innate biocompatibility by electroporation and delivered to target cells, the Dicer mediated substrate cleavage was effectively monitored by the fluorescence recovery, providing a powerful tool for Dicer analysis. Importantly, the cleaved product exhibited significant suppression toward tumor cell growth and regulated cancer cells cycle. This work might open a new avenue for Dicer analysis and Dicer-related clinical application.

Overexpressed miR-200a promotes bladder cancer invasion through direct regulating Dicer/miR-16/JNK2/MMP-2 axis

Invasive bladder cancer (BC) is one of the most lethal malignant urological tumors. Although miR-200a has been reported as an onco-miRNA that targets the PTEN gene in endometrioid carcinoma, its biological significance in BC invasion has been poorly explored. In the current study, we found that miR-200a was markedly overexpressed in both human BC tissues and BBN-induced muscle-invasive BC tissues. We further showed that miR-200a overexpression specifically promoted human BC cell invasion, but not migration, via transcriptional upregulation of matrix metalloproteinase (MMP)-2. Mechanistic studies indicated that the increased phosphorylation of c-Jun mediated the increasing levels of MMP-2 mRNA transcription. Further investigation revealed that Dicer was decreased in miR-200a overexpressed BC cells; this resulted in inhibition of miR-16 maturation and consequently led to increased JNK2 protein translation and c-Jun activation. Taken together, the studies here showed that miR-200a overexpression inhibited Dicer expression, in turn, resulted in inhibition of miR-16 maturation, leading to upregulation of JNK2 expression, c-Jun phosphorylation, MMP-2 transcription and, ultimately, BC invasion. Collectively, these results demonstrate that miR-200a is an onco-miRNA that is a positive regulator for BC invasion. This finding could be very useful in the ongoing development of new strategies to treat invasive BC patients.

113P - Dicer contributes to chemoresistance in colorectal cancer via regulating a set of miRNAs and their downstream mRNAs

BackgroundChemotherapy has a good success rate in colorectal cancer (CRC); however, recurrence of CRC is still frequent due to acquired drug resistance. Dicer, one of the key enzymes of the microRNA (miRNA) biogenesis pathway, may be involved in chemoresistance through regulating the expression of miRNAs. Until now, the role of Dicer in regulating chemoresistance is still unclear. MethodsOxaliplatin- or 5’FU-resistant cell lines were established to investigate the molecular mechanism of chemoresistance. Western blot was performed to detect the expression of Dicer; next-generation sequencing (NGS) and cDNA array were performed to identify the alternative expression of miRNAs and mRNAs; and RT-qPCR or TaqMan PCR was used to validate the expression of potential miRNAs or mRNAs. Functional characterization of drug-resistant cells was performed by cell proliferation, colony formation, sphere formation, migration and invasive assays. ResultsFrom the TCGA database, the expression level of Dicer mRNA is increased in colorectal cancer; the level of Dicer protein is increased in oxaliplatin- or 5’FU-resistant cell lines. The drug resistant cells possess increased abilities in cell migration, invasion and sphere formation. Knocked-down expression of Dicer can enhance the drug sensitivity in drug resistant cells; in contrast, overexpression of Dicer increases drug resistance in parental drug sensitive cells. By next-generation sequencing and cDNA array, we found that the expression of a set of miRNAs and their downstream mRNAs is changed in oxaliplatin resistant cells. Dicer overexpression enhances the expression of those miRNAs and mRNAs in drug sensitive parental cells. Clinical evaluation further confirmed the increased expression of those miRNAs in the plasma of CRC patients is positively correlated with a poor response to oxaliplatin. In addition, NF-kB and Wnt pathways were found to be activated in drug resistant cells. ConclusionsOur results suggest that those miRNAs may act as biomarkers to predict oxaliplatin response in colorectal cancer by detecting their plasma miRNAs. The molecular mechanism of those miRNAs in regulating the drug response is currently under our investigation. Legal entity responsible for the studyThe author. FundingMinistry of Science and Technology. DisclosureThe author has declared no conflicts of interest.

The Effects on Ishikawa Endometrial Cancer Cell Lines upon Treatment with Hormonal Agents and Novel Drug Target

Background and Hypothesis: Endometrial cancer is the most common gynecological malignancy in the US and will claim the lives of over 12,000 women in 2019. Women with high-risk histologic tumors, such as high-grade endometrial adenocarcinoma, represent over 50% of these deaths. Novel treatments are needed to treat these aggressive tumors. Decreased expression of the ribonuclease DICER is associated with high-grade and recurrent endometrial adenocarcinoma. To study the role of DICER in endometrial cancer, we used CRISPR/Cas9 to delete DICER in an endometrial cancer cell line. RNA sequencing revealed dysregulation of steroid hormone receptor signaling and high expression of APEX1 [apurinic/apyrimidinic endonuclease 1 (Ref-1/APE1)]. Elevated levels of Ref-1/APE1 have been found in various tumor types, and Ref1/APE1 inhibitors are in clinical trials. We hypothesize that our DICER deleted cell lines will be hormone insensitive yet respond to Ref-1/APE1 inhibitors by decreasing cellular proliferation.&#x0D; Project Methods: Ingenuity Pathway Analysis provided an unbiased approach to determine dysregulated genes in steroid hormone signaling in our RNA transcriptomic data. QPCR was used to confirm gene expression in independent samples. Cells were treated with steroid hormones or Ref-1/APE1 inhibitors and proliferation was assessed using MTS.&#x0D; Results: DICER deleted cell lines had downregulated expression of steroid hormone receptors. Treatment with steroid hormones did not have a significant effect on proliferation. However, treatment with Ref-1/APE1 inhibitors resulted in a significant decrease in proliferation.&#x0D; Conclusion and Potential Impact: Ref-1/APE1 inhibitors affect cellular proliferation of endometrial cancer cell lines. Because survival has actually worsened for women with endometrial cancer, novel therapies such as Ref1/APE1 inhibitors deserve future study.

CyclinD1 inhibits dicer and crucial miRNA expression by chromatin modification to promote the progression of intrahepatic cholangiocarcinoma

BackgroundCyclinD1 is crucial for cell cycling and can regulate the expression of Dicer, a crucial regulator of microRNA maturation. However, little is known on how CyclinD1 regulates Dicer and miRNA expression, and the progression of intrahepatic cholangiocarcinoma (ICC).MethodsThe expression of CyclinD1 and Dicer in non-tumor cholangiocytes, ICC cells and tissues as well as their association with clinicopathological characteristics and survival were examined. The potential mechanisms by which CyclinD1 regulates Dicer and relative miRNA expression were determined by immunoprecipitation, ChIP sequence, BSP and luciferase reporter assays following induction of CyclinD1 over-expression or silencing and Dicer silencing. The impact of CyclinD1 and/or Dicer silencing on the growth of ICC was tested in vivo.ResultsUp-regulated CyclinD1 was associated with down-regulated Dicer expression in ICC tissues and poorer overall survival in patients with ICC. CyclinD1 interacted with the nuclear H3K9me3 and SUV39H1 and bound to the Dicer promoter to increase its CpG island methylation in ICC cells. Functionally, CyclinD1 silencing inhibited the malignancy of ICC cells, which were mitigated partially by Dicer silencing in ICC cells. Dicer silencing down-regulated miR-1914-5p and miR-541-5p expression, which targeted and promoted CyclinD1 and CDK6 expression in ICC cells.ConclusionsOur findings uncover that CyclinD1 inhibits Dicer expression by chromatin modification to reduce miR-1914-5p/miR-541-5p expression, which positively-feedback enhances CyclinD1 and CDK6 expression and progression of ICC.

Dietary sulfur amino acid restriction upregulates DICER to confer beneficial effects

ObjectiveDietary restriction (DR) improves health and prolongs lifespan in part by upregulating type III endoribonuclease DICER in adipose tissue. In this study, we aimed to specifically test which missing dietary component was responsible for DICER upregulation. MethodsWe performed a nutrient screen in mouse preadipocytes and validated the results in vivo using different kinds of dietary interventions in wild type or genetically modified mice and worms, also testing the requirement of DICER on the effects of the diets. ResultsWe found that sulfur amino acid restriction (i.e., methionine or cysteine) is sufficient to increase Dicer mRNA expression in preadipocytes. Consistently, while DR increases DICER expression in adipose tissue of mice, this effect is blunted by supplementation of the diet with methionine, cysteine, or casein, but not with a lipid or carbohydrate source. Accordingly, dietary methionine or protein restriction mirrors the effects of DR. These changes are associated with alterations in serum adiponectin. We also found that DICER controls and is controlled by adiponectin. In mice, DICER plays a role in methionine restriction-induced upregulation of Ucp1 in adipose tissue. In C. elegans, DR and a model of methionine restriction also promote DICER expression in the intestine (an analog of the adipose tissue) and prolong lifespan in a DICER-dependent manner. ConclusionsWe propose an evolutionary conserved mechanism in which dietary sulfur amino acid restriction upregulates DICER levels in adipose tissue leading to beneficial health effects.

Bioencapsulation efficacy of sulfated galactans in adult Artemia salina for enhancing immunity in shrimp Litopenaeus vannamei

Live food organisms like Artemia have been used for delivery of different substances such as nutrients, probiotics and immune-stimulants to aquatic animals. Previously, we reported that sulfated galactans (SG) from the red seaweed Gracilaria fisheri (G. fisheri) increased immune activity in shrimp. In the present study we further investigated the capacity and efficiency of bioencapsulation of SG in adult Artemia for delivery to tissues and potentially boosting the expression of immune genes in post larvae shrimp. SG were labelled with FITC (FITC-SG) for in vivo tracking in shrimp. Bioencapsulation of adult Artemia with FITC-SG (0–100 μg mL−1) was performed and the fluorescence intensity was detected in the gut lumen after enrichment periods of 30 min, 1 h, 2 h, 6 h and 24 h. The results showed the Artemia took up SG over time in a concentration-dependent manner. Shrimp were fed with the bioencapsulated Artemia (FITC-SG, 20 μg mL−1) and the shrimp were evaluated under a stereo-fluorescent microscope. At 24 h after administration, FITC-SG was located in gills and hepatopancreas and also bound with haemocytes. With daily SG administration, the genes IMD, IKKβ were up-regulated (after 1 day) while genes dicer and proPO-I were up-regulated later (after 7 days). Moreover, continued monitoring of shrimp fed for 3 consecutive days only with SG at the dose of 0.5 mg g−1 BW showed increases in the expression of IMD, IKKβ genes on day 1 and which gradually declined to normal levels on day 14, while the expression of dicer and proPO-I was increased on day 3 and remained high on day 14. These results demonstrate that bioencapsulation of SG in adult Artemia successfully delivers SG to shrimp tissues, which then bind with haemocytes and subsequently activate immune genes, and potentially increase immunity in shrimp. In addition, the present study suggests that a 3-consecutive-day regimen of SG supplemented in Artemia (0.5 mg g−1 BW) may boost and sustain the enhanced immune functions in post larvae shrimp.

Effect of estrogen-related receptor silencing on miRNA protein machinery expression, global methylation, and deacetylation in bank vole (Myodes glareolus) and mouse tumor Leydig cells

The function of estrogen-related receptor (ERR) in testicular cells is at the beginning of exploration. Our previous findings showed that expression pattern of estrogen-related receptor (ERR) in mouse Leydig cell depends on physiological status of the cell. Exogenous hormones/hormonally active chemicals affect ERR expression. In Leydig cells in vitro, ERRα and ERRγ show opposing regulatory properties.The aim of this study was to examine the role of ERR in epigenetic processes in cells with altered level of secreted estrogens; mouse tumor Leydig cells and bank vole Leydig cells, respectively.In Leydig cells, ERRα and ERRγ were silenced via siRNA. mRNA and protein expression and protein localization of molecules required for miRNA biogenesis and function (Exportin 5, Dicer, Drosha and Argonaute 2; Ago2) were studied with the use of qRT-PCR, Western blotting, and immunohistochemistry. Global DNA methylation and histone deacetylation status together with estradiol secretion were determined with fluorometric, and immunoenzymatic assays.Regardless of ERR type knockdown in tumor Leydig cells, downregulation (P < 0.05; P < 0.01; P < 0.001) of Exportin5, Dicer, Drosha but not Ago2 was revealed while at protein level only Drosha was downregulated (P < 0.01) by both ERRα and ERRγ. Oppositely, Exportin5, Dicer and Ago2 showed ERR type-dependent regulation (downregulation; P < 0.01 by ERRα and upregulation; P < 0.01; P < 0.001 by ERRγ). In ERR-silenced vole Leydig cells, expression of Exportin5, endonucleases and Ago2 was not changed. Immunolocalization of Dicer and Ago2 was independent of the cell origin in contrast to localization of Exportin5 and Drosha which was dependent on the cell origin and ERR type knockdown. Absence of ERR effected on cell methylation status (ERRα increased it; P < 0.01 while ERRγ decreased it; P < 0.01, P < 0.001) but it not changed histone deacetylates activity. ERRα and ERRγ silencing decreased (P < 0.01, P < 0.001) estradiol secretion in both tumor and vole Leydig cells.In mouse and bank vole Leydig cell, Exportin5, Dicer, Drosha and Ago2 expression as well as methylation status are regulated by ERR in a manner related to receptor type, molecule type, cell origin and level of secreted estrogen.

Abstract 2355: IKKa serves as a molecular switch linking miRNA biogenesis and inflammation in skin tumorigenesis

Abstract Our previous studies have shown that the absence of tumor suppressor IKKα leads to development of spontaneous mouse squamous cell carcinomas (SCCs) in the skin, lungs, and esophagus, which is partially mediated by aberrant epigenetic alterations and inflammation. However, it remains unknown whether there is a crosstalk between the above-mentioned mechanisms. As a part of the epigenetic machinery, miRNAs regulate many genes post-transcriptionally. Here we show that IKKα deletion in the mouse epidermis results in impaired miRNA processing with markedly reduced expression of ribonuclease Dicer, an enzyme required for miRNA maturation. We found that the lack of IKKα causes Dicer gene silencing through DNA methylation. As a result, the maturation of miR-203-3p, which is the most abundant miRNA in keratinocytes, is blocked in Ikkα-null keratinocytes. Next, we identified Myd88, an important component of innate immunity and inflammation, as one of miR-203-3p targets. The level of Myd88 expression is robustly increased in Ikkα-null keratinocytes due to the lack of miR-203-3p. We also observed increased activity of caspase 1 cleavage, an indicator of inflammasome activation, in Ikkα-null keratinocytes. Interestingly, Myd88 deletion or 5-aza-2’-deoxycytidine (DNA methyltransferase inhibitor) treatment rescued the survival of Ikkαf/f;K5Cre mice, which usually die within three weeks after birth. Ikkαf/f;Myd88f/f;K5Cre mice also develop much fewer spontaneous skin tumors. By analyzing a cohort of human head and neck SCCs from TCGA database, we found that Dicer expression is concurrently downregulated among 32% of patients with low IKKα expression. Taken together, our finding illustrated how IKKα loss mediates Dicer gene silencing, which turns off miRNA processing, including miR-203-3p maturation, and eventually triggers Myd88-dependent inflammation. Thus, our study revealed a novel IKKα/Dicer/miR-203-3p/Myd88 signaling loop, in which IKKα serves as a molecular switch linking microRNA biogenesis and inflammation in skin tumorigenesis. Citation Format: Feng Zhu, Jian Zhang, Jami Willette-Brown, Ling Su, Xiaolin Wu, Yinling Hu. IKKa serves as a molecular switch linking miRNA biogenesis and inflammation in skin tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2355.

Abstract 3016: Dicer overexpression contributes to chemoresistance in colorectal cancer via up-regulating a set of miRNAs

Abstract Chemotherapy has a good success rate in the treatment of colorectal cancer; however, recurrence of colorectal cancer is still frequent due to acquired resistance. Aurora-A, a cell cycle-regulated kinase, plays an important role in colorectal cancer development and drug resistance. Dicer, one of the key enzymes of the microRNA (miRNA) biogenesis pathway, may be involved in chemoresistance through regulating the expression of miRNAs. According to the TCGA database, the expression levels of Dicer and Aurora-A were increased in colorectal cancer, which may contribute to chemoresistance. Our results showed that the protein levels of Dicer and Aurora-A are increased in oxaliplatin resistant cell lines. Knocked-down expression of Dicer or Aurora-A can enhance drug sensitivity in oxaliplatin-resistant cells; in contrast, overexpression of Dicer or Aurora-A increased drug resistance in parental drug sensitive cells. By next-generation sequencing, we found that the expression of a set of miRNAs is increased in oxaliplatin resistant cells. Dicer overexpression enhances the expression of those miRNAs in drug sensitive parental cells. Clinical evaluation further confirmed the increased expression of those miRNA in the plasma of CRC patients with a poor response to oxaliplatin. Our results suggest that those miRNAs may act as biomarkers to predict oxaliplatin response in colorectal cancer by detecting their plasma miRNAs. The molecular mechanism of those miRNAs in regulating the drug response is currently under our investigation. Citation Format: Li Jyuan Lin, Liang-Yi Hung. Dicer overexpression contributes to chemoresistance in colorectal cancer via up-regulating a set of miRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3016.

Low Drosha protein expression in cutaneous T-cell lymphoma is associated with worse disease outcome.

Dysregulation of microRNAs (miRNAs) key regulators may contribute to the pathogenesis of malignancies. miRNA machinery genes such Dicer and Drosha have been reported to be biomarkers in different cancer types. We aimed to evaluate Drosha and Dicer protein expression in cutaneous T-cell lymphoma (CTCL). We performed Drosha and Dicer immunohistochemistry in 45 patients with mycosis fungoides and subtypes. Drosha and Dicer expression scores were correlated with clinical parameters including disease-specific death (DSD), stage of disease and different laboratory data. Uni- and multivariate statistics were performed. On univariate analysis, elevated serum LDH and low Drosha expression were significantly associated with advanced stage (P=0.032 and 0.0062, respectively) and lymphoma-specific death (LSD; P=0.017 and P=0.005, respectively). Moreover, elevated circulating CD4+/CD26- lymphocytes were significantly associated with advanced stage (P=0.032) and DSD (P=0.0098). On multivariate analysis, low Drosha expression remained in the logistic regression model as significant independent predictor for advanced disease stages [P=0.013; odds ratio: 5 (confidence interval) CI 1.3-19.3]. Moreover, low Drosha expression (P=0.026) and elevated LDH (P=0.025) remained as significant independent predictors for DSD with odds ratios of 13.5 (CI 1.3-134.4 and 8.7 CI 1.3-57.2, respectively). Low Drosha expression is an independent predictor for advanced stage as well as LSD in CTCL patients indicating a tumour suppressor gene function of Drosha in this disorder.

Oleanolic acid inhibits RANKL-induced osteoclastogenesis via ER alpha/miR-503/RANK signaling pathway in RAW264.7 cells

Oleanolic acid (OA) has recently become a research hotspot in the treatment of many human diseases, especially osteoporosis and arthritis. However, the mechanisms are not elucidated completely. We aimed to elucidate the target and the mechanism via which OA inhibited osteoclast differentiation. We used TRAP staining and toluidine blue dye to test OA effect on osteoclastogenesis and bone resorption respectively. We detected the expression level of osteoclast differentiation related genes, estrogen receptor alpha (ERα) and miR-503. We blocked ERα with its specific blocker, methylpiperidino pyrazole (MPP). We antagonized the function of miR-503 with antagomir-503-5p. RT-PCR and ELISA kits were used to investigate the effects of OA on miR-503 formation and maturation-relevant enzymes Dicer and Drosha at gene and protein levels. The data suggested that OA inhibited osteoclastogenesis and bone resorption. OA upregulated ERα and miR-503 expression levels, inhibited RANK expression. MPP significantly attenuated the OA effect including inhibiting osteoclastogenesis, inhibiting bone resorption and up-regulating miR-503 expression. It showed that ERα was the target of OA and OA up-regulated miR-503 expression through ERα. Antagomir-503-5p inhibited the function of miR-503 and attenuated the inhibition of OA on osteoclastogenesis, suggesting that OA inhibited osteoclast by up-regulating miR-503 expression. In addition, OA up-regulated miR-503 by up-regulating Dicer expression. In conclusion, OA inhibits RANKL-induced osteoclastogenesis via ERα/miR-503/RANK signaling pathway in RAW264.7 cells.

The impact of variations in transcription of DICER and AGO2 on exacerbation of childhood B-cell lineage acute lymphoblastic leukaemia.

The expression of microRNA in eukaryotic cells is subject to tightly regulated processing. The altered expression of microRNAs in a number of cancers suggests their contribution to disease pathogenesis, where processing pathways may be involved in disease pathogenesis. In the present study, we evaluated changes in the profile of two main components of microRNA biogenesis, AGO2 and DICER, and assessed their correlation with disease progression in childhood acute lymphoblastic leukaemia (ALL). To achieve this aim, 25 patients afflicted with ALL were included in the study along with 25 healthy subjects as control. The expression level of AGO2 and DICER was evaluated by real-time PCR. The results revealed an increase in the expression of DICER and a decrease in AGO2 in patients. The correlation between the alteration levels of these genes with pathologic events was also studied. This increase or decrease proved to be directly correlated with the progression of the disease particularly in L1 to L2. According to the obtained results, it can be deduced that dysregulation in transcription of DICER and AGO2, involved in the formation of mature microRNAs in cytoplasm of ALL cancer cells, is a part of the pathological molecular mechanism implicated in the exacerbation of this malignancy. Therefore, the genes involved in microRNAs biogenesis that have been studied here could be considered as candidate prognostic markers especially in childhood ALL which will help towards a better understanding of the molecular basis of ALL.

Exosomal miRNA-107 induces myeloid-derived suppressor cell expansion in gastric cancer.

Background: Myeloid-derived suppressor cells (MDSCs) promote immunosuppression in the tumor microenvironment, support tumor growth and survival, and may contribute to immunotherapy resistance. Recent studies showed that tumor-derived exosomes (TDEs) can induce MDSCs accumulation and expansion, the mechanisms of which are largely unknown.Methods: The morphologies and sizes of the exosomes was observed by using a JEM-1400 transmission electron microscope. MicroRNA(miR)-107 and ARG1, DICER1, PTEN, PI3K, AKT, mTOR, and NF-kB mRNAs were quantified by quantitative reverse tanscription PCR. Dual-Luciferase Reports Assay were used to examine the expression of genes which was targeted by miR-107. The expression of proteins were analyzed by using western blot.Results: MiR-107 was not only overexpressed in gastric cancer cells but also enriched in their secreted TDEs. Also, these miR-107 enriched TDEs could be taken up by HLA-DR-CD33+MDSCs, where miR-107 was able to target and suppress expression of DICER1 and PTEN genes. Dampened DICER1 expression supported expansion of MDSCs , while decreased PTEN led to activation of the PI3K pathway, resulting in increased ARG1 expression. Furthemore, gastric cancer-derived miR-107 TDEs, when dosed intravenously into mice, were also capable of inducing expansion of CD11b+Gr1+/high MDSCs in mouse peripheral blood and altering expression of DICER1, PTEN, ARG1, and NOS2 in the MDSCs.Conclusions: Our findings demonstrate for the first time that gastric cancer-secreted exosomes are able to deliver miR-107 to the host MDSCs where they induce their expansion and activition by targeting DICER1 and PTEN genes, thereby may provide novel cancer therapeutics target for gastric cancer.

Diagnostic value of plasma expression of microRNAs complementary to Drosha and Dicer in lung cancer patients.

Lung cancer (LC) is diagnosed mostly in advanced, non-operable stage, with poor prognosis. The analysis of microRNAs may be a useful tool for early and non-invasive detection of cancer. Dicer and Drosha are enzymes with an essential role for microRNA biogenesis. The aim of our study was to analyze the expression of miRNA-27a-3p, miRNA-31, miRNA-182, miRNA-195 with the ability to reciprocal regulation of Dicer and Drosha expression in lung cancer patients. The relative expression of microRNAs was detected by qPCR in plasma of 160 LC patients. The U-Mann Whitney test was used to compare the relative expression between particular groups of lung cancer patients and healthy individuals. The diagnostic value of microRNAs examination was analyzed using a receiver operating curve. We demonstrated that the plasma levels of miRNA-27, miRNA-31 and miRNA-182 were significantly higher and miRNA-195 significantly lower in the whole group of LC patients and in patients with early stages of NSCLC, in comparison with healthy donors. ROC analysis showed that four studied microRNAs have a potential diagnostic value for early stages of NSCLC with AUC=0.95 for miRNA-27a (94% sensitivity and 81% specificity, p=0.0001), 0.71 for miRNA-31 (73% sensitivity and 61% specificity, p=0.001) 0.77 for miRNA-182 (70% sensitivity and 79% specificity, p=0.0001) and 0.82 for miRNA-195 (74% sensitivity and 80% specificity, p=0.0001). We have proved that the expression of miRNA-27a-3p, miRNA-31, miRNA-182, and miRNA-195 in patients with LC is different from the expression of these molecules in healthy people. The examination of these microRNAs in plasma could be used in non-invasive lung cancer diagnosis.

Cloning, expression, and characterization of the zebrafish Dicer and Drosha enzymes

The biogenesis of animal microRNAs (miRNAs) involves transcription followed by a series of processing steps, with Drosha and Dicer being two key enzymes that cleave primary miRNA (pri-miRNA) and precursor miRNA (pre-miRNA) transcripts, respectively. While human and fly Dicer and human Drosha are well studied, their homologs in other organisms have not been biochemically characterized, leaving open the question of whether their miRNA substrate specificities and regulatory functions are conserved. Zebrafish is a widely used model organism, but its miRNA processing enzymes have never been reconstituted and analyzed. In this study we cloned and constructed expression plasmids encoding zebrafish Dicer, Drosha, and their accessory proteins TARBP2 and DGCR8. After transfection of human cell cultures, we isolated the recombinant protein complexes. We found that zebrafish Dicer bound TARBP2, but Dicer alone exhibited significant pre-miRNA processing activity. On the other hand, zebrafish Drosha associated with DGCR8, and both were required to cleave pri-miRNAs. The Drosha/DGCR8 holoenzyme preferred pri-miRNAs with a large terminal loop, an extended duplex region, and flanking single stranded RNAs. These results lay the foundation for future studies of the regulatory roles and conserved mechanisms of Drosha and Dicer.

SUN-194 Erasing the Methyl Mark: Mechanism of Androgen Action in the Ovary

Androgen actions through androgen receptor (AR) are critical for follicle growth and female fertility. While excess androgen level leads to polycystic ovary syndrome (PCOS), a certain amount of direct androgen action is critical for normal ovarian function. Thus, with respect to androgen actions in the ovary, balance is key. However, how androgens regulate ovarian physiology or its underlying mechanism is still poorly understood. Androgen actions are mediated through ‘nuclear/genomic’ or ‘extra-nuclear/non-genomic’ actions of AR. Recently we reported that androgens through both the extra-nuclear and nuclear pathways inhibit the expression and activity of Ezh2, (a histone methyl-transferase involved in gene repression mark, H3K27me3) to induce different genes that are imperative for ovarian function. Here, we further extended our study to elucidate other histone modifications induced by androgens and its downstream physiological effect in follicular development. In addition to Ezh2, we have found that androgens also regulated the expression of a histone demethylase called Jumonji domain containing protein 3 (JMJD3/KDM6B), that is responsible of removing H3K27me3 mark. Results showed that DHT in GCs increased hypoxia-inducible factor 1 alpha (HIF1α) protein levels through the PI3K/Akt pathway, which in turn induced JMJD3 expression. We found that DHT had not affect HIF1α mRNA but increased HIF1α protein levels. This effect of DHT on HIF1α protein level was blocked by flutamide (AR inhibitor), LY294002 (PI3K inhibitor) as well as cyclohexamide (protein translational inhibitor). Interestingly the 5’ prime end of JMJD3 have HIF1α response elements (HRE) and it is well established that several JMJDs are induced by HIF1α under both normal conditions and hypoxic stress. Our studies showed that DHT treatment induced JMJD3 expression and decreased H3K27me3 levels. Inhibition of AR, PI3K/AKT pathway and siRNA-mediated knockdown of HIF1α attenuated androgen-induced expression of JMJD3. Moreover, ChIP studies showed increased binding of HIF1α on the JMJD3 promoter region with DHT treatment. In our efforts to understand the downstream effects of androgen-induced JMJD3 expression, we found Dicer1 was upregulated upon DHT treatment to mouse granulosa cells. Given the critical role of Dicer in follicular development, ovarian physiology and female fertility, in general, along with the importance of androgens in ovarian physiology, we propose that regulation of Dicer is one of the underlying mechanisms of androgen actions in the ovary. Androgens in a transcription-independent manner, through the PI3K/Akt signaling pathway, enhance HIF1α protein synthesis under normoxic conditions which in turn regulate the expression of Dicer through JMJD3. Altogether, this study provides a mechanistic insight of androgen actions in ovarian function by epigenetic modulation.

MON-100 Unique miRNA Signature in HIV Lipodystrophy with Reduced Adipose Dicer Expression

Suppression of Dicer, an endoribonuclease that regulates microRNAs(miRNA), has evolved as a viral mechanism to enhance host HIV infectivity and may have unintended metabolic consequences. Animal knockout models of adipose-specific dicer(ADicer) acquire lipodystrophy accompanied by severe metabolic abnormalities. Data show adipose is a source of exosomal miRNAs, functioning as adipokines influencing metabolic homeostasis. We hypothesized a unique miRNA profile among individuals well-phenotyped for HIV lipodystrophy and reduced ADicer expression. We evaluated >1000 miRNAs from exosomes derived from sera among the 27 male individuals [9 HIV lipodystrophy(HIV/lipo), 9 HIV without lipodystrophy(HIV/non-lipo), 9 non-HIV] whom we previously showed variations in ADicer: most suppressed among HIV lipo, followed by HIV non-lipo and non-HIV (2.49[0.02,4.88] vs. 11.20[4.83,21.45] vs. 17.69[10.72,47.91],P=.002). To estimate miRNA abundance, data was normalized using the geomean of expressed Ct values. A false discovery rate analysis(FDR) was applied. We investigated whether significant exosomal miRNAs demonstrated a similar pattern in corresponding subcutaneous adipose tissue(SAT) samples. Using target prediction databases (TargetScan, miRDB, Diana), we identified genes related to fat biology (target score ≥85%) with clinical relevance to lipodystrophic phenotypes and further evaluated these in the adipose depot. HIV/lipo individuals(age 56±3years, BMI 30±1kg/m2, duration HIV 24±2years, duration ART 20±2years, CD4+ count 482±90cells/μl, undetectable VL 67%) were similar to HIV/non-lipo(age 52±3 years, BMI 30±1 kg/m2) and non-HIV(age 55±3years, BMI 30±1kg/m2) individuals. Reduced ADicer expression was significantly related to reduced CD4+ count(r=0.55,P=.02), duration ART use(r=-0.70,P=.001) and tended to be related to duration HIV(r=-0.44,P=.07) and reduced CD8+ count(r=0.42,P=.08). Applying FDR, we detected exosomal miRNA-20a-3p(P=.0026), 324-5p(P=.0059), and 186-5p(P=.0977) were expressed differentially in HIV/lipo vs. non-HIV and 324-5p(P=.0348) in HIV/lipo vs. HIV/non-lipo. We further confirmed that miRNA-20a-3p was significantly and differentially expressed in the SAT, expressed highest in HIV lipo followed by HIV non-lipo and non-HIV [9.73±12.38 vs. 2.24±1.84 vs. 1.02±0.19, P<0.05] in a pattern similar to the exosomal profiles. Relevant miRNA and their target genes which tended to be differentially expressed in the SAT of HIV lipo vs. HIV non-lipo include: 20a-3p (TDG, P=0.07) and 186-5p (PDE10A, PID1; P<0.05). Altered ADicer expression and specific exosomal miRNAs may affect gene expression of regulators important to fat biology and metabolic homeostasis in HIV. In particular, miRNA-20a-3p may function as an adipokine demonstrating similar expression profiles in exosomes and adipose tissue of individuals with HIV lipodystrophy.