Inhibition Of Luciferase Activity Research Articles

DSCAM-AS1 promotes cervical carcinoma cell proliferation and invasion via sponging miR-338-3p.

Deregulated lncRNA DSCAM-AS1 expression was found in several tumors. However, mechanism and functional role of DSCAM-AS1 in cervical carcinoma remain unknown. DSCAM-AS1 was detected in cervical carcinoma specimens and cells by RT-qPCR. CCK-8, Matrigel transwell, and flow cytometry were conducted to determine cell functions. In this research, we firstly we explored DSCAM-AS1 expression in cervical carcinoma cells and specimens. We revealed that DSCAM-AS1 was upregulated in cervical carcinoma lines (C4-1, Caski, Hela, and Siha) compared to GH329 cells. DSCAM-AS1 was upregulated in cervical carcinoma specimens compared to control no-tumor specimens. Overexpression of DSCAM-AS1 induced cervical carcinoma cell growth and cycle. Moreover, our data revealed that miR-338-3p expression was downregulated in cervical carcinoma cells and specimens. There was a negative correlation between miR-338-3p expression and DSCAM-AS1 expression in cervical carcinoma specimens. Elevated expression of miR-338-3p decreased cervical carcinoma cell growth and cycle and invasion. Furthermore, luciferase reporter analysis revealed that miR-338-3p overexpression suppressed luciferase activity of WT-DSCAM-AS1 vector but not the mut-DSCAM-AS1. Ectopic expression of DSCAM-AS1 decreased miR-338-3p expression in the Siha cell. Overexpression of DSCAM-AS1 promoted cervical carcinoma cell growth and cycle via regulating miR-338-3p. These results suggested that DSCAM-AS1 functions as one oncogene through sponging miR-338-3p in cervical carcinoma.

MiR-139-5p Targeting CCNB1 Modulates Proliferation, Migration, Invasion and Cell Cycle in Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most frequent histological subtype of non-small cell lung cancer. Cyclin B1 (CCNB1) is the vital initiator and controller of mitosis. Studies have indicated that CCNB1 overexpression is closely associated with cell proliferation and tumorigenesis in many cancers. Thus, discovery of molecular mechanism of CCNB1 in LUAD is conducive to developing new diagnostic or therapeutic targets for LUAD. We acquired mature miRNA and mRNA expression information of LUAD from TCGA database, as well as related clinical data. CCNB1 expression in normal and LUAD tissue was analyzed. Relationship between CCNB1 and patient's survival and clinical stage was analyzed. Upstream regulatory gene miRNA of CCNB1 was predicted. qRT-PCR and western blot examined expression levels of CCNB1 and miR-139-5p in cells. CCK-8 tested cell proliferation. Scratch healing and Transwell determined cell migration and invasion. Flow cytometry analyzed the cell cycle. Dual-luciferase verified targeting relationship between the two genes. Compared to controls, CCNB1 expression was prominently high in LUAD patient samples, and associated with advanced tumor stages and shorter overall survival. MiR-139-5p expressed an evidently negative correlation with CCNB1 and was predicted to target CCNB1. MiR-139-5p mimics reduced CCNB1 mRNA and protein expression, and suppressed luciferase activity in a target-specific manner, as confirmed by a control construct with a mutated miR-139-5p binding site. CCNB1 overexpression fostered progression of LUAD cells. Mechanistically, miR-139-5p might negatively regulate CCNB1 in LUAD, thereby suppressing cell proliferation, migration, invasion and cell cycle.

I.p.-injected cationic liposomes are retained and accumulate in peritoneally disseminated tumors

Intraperitoneal (i.p) chemotherapy is an attractive approach to treat peritoneally disseminated cancers by delivering therapeutic agents directly to the peritoneal cavity where some disseminated tumors are located. Cationic liposomes (CLs) have been used as a viable delivery carrier for i.p. chemotherapy to improve the peritoneal retention of anticancer agents. However, there are no reports on the fate of CLs following i.p. administration to the peritoneal cavity in the presence of disseminated tumors. We prepared a tumor xenograft murine model of peritoneally disseminated gastric cancer by i.p. inoculation of human gastric cancer cells and followed the fate of either CLs or PEGylated CLs (PEG-CLs) after i.p. injection in the model. I.p.-injected CLs were retained in peritoneal cavity for at least 3 days post-injection as a result of clustering with ascites fluid proteins, mainly albumin, while i.p. PEG-CLs was rapidly cleared from the peritoneal cavity to the circulation within 3 h post-injection. Importantly, i.p. CLs efficiently accumulated in the targeted disseminated tumor cells, but not in other abdominal organs including liver, spleen, and kidney. The tumor selectivity upon i.p. administration of CLs may be associated with the lymphatic drainage system. A lipoplex formulation composed of CLs with short hairpin RNA (shRNA) against luciferase, a model therapeutic agent, suppressed luciferase activity in peritoneally disseminated tumors by 80%, with no cytokine secretion in serum. This suggests that i.p. CLs can efficiently deliver a therapeutic agent to peritoneally disseminated tumors with few systemic adverse events. These results suggest that i.p. treatment with CLs or non-PEGylated lipoplexes may be a promising approach for the treatment of peritoneally disseminated cancers through their ability to selectively deliver therapeutic agents to i.p. target sites with minimal systemic adverse events.

A putative protein-RNA complex regulates posttranscriptional processing of cytochrome P450 aromatase (CYP19A1) in bovine granulosa cells.

Follicle growth and granulosa cell health are dependent on the secretion of estradiol from granulosa cells. Estradiol is synthesized from androgen precursor by cytochrome P450 aromatase (CYP19A1), and in cattle CYP19A1 messenger RNA has a short half-life but a long (3.5 kb) 3'-untranslated region (3'UTR), suggesting that posttranscriptional regulation may be important for control of enzyme activity. We tested this hypothesis by inserting the CYP19A1 3'UTR and fragments thereof into a reporter vector between the end of the luciferase coding region and the polyadenylation signal. The full-length aromatase 3'UTR suppressed luciferase activity to 10% of control levels, and smaller fragments showed that this inhibitory activity lies between +926 and +1134 of the 3'UTR. Protein-RNA cross-linking experiments revealed that these 3'UTR fragments formed an RNA-protein complex of approximately 70 kDa that was present in granulosa cells but not in corpus luteum, lung, liver, kidney, pancreas, or bladder extracts. The RNA-binding activity was specific to the 3'UTR, as shown by competition experiments with unlabeled RNA, and was present only in 3'UTR constructs that inhibited luciferase activity. These data suggest that posttranscriptional regulation is an important component of the control of CYP19A1 expression and involves protein binding to a specific sequence in the 3'UTR.

Downregulation of miR‑143 modulates KRAS expression in colorectal carcinoma cells.

MicroRNAs (miRs) are a class of non‑coding small RNAs that have been demonstrated to be involved in the pathogenesis of human cancer. There is even evidence that microRNAs can act as oncogenes or tumor suppressors. Although microRNA expression profiles have been characterized in colorectal carcinoma, their precise physiological functions are largely unknown. It has become clear that the activated KRAS Proto‑Oncogene, GTPase (KRAS) oncogene plays an important role in colorectal carcinogenesis. In the present study, it was found that the level of mature miR‑143 was lower in colorectal carcinoma tissues compared with that observed in normal adjacent tissues. A lack of miR‑143 was detected in human colorectal carcinoma cell lines, SW480, LoVo and HT‑29, compared to the high expression observed in normal colon epithelial cell line NCM460. pcDNA3.1‑pri‑miR‑143 and its mutant were successfully constructed and transfected into colorectal carcinoma cells. Increased accumulation of mature miR‑143 was observed in the pcDNA3.1‑pri‑miR‑143‑transfected cells. In SW480 cells, transfection of pcDNA3.1‑pri‑miR‑143 resulted in a 35 and 47% reduction in cell growth after incubation for 4 and 5 days, respectively, compared with transfection of the pcDNA3.1‑pri‑miR‑143 mutant; while in LoVo cells, transfection of pcDNA3.1‑pri‑miR‑143 resulted in a 33 and 46% reduction in cell growth respectively. In contrast, transfection of pcDNA3.1‑pri‑miR‑143 had no significantly effects on HT‑29 cell growth. We also found that transfection of pcDNA3.1‑pri‑miR‑143 had no effect on levels of KRAS mRNA, but resulted in a 58% decrease in the KRAS protein level in the transfected SW480 cells, while an approximate 54 and 43% KRAS protein reduction in LoVo and HT‑29 cells, respectively, compared with the pcDNA3.1‑pri‑miR‑143 mutant. Two fragments containing the putative complementary site were cloned into the pGL3 vector, constructing the luciferase reporter pGL3‑KRAS‑CS1 and pGL3‑KRAS‑CS2. Cotransfection of pcDNA3.1‑pri‑miR‑143 with pGL3‑KRAS‑CS1 and pGL3‑KRAS‑CS2 respectively resulted in 4.6‑ and 3.3‑fold inhibition of luciferase activity in the SW480 cells, while a 4.0‑ and 3.2‑fold inhibition of luciferase activity in the LoVo cells, 3.7‑ and 3.1‑fold inhibition in the HT‑29 cells. Differences in pGL3‑KRAS‑CS1 and pGL3‑KRAS‑CS2 activity were not significant. Our results revealed that increased accumulation of miR‑143 is likely to modulate levels of KRAS protein expression at the post‑transcriptional level by interacting specifically with the complementary site, and consequently inhibiting proliferation of the transfected cells.

MiR3609 sensitizes breast cancer cells to adriamycin by blocking the programmed death-ligand 1 immune checkpoint

Chemoresistance is the leading cause of breast cancer therapy failure, and studies of the mechanisms underlying chemoresistance and the treatment of drug-resistant tumors remain challenging. In the present study, we discovered a novel microRNA, miR3609, that influences the malignancy of breast cancer. Our results showed miR-3609 expression was lower in resistant breast cancer cells than in sensitive breast cancer cells (MCF-7), while PD-L1 expression was higher in resistant breast cancer cells than in sensitive breast cancer cells. Co-transfection of a miR-3609 plasmid with a luciferase construct containing the PD-L1 3′-untranslated region suppressed luciferase activity. Transfection of a miR-3609 mimic markedly suppressed PD-L1 protein expression in MDA-MB-231 and MDA-MB-468 cells in a dose-dependent manner and increased the sensitivity of MCF7/ADR cells to adriamycin, whereas transfection of a miR-3609 inhibitor enhanced PD-L1 protein expression in HBL-100 and MCF-7 cells. In addition, knockdown of PD-L1 by siRNA restored the sensitivity of MCF7/ADR cells to adriamycin. Mice injected with breast cancer cells stably overexpressing miR3609 survived markedly longer and had fewer tumors than mice injected with control miRNA (miR-sc)-transfected cells. Treatment with a CD8+ blocking antibody (anti-CD8) eliminated these effects, suggesting that CD8+ T cells are required for the efficacy of miR3609 in breast cancer. Further, low miR-3609 expression and high PD-L1 expression were correlated with poor prognosis in breast cancer patients. Therefore, restoration of miR-3609 expression may sensitize breast cancer to adriamycin by blocking PD-L1 expression.

MicroRNA-144 regulates the NF-κB signaling in miiuy croaker via targeting IL1β

microRNAs have been demonstrated to be critical regulators of the immune responses. While, the miRNA-mediate the detail regulatory mechanism response is still not clear in fish species. In this research, the regulation of miRNA to the NF-κB signaling through decreasing the target gene mRNAs was discussed in miiuy croaker. We first used the bioinformatics predicted miR-144 has a direct negative regulatory affect on IL1β in miiuy croaker, further the luciferase assays were used to probe the functions of miR-144. The overexpression of miR-144 mimics and pre-miR-144 plasmid all showed the dose-dependent pattern on IL1β. Moreover, the inhibition of luciferase activity was attenuated after co-transfected with miR-144 inhibitors. In addition, we observed that the miR-144 could negative regulate to the nuclear factor kappaB (NF-κB) signaling in miiuy croaker by targeting IL1β. In conclusion, our studies on miR-144 will enlarge knowledge of its functions in regulation of immune response, further provide a new insight to research on the immune regulation mechanism in teleost fish.

The 9H-Fluoren Vinyl Ether Derivative SAM461 Inhibits Bacterial Luciferase Activity and Protects Artemia franciscana From Luminescent Vibriosis.

Vibrio campbellii is a major pathogen in aquaculture. It is a causative agent of the so-called “luminescent vibriosis,” a life-threatening condition caused by bioluminescent Vibrio spp. that often involves mass mortality of farmed shrimps. The emergence of multidrug resistant Vibrio strains raises a concern and poses a challenge for the treatment of this infection in the coming years. Inhibition of bacterial cell-to-cell communication or quorum sensing (QS) has been proposed as an alternative to antibiotic therapies. Aiming to identify novel QS disruptors, the 9H-fluroen-9yl vinyl ether derivative SAM461 was found to thwart V. campbellii bioluminescence, a QS-regulated phenotype. Phenotypic and gene expression analyses revealed, however, that the mode of action of SAM461 was unrelated to QS inhibition. Further evaluation with purified Vibrio fischeri and NanoLuc luciferases revealed enzymatic inhibition at micromolar concentrations. In silico analysis by molecular docking suggested binding of SAM461 in the active site cavities of both luciferase enzymes. Subsequent in vivo testing of SAM461 with gnotobiotic Artemia franciscana nauplii demonstrated naupliar protection against V. campbellii infection at low micromolar concentrations. Taken together, these findings suggest that suppression of luciferase activity could constitute a novel paradigm in the development of alternative anti-infective chemotherapies against luminescent vibriosis, and pave the ground for the chemical synthesis and biological characterization of derivatives with promising antimicrobial prospects.

MiR-362 regulates the proliferation and apoptosis of porcine immature Sertoli cells through targeting the ZNF644 gene

In testicular tissue, immature Sertoli cell proliferation ability determines the size of mature Sertoli cell populations, which further regulates the spermatogenesis in the adult male animals. Studies have demonstrated that microRNAs (miRNAs) participate in the regulation of the immature Sertoli cell proliferation and apoptosis, but the functions of most identified miRNAs remain unclear. In this study, based on previous RNA-seq results, we analyzed the regulatory role (s) of miR-362 in proliferation and apoptosis of porcine immature Sertoli cells. The ZFN644 gene was predicted to be a target gene of miR-362 using bioinformatics methods. The expression levels of miR-362 and ZNF644 gene were measured using qRT-PCR assay in developing porcine testicular tissues and in immature Sertoli cells transfected with either miR-362 mimic or miR-362 inhibitor. The dual luciferase reporter assay was used to determine the regulatory relationship between miR-362 and ZNF644. The results showed that a putative target site of miR-362 was located in the 3'UTR of ZNF644. The expression of miR-362 was significantly and negatively correlated with ZNF644 expression in the developing porcine testicular tissues. Co-transfection of miR-362 and psiCHECK2-ZNF644-WT 3'UTR luciferase vector significantly suppressed luciferase activity. The ZNF644 gene expression level was significantly regulated by miR-362, demonstrating that miR-362 targets ZNF644 gene and inhibits its expression in porcine immature Sertoli cells. Flow cytometry, CCK8, and EdU assays were used to measure the effects of over-expression of miR-362 or knockdown of ZNF644 on porcine immature Sertoli cell proliferation; Annexin V-FITC/PI staining assays and qRT-PCR technology were used to test the apoptosis and the expression levels of cell survival-related genes, respectively. Over-expression of miR-362 and knockdown of ZNF644 arrested the porcine immature Sertoli cells in G1 and G2 phases of the cell cycle, respectively, and inhibited proliferation, enhanced apoptosis in the porcine immature Sertoli cells, and significantly regulated the expression levels of cell survival-related genes. Taken together, these data indicate that miR-362 inhibits proliferation and promotes apoptosis in porcine immature Sertoli cells by targeting the ZNF644 gene, thereby providing the scientific basis for further study on the function(s) of miR-362 in the porcine spermatogenesis.

Abstract 2013: microRNAs targeting EMT transcription factors in breast cancer and their relation to lymph node metastasis

Abstract Metastases derived from primary tumors are responsible for the high cancer-associated death rates. Lymph nodes near to the primary breast tumor have a high chance to develop a secondary tumor, representing one of the first signs of metastasis. For this reason, inhibition of metastasis should be a major goal for breast cancer treatment. Metastasis is a non-random process that initiates with a phenotypic change of the tumor cells, named Epithelial-Mesenchymal Transition (EMT), and is leaded by the transcription factors SNAIL, SLUG, ZEB and TWIST. Because these changes in tumor cells must be plastic and reversible, epigenetic alterations are necessary for EMT. In this sense, microRNAs have emerged as candidate molecular biomarkers and novel therapeutic targets associated to metastasis. The aim of this study is to identify microRNAs that regulate the expression of EMT-transcription factors in breast cancer tumors, and that are involved in lymph node metastasis. For this purpose, we used microRNA microarray data from 50 fresh frozen breast tumors, 28 from patients with lymph node metastasis. Transcription factor expression was assayed by immunohistochemistry. Microarray data analysis using RankProd (R package) revealed approximately 40 microRNAs down-regulated in breast tumors expressing EMT-transcription factors (p<0.05). Using in silico analysis, we predicted 28 microRNAs to be regulators of SNAIL, SLUG and/or TWIST. Four not previously validated microRNAs were selected: miR-202, miR-210, miR-331 and miR-34b. We used luciferase reporter assays to assess the regulation of the 3´UTR of selected transcription factors by each microRNA, at two different concentrations: 10nM and 50nM. We found that miR-210 caused a 50% decrease in luciferase activity through SNAIL and SLUG 3'UTR when tested at 50nM. miR-331, predicted as regulator of SLUG 3'UTR, decreased luciferase activity at both evaluated concentrations in a dose-dependent manner, in a 20% and a 37%. In the case of TWIST, miR-34b at 50nM, decreased luciferase activity in a 65%. No inhibition of luciferase activity was found with mir202. In addition to transcription factors, two of these microRNAs have been predicted as regulators of chemokine receptors CXCR4 and CCR7. Both receptors direct lymphocyte migration to lymphoid organs, and their role has been also implicated in cancer. Our results suggest that down regulation of specific microRNAs, and up regulation of their targets, may induce a metastatic behavior of primary breast tumor cells, promoting invasion and colonization of lymph nodes. In this sense, changes in the expression of microRNAs may serve as biomarkers of breast cancer prognosis in patients. Citation Format: Elisa Pérez-Moreno, Valentina Zavala, Gabriela Valarezo, Wanda Fernández, Pilar Carvallo. microRNAs targeting EMT transcription factors in breast cancer and their relation to lymph node metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2013.

Atrial overexpression of microRNA-27b attenuates angiotensin II-induced atrial fibrosis and fibrillation by targeting ALK5.

Atrial fibrosis influences atrial fibrillation (AF) development by transforming growth factor beta 1 (TGF-β1)/Smad pathway. Although microRNAs are implicated in the pathogenesis of various diseases, information regarding the functional role of microRNAs in atrial dysfunction is limited. In the present study, we found that microRNA-27b (miR-27b) was the dominant member of miR-27 family expressed in left atrium. Moreover, the expression of miR-27b was significantly reduced after angiotensin II (AngII) infusion. Masson's trichrome staining revealed that delivery of miR-27b adeno-associated virus to left atrium led to a decrease in atrial fibrosis induced by AngII. The increased expression of collagen I, collagen III, plasminogen activator inhibitor type 1 and alpha smooth muscle actin was also inhibited after miR-27b upregulation. In isolated perfused hearts, miR-27b restoration markedly attenuated AngII-induced increase in interatrial conduction time, AF incidence and AF duration. Furthermore, our data evidence that miR-27b is a novel miRNA that targets ALK5, a receptor of TGF-β1, through binding to the 3' untranslated region of ALK5 mRNA. Ectopic miR-27b suppressed luciferase activity and expression of ALK5, whereas inhibition of miR-27b increased ALK5 luciferase activity and expression. Additionally, miR-27b inhibited AngII-induced Smad-2/3 phosphorylation without altering Smad-1 activity. Taken together, our study demonstrates that miR-27b ameliorates atrial fibrosis and AF through inactivation of Smad-2/3 pathway by targeting ALK5, suggesting miR-27b may play an anti-fibrotic role in left atrium and function as a novel therapeutic target for the treatment of cardiac dysfunction.

Relevance of the N-terminal and major hydrophobic domains of non-structural protein 3A in the replicative process of a DNA-launched foot-and-mouth disease virus replicon.

A foot-and-mouth disease virus (FMDV) DNA-launched reporter replicon containing a luciferase gene was used to assess the impact of non-structural (NS) protein 3A on viral replication. Independent deletions within the N-terminal region (amino acid [aa] residues 6 to 24) and the central hydrophobic region (HR, aa 59 to 76) of FMDV NS protein 3A were engineered, and luciferase activity in lysates of control and mutated replicon-transfected cells was measured. Triple alanine replacements of the N-terminal triplet Arg 18- His 19 -Glu 20 and a single alanine substitution of the highly charged Glu 20 residue both resulted in a 70-80% reduction in luciferase activity when compared with wild-type controls. Alanine substitution of the 17 aa present in the central HR, on the other hand, resulted in complete inhibition of luciferase activity and in the accumulation of the mutated 3A within the cell nucleus according to immunofluorescence analysis. Our results suggest that both the aa sequence around the putatively exposed hydrophilic E20 residue at the N-terminus of the protein and the hydrophobic tract located between aa 59 and 76 are of major relevance for maintaining the functionality of the 3A protein and preventing its mislocalization into the cell nucleus.

Long non-coding RNA and microRNA-675/let-7a mediates the protective effect of melatonin against early brain injury after subarachnoid hemorrhage via targeting TP53 and neural growth factor

The objective of this study was to identify the protective effect of melatonin (MT) against early brain injury (EBI) following subarachnoid hemorrhage (SAH) and explore the underlying molecular mechanism. Real-time polymerase chain reaction (PCR) and luciferase assay were utilized to detect the effect of MT on H19 expression level, computation analysis and luciferase assay were conducted to the underlying mechanism of let-7a and miR-675. Real-time PCR, western blot analysis, immunohistochemistry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and flow cytometry analysis were performed to detect the effect of MT on H19, miR-675, let-7a, TP53, neural growth factor (NGF) levels, cell viability, and apoptosis status. Melatonin increased H19 expression level by enhancing H19 transcriptional efficiency in a concentration-dependent manner. MiR-675 and let-7a directly targeted P53 and NGF, respectively, and miR-675 reduced luciferase activity of wild-type but not mutant TP53 3′UTR. Meanwhile, let-7a suppressed luciferase activity of wild-type but not mutant NGF 3′UTR. H2O2 increased number of SA-b-gal, and while MT administration repressed the premature senescence. H2O2 obviously upregulated expressions of H19, miR-675, and NGF, and downregulated let-7a and TP53 levels; however, MT treatment reduced expressions of H19, miR-675, and NGF, and improved let-7a and TP53 levels. Treating with MT attenuated the neurological deficits and reduced the brain swelling. MT treatment repressed apoptosis of neurons caused by SAH. Levels of H19, miR-675, and NGF were much higher in the SAH + MT group, while there were even higher levels of H19, miR-675, and NGF in the SAH group than in the sham group; levels of let-7a and TP53 were much lower in the SAH + MT group, while they were even lower in the SAH group than in the sham group. Our study revealed that treatment with MT protected against EBI after SAH by modulating the signaling pathways of H19-miR-675-P53-apoptosis and H19-let-7a-NGF-apoptosis.

Role of miR-148a in Mitigating Hepatic Ischemia-Reperfusion Injury by Repressing the TLR4 Signaling Pathway via Targeting CaMKIIα in Vivo and in Vitro

Background/Aims: Hepatic ischemia-reperfusion (I/R) injury, which is mainly induced by inflammation and unstable intracellular ions, is a major negative consequence of surgery that compromises hepatic function. However, the exact mechanisms of liver I/R injury have not been determined. Positive crosstalk with the Ca²⁺/CaMKII pathway is required for complete activation of the TLR4 pathway and inflammation. We previously found that miR-148a, which decreased in abundance with increasing reperfusion time, targeted and repressed the expression of CaMKIIα. In the present study, we examined the role of the miR-148a machinery in I/R-induced Ca²⁺/CaMKII and TLR4 signaling changes, inflammation, and liver dysfunction in vivo and in vitro. Methods: Liver function was evaluated by serum aminotransferase levels and hematoxylin-eosin (HE) staining. Inflammatory factors were detected by enzyme-linked immunosorbent assay. Gene and protein expression were assessed by RT-PCR and western blot. Small interfering RNA was used to silence target gene expression. HE staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were used to measure hepatic tissue apoptosis. These assays were performed to identify factors upregulated in hepatic I/R injury and downregulated by miR-148a. Results: We manifested that expression of CaMKIIα and phosphorylation of TAK1 and IRF3 were elevated in hypoxia/reoxygenation (H/R)-treated primary Kupffer cells (KCs) and liver tissue of I/R-treated mice, but these effects were attenuated by treatment with miR-148a mimic and were accompanied by the alleviation of liver dysfunction and hepatocellular apoptosis. Luciferase reporter experiments showed that miR148a suppressed luciferase activity by almost 60%. Moreover, knockdown of CaMKIIα in H/R KCs led to significant deficiencies in p-TAK1, P-IRF3, IL-6, and TNF-α, which was consistent with the effects of miR-148a overexpression. Otherwise, the same trend of activation of TAK1 and IRF3 and inflammatory factors in vitro was observed in the siTAK1 + siIRF3 group compared with the siCaMKIIα group. Conclusion: Taken together, we conclude that miR-148a may mitigate hepatic I/R injury by ameliorating TLR4-mediated inflammation via targeting CaMKIIα in vitro and in vivo.

Abstract A046: miR-199a-3p induces BRCA1 dysfunction in triple-negative breast cancer

Abstract Background: BRCA1 mutations are highly susceptible to familial breast and ovarian cancer, as well as the more aggressive triple-negative breast cancer (TNBC). However, DNA repair defects caused by BRCA dysfunction were reported to have better response to platinum-based chemotherapy and PARP inhibitors. Increasing evidence suggest the roles of miRNAs in regulating BRCA1 functions, with emphasis on cancer progression and therapeutic sensitivity. We previously identified miR-199a-3p as a potential TNBC biomarker, and BRCA1 is the putative downstream target based on in silico prediction. In this study, we aim to investigate the functionalities of miR-199a-3p in TNBC. Methods: The correlation between BRCA1 and miR-199a-3p was investigated through expression analysis of 32 TNBC tumor specimens, and further confirmed by dual-luciferase reporter assay. The functionalities of miR-199a-3p in TNBC cells were assessed by MTT cell proliferation, migration, and clonogenic assays. The effects of miR-199a-3p on cisplatin and PARP inhibitor veliparib sensitivities were determined by MTT assays, followed by apoptosis and cell cycle analyses. Results: An inverse correlation between miR-199a-3p and BRCA1 expressions was observed in TNBC tumor specimens. miR-199a-3p was demonstrated to directly target BRCA1, and caused downregulation of BRCA1 protein expression, and suppressed luciferase activity mediated by BRCA1-3’-UTR in reporter assays. Overexpression of miR-199a-3p in TNBC cell lines (MDA-MB-231 and MDA-MB-468) was shown to inhibit cell proliferation, migration, and clonogenic ability, suggesting tumor-suppressive functions. On the other hand, cisplatin treatment induced BRCA1 expression, concurrent with reduced miR-199a-3p, in TNBC cells. Restoration of miR-199a-3p expression sensitized the cells to cisplatin and PARP inhibitor veliparib. Ectopic expression of miR-199a-3p also suppressed aldehyde dehydrogenase (ALDH)-positive stem cell-like population in cisplatin-resistant TNBC cells. Conclusions: Our results suggest that miR-199a-3p is involved in TNBC progression and chemotherapeutic sensitivity, at least partially through regulation of BRCA1. Citation Format: John C. Ho, Vivian Y. Shin, Jiawei Chen, Man T. Siu, Isabella Cheuk, Ava Kwong. miR-199a-3p induces BRCA1 dysfunction in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A046.

Effect of mmu-miR-30b on mouse FoxO3 mRNA expression

Objective To analyze the direct interaction between mmu-miR-30b and mouse FoxO3 (mFoxO3) mRNA. Methods Three target gene fragments, which were respectively 402, 123 and 299 bp in length, were amplified from mouse cDNA by PCR using specific primers and site-direct mutant primers. A complete mutant fragment was obtained by joining together the 123 and 299 bp fragments. Recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed through inserting wild and mutant fragments of mFoxO3 into pmirGLO vector, respectively. HEK 293T cells were respectively co-transfected with the constructed recombinant plasmids and mmu-miR-30b/mmu-miR-30b inhibitor. Dual-luciferase reporter assay system was used to determine the Firefly-Renilla luciferase activity in different groups. Western blot assay was performed to evaluate the regulatory effect of mmu-miR-30b on mFoxO3 expression. Results Restriction enzyme analysis and gene sequencing showed that the two recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed successfully. The activity of Firefly-Renilla luciferase in HEK 293T cells transfected with pmirGLO, pmirGLO+ mmu-miR-30b, pmirGLO+ mmu-miR-30b inhibitor, pmirGLO-mFoxO3+ mmu-miR-30b, pmirGLO-mFoxO3+ mmu-miR-30b+ mmu-miR-30b inhibitor, or pmirGLO-mFoxO3 mt+ mmu-miR-30b was 13.18±0.97, 14.35±0.99, 12.46±1.20, 9.55±1.11, 13.71±0.89 and 10.99±0.92, respectively. Compared with pmirGLO+ mmu-miR-30b group, the luciferase activity was significantly decreased in pmirGLO-mFoxO3+ mmu-miR-30b group (P 0.05). In addition, the suppressed Firefly-Renilla luciferase activity in pmirGLO-mFoxO3+ mmu-miR-30b group was restored by mmu-miR-30b inhibitor treatment (P<0.05). Enhancing the expression of mmu-miR-30b could markedly inhibit the expression of mFoxO3 at protien level (P<0.05), and that could be significantly attenuated by mmu-miR-30b inhibitor treatmeat (P<0.05). Conclusion mFoxO3 mRNA is a novel target gene of mmu-miR-30b. There is a direct interaction between mmu-miR-30b and mFoxO3 mRNA. Key words: mmu-miR-30b; Mouse FoxO3; Target gene

AKT-targeted anti-inflammatory activity of Panax ginseng calyx ethanolic extract

BackgroundKorean ginseng (Panax ginseng) plays an anti-inflammatory role in a variety of inflammatory diseases such as gastritis, hepatitis, and colitis. However, inflammation-regulatory activity of the calyx of the P. ginseng berry has not been thoroughly evaluated. To understand whether the calyx portion of the P. ginseng berry is able to ameliorate inflammatory processes, an ethanolic extract of P. ginseng berry calyx (Pg-C-EE) was prepared, and lipopolysaccharide-activated macrophages and HEK293 cells transfected with inflammation-regulatory proteins were used to test the anti-inflammatory action of Pg-C-EE. MethodsThe ginsenoside contents of Pg-C-EE were analyzed by HPLC. Suppressive activity of Pg-C-EE on NO production, inflammatory gene expression, transcriptional activation, and inflammation signaling events were examined using the Griess assay, reverse transcription-polymerization chain reaction, luciferase activity reporter gene assay, and immunoblotting analysis. ResultsPg-C-EE reduced NO production and diminished mRNA expression of inflammatory genes such as cyclooxygenase-2, inducible NO synthase, and tumor necrosis factor-α in a dose-dependent manner. This extract suppressed luciferase activity induced only by nuclear factor-κB. Interestingly, immunoblotting analysis results demonstrated that Pg-C-EE reduced the activities of protein kinase B (AKT)1 and AKT2. ConclusionThese results suggest that Pg-C-EE may have nuclear-factor-κB-targeted anti-inflammatory properties through suppression of AKT. The calyx of the P. ginseng berry is an underused part of the ginseng plant, and development of calyx-derived extracts may be useful for treatment of inflammatory diseases.

MiR-503 inhibits platelet-derived growth factor-induced human aortic vascular smooth muscle cell proliferation and migration through targeting the insulin receptor.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMC) is a common feature of disease progression in atherosclerosis. Here, we investigated the potential role of miR-503 in platelet-derived growth factor (PDGF)-induced proliferation and migration of human aortic smooth muscle cells and the underlying mechanisms of action. miR-503 expression was significantly downregulated in a dose- and time-dependent manner following PDGF treatment. Introduction of miR-503 mimics into cultured SMCs significantly attenuated cell proliferation and migration induced by PDGF. Bioinformatics analyses revealed that the insulin receptor (INSR) is a target candidate of miR-503. miR-503 suppressed luciferase activity driven by a vector containing the 3'-untranslated region of INSR in a sequence-specific manner. Downregulation of INSR appeared critical for miR-503-mediated inhibitory effects on PDGF-induced cell proliferation and migration in human aortic SMCs. Based on the collective data, we suggest a novel role of miR-503 as a regulator of VSMC proliferation and migration through modulating INSR.

In vitro and in vivo anti-inflammatory activities of Korean Red Ginseng-derived components

BackgroundAlthough Korean Red Ginseng (KRG) has been traditionally used for a long time, its anti-inflammatory role and underlying molecular and cellular mechanisms have been poorly understood. In this study, the anti-inflammatory roles of KRG-derived components, namely, water extract (KRG-WE), saponin fraction (KRG-SF), and nonsaponin fraction (KRG-NSF), were investigated. MethodsTo check saponin levels in the test fractions, KRG-WE, KRG-NSF, and KRG-SF were analyzed using high-performance liquid chromatography. The anti-inflammatory roles and underlying cellular and molecular mechanisms of these components were investigated using a macrophage-like cell line (RAW264.7 cells) and an acute gastritis model in mice. ResultsOf the tested fractions, KGR-SF (but not KRG-NSF and KRG-WE) markedly inhibited the viability of RAW264.7 cells, and splenocytes at more than 500 μg/mL significantly suppressed NO production at 100 μg/mL, diminished mRNA expression of inflammatory genes such as inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α, and interferon-β at 200 μg/mL, and completely blocked phagocytic uptake by RAW264.7 cells. All three fractions suppressed luciferase activity triggered by interferon regulatory factor 3 (IRF3), but not that triggered by activator protein-1 and nuclear factor-kappa B. Phospho-IRF3 and phospho-TBK1 were simultaneously decreased in KRG-SF. Interestingly, all these fractions, when orally administered, clearly ameliorated the symptoms of gastric ulcer in HCl/ethanol-induced gastritis mice. ConclusionThese results suggest that KRG-WE, KRG-NSF, and KRG-SF might have anti-inflammatory properties, mostly because of the suppression of the IRF3 pathway.

Abstract 1619: MicroRNA-466 regulates bone metastasis by targeting RUNX2 in prostate cancer

Abstract MicroRNAs are small, non-coding RNAs that regulate gene expression by targeted repression of transcription and translation. The main objective of this study was to investigate the role of microRNA-466 (miR-466) in prostate carcinogenesis (PC). The expression of miR-466 was significantly suppressed in PC tissue samples and cell lines (PC3, Du145, LNCaP, MDaPCa2b and LAPC4) when compared with normal tissues and a non-malignant cell line (RWPE1). Receiver operating curve analysis showed that miR-466 expression is an independent diagnostic predictor to discriminate between normal and tumor cases. Functionally ectopic expression of miR-466 caused a robust decrease in cell migration, invasion and proliferation of prostate cancer cells. We found that miR-466 exerted these functional effects by directly targeting the oncogenic RUNX2, a key transcription factor in PC. Over-expression of miR-466 in prostate cancer cells suppressed luciferase activity of reporter plasmids containing wild type 3’UTR sequences complementary to RUNX2, which was abolished by 3’UTR mutations. miR-466 reduced RUNX2 regulated downstream molecules such as ANGPT1, ANGPT4, MMP11, Osteopontin (SPP1), and Osteocalcin (OC). MiR-466 also inhibited genes involved in cell proliferation, migration and invasion such as pAKT, FYN and FAK. To determine the biological significance of miR-466 in prostate cancer, we performed parallel experiments in the non-malignant cell line RWPE1 followed by functional assays. Attenuation of miR-466 induced oncogenic characteristics such as increased proliferation, migration and invasion in RWPE1 cells. To examine the in vivo antitumor effects of miR-466, we performed primary and metastatic mouse model experiments. Intra-prostatic implantation of stable cells expressing miR-466 significantly attenuated primary tumor growth. Whereas, bone metastatic dissemination of PC cells was significantly suppressed by intra-cardiac inoculation of stable miR-466 expressing cells compared to control cells in nude mice. We also monitored the overall survival of mice in the metastatic model. Kaplan-Meier survival analysis indicated higher overall survival in the miR-466 group compared to controls. This is the first study demonstrating that: i) miRNA-466 is downregulated, acts as metastasis suppressor and has diagnostic implications in PC; ii) miR-466 has biological significance in PC as its attenuation induced oncogenic characteristics in normal cells; iii) miR-466 directly targeted RUNX2 and inhibited its downstream genes and iv) stable expression of miR-466 inhibited primary tumor growth and bone metastasis in in vivo models. The use of RNAi is currently being implemented as a gene-specific approach for molecular medicine. By the same principle, it is possible that restoration of silenced miR-466 may lead to down-regulation of target oncogenes thereby contributing to novel therapeutic approaches in the treatment of prostate cancer metastasis. Citation Format: Shahana Majid, Hanna Nip, Altaf A. Dar, Sharanjot Saini, Varahram Shahryari, Soichiro Yamamura, Yozo Mitsui, Nathan Bucay, Guoren Deng, Rajvir Dahiya, Yuichiro Tanaka. MicroRNA-466 regulates bone metastasis by targeting RUNX2 in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1619.