Luciferase Activity Of Wild-type Research Articles

Curcumin inhibits expression of brain derived neurotrophic factor (BDNF) in hippocampus of mice with sleep deprivation

Purpose: To determine the inhibitory effect of curcumin on the expression of brain-derived neurotrophic factor (BDNF) in hippocampus of mice with sleep deprivation.
 Methods: Expressions of BDNF and miRNA-206 were evaluated using quantitative real time-polymerase chain reaction (qRT-PCR) in a sleep-deprived mouse model. Interaction of BDNF and miRNA-206 was assessed by dual luciferase and qPCR) assays. The effect of curcumin on BDNF and miRNA-206 was also determined by qRT-PCR.
 Results: Expressions of BDNF and miRNA-206 were significantly suppressed in hippocampus of mice in sleep deprivation group compared with control mice (p < 0.05). Treatment with miRNA-206 significantly suppressed the luciferase activity of wild type (WT) BDNF reporter gene vector (p < 0.05) rather than the mutated (mut) vectors. Inhibition of miRNA-206 upregulated RNA and protein levels of BDNF (p < 0.05), while curcumin treatment inhibited BDNF expression (p < 0.01) and upregulated miRNA-206 level (p < 0.01).
 Conclusion: Curcumin inhibits BDNF expression by upregulating miRNA-206 level in hippocampus tissues of mice in the sleep deprivation model. The relationship between BDNF and miRNA-206 might provide a new direction for the development of new therapeutics for sleep deprivation.

The minor allele of rs17427875 in long non-coding RNA-HOXA11-AS influences the prognosis of subarachnoid hemorrhage (SAH) via modulating miR-15a and STAT3 expression

Background: HOAX11-AS was reported to promote the progression of liver cancer via the signaling pathway of miR-15a-3p/STAT3. In this study, we investigated the effect of rs17427875 on the prognosis of subarachnoid hemorrhage (SAH) and its underlying molecular mechanisms.Methods: 158 SAH patients were recruited and grouped according to their genotypes rs17427875. Peripheral blood and cerebrospinal fluid (CSF) samples were collected for subsequent analysis. Quantitative real-time PCR, luciferase assays, Western blot and ELISA were performed to analyze the correlations between the expression of lncRNA-HOXA11-AS, miR-15a, TNF-α and NF-κB.Results: The survival rate was remarkably higher in SAH patients carrying the AA genotype of rs17427875 when compared with those carrying the AT genotype. The expression of miR-15a was significantly repressed in the peripheral blood and CSF of SAH patients carrying the AT allele when compared with that in patients carrying the AA allele. MiR-15a showed a remarkable efficacy in inhibiting the luciferase activity of wild type lncRNA-HOXA11-AS and STAT3 in THP-1 cells. P-HOXA11-AS-T showed a stronger ability to suppress the expression of miR-15a and activate the expression of STAT3, TNF-α and NF-κB in THP-1 cells when compared with P-HOXA11-AS-A.Conclusions: The findings demonstrated that the presence of the minor allele of rs17427875 in lncRNA-HOXA11-AS could increase the expression level of lncRNA-HOXA11-AS, thus elevating the expression level of STAT3 via down-regulating miR-15a, and increased STAT3 expression could aggravate inflammation to cause poor prognosis of SAH. Therefore, the rs17427875 polymorphism can be used as a potential biomarker for the prognosis of SAH.

Magnesium isoglycyrrhizinate suppresses bladder cancer progression by modulating the miR-26b/Nox4 axis

ABSTRACT Magnesium isoglycyrrhizinate (MI), a magnesium salt of 18α-GA stereoisomer, has been reported to exert efficient hepatoprotective activity. However, its effect on bladder cancer remains unclear. The study explored the effects of MI on the growth, colony formation, apoptosis, invasion, and migration of bladder cancer cells (HTB9 and BIU87 cells). Typical apoptotic changes of bladder cancer cells such as nuclear concentration and fragmentation were observed using Hoechst staining. The effects of MI on the expression levels of microRNA-26b (miR-26b), NADPH oxidase 4 (Nox4), nuclear transcription factor-κB (NF-κB), and hHypoxia inducible factor-1α (HIF-1α) were detected using qRT-PCR and Western blot. The potential targets of miR-26b were predicted using Targetscan, and their interactions were determined by luciferase reporter assay. A xenograft mouse model was established to evaluate the anti-tumor effects of MI in vivo. MI significantly suppressed the proliferation, colony formation, invasion, and migration and induced apoptosis of human bladder cancer cells, and MI significantly increased miR-26b expression. Nox 4 was identified to be a direct target of miR-26b. MiR-26b mimics significantly decreased the relative luciferase activity of wild type (WT) Nox 4 but not mutant type (MUT) Nox4. Meanwhile, MI markedly downregulated the expression levels of Nox4, NF-κB, and HIF-1α both in vitro and in vivo. Moreover, MI inhibited xenograft tumor growth in vivo and decreased the expression of Nox4, NF-κB, and HIF-1α. Overall, MI showed a potent anti-tumor effect against bladder cancer partially via modulating the miR-26b/Nox4 axis.

Sa1983 Long Non-Coding RNA MALAT-1 Promote Invasion and Migration of Gastric Cancer

G A A b st ra ct s by qPCR and western blot. Results. 5 miRNAs were upregulated by 4-fold or more in Snailknockdown cells. Among them, miR-133b and -203 were involved in human biological process (GO 0008150) and molecular function (GO 000674). However, the mechanism of miR-133b in CRC ismuch less known. Increased expression ofmiR-133b in Snail-knockdown cells was confirmed by qPCR. Transactivity of pluc790 was significantly decreased by Snail knockdown. MiR-133b was dramatically decreased in CRC vs normal tissues (N=55). MiR133b overexpression led to reduction of CRC growth and metastasis in vivo as well as cell proliferation, invasion and migration in vitro, but no apoptosis. 59 targets of miR-133b were predicted and 4 were present in GO 0000988 (binding transcription factor activity, GO Level 3). MiR-133b negatively regulates MAML1 expression. After cotransfection of miR133b mimics, pmiR-MAML1-wt and -Mut into HEK293 cells, luciferase activity of wild type, but no mutant, was significantly decreased with miR-133b overexpression compared with control group, confirming the specificity of the interaction between miR-133b and MAML13'UTR mRNA. Similar to Snail, MAML1 expression was significantly increased in CRC. Positive correlation between MAML1 and Snail were found in both clinical specimens and cells.Conclusion.Our findings reveal a newmechanism of Snail-inducing tumor progression via miR-133b. We also describe the tumor-suppressing role of miR-133b in CRC, thus providing a preclinical rationale to explore miR-133b as an effective therapeutic strategy to eradicate CRC.

Bone Marrow Niche Down-Regulates Mir-30 In Multiple Myeloma Cells to Promote Cancer Progression and Cancer Initiation by Targeting BCL9/Wnt Pathway.

AbstractAbstract 1569“Niche” is a specialized microenvironment which controls the fate specification and development of stem and progenitor cells. The bone marrow (BM) niche is composed of osteoblasts, osteoclasts, bone marrow endothelial cells, stromal cells, adipocytes, and extracellular matrix proteins (ECM). These elements provide an optimal growth environment for multiple hematological malignancies, including multiple myeloma (MM) cancer stem cells (CSCs). For example, the MM bone marrow stromal cells (BMSCs) confer survival and chemoresistance of MM cells to current therapies. A better and more detailed understanding of the neoplastic MM niche will therefore provide a model for identifying and validating novel targeted therapies directed against MM. Our previous data in 78 MM patient samples showed that miR-30 family members were more weakly expressed in patients samples compared with normal plasma cells. In our present study, we showed that miR-30 is downregulated by co-culture of either MM cell line or MM patients sample with BMSCs. Bioinformatics analysis showed that Bcl9 is a common target of miR-30, with two different binding sites in 3'UTR region of Bcl9 mRNA predicted by 3 different web-based softwares. Importantly, we confirmed Bcl9 as a direct target of miR-30 in MM cells by both gain of function and loss of function studies. Specifically, ectopic expression of miR-30 by using HIV based lentivirus (V-miR-30) downregulates Bcl9 gene expression by mRNA degradation in either 293T or H929 cells compared with GFP control cells (V-GFP). Conversely, knockdown of miR-30 family expression upregulates Bcl9 mRNA and protein level in MMS1 cells, which have relatively higher miR-30 and lower Bcl9 levels. To prove a direct interaction between miR-30 and the 3'UTR of Bcl9, two wild type Bcl9-3'UTR reporter vectors (pmiR-Bcl9-30-1W and pmiR-Bcl9-30-2W) and two mutant Bcl9-3'UTR reporter vectors (pmiR-Bcl9-30-1M and pmiR-Bcl9-30-2M) were cotransfected into H929 cells, together with V-miR-30 or V-GFP. Luciferase activity of wild type, but not mutant, was significantly decreased with V-miR-30 with respect to V-GFP. Downregulation of miR-30 induced overexpression of Bcl9 in MM cell line or patients samples, which in turn transcriptionally activates WNT pathway downstream target genes such as Axin2 and CD44. As expected, WNT pathway TOP/FOP luciferase activity was induced by knockdown miR-30 in MMS1 cells and suppressed by ectopic expression of miR-30 in H929 cells. In addition, the stem cell population of H929 V-miR-30 dramatically decreased, identified using functional Hoechst 33342 stem cell staining assay. Moreover, in experiments using stem cell medium to culture sorted CSCs, the sphere number and size is less in H929 V-miR-30c CSCs compared with H929 V-GFP control CSCs. In addition, proliferation and tumor formation were inhibited in vitro and in vivo in H929 V-miR-30 stable cells compared with H929 V-GFP cells. Finally, H929 V-miR-30 stable cells were more sensitive to bortezomib treatment than GFP control cells in the presence or absence of BMSCs. Our studies therefore demonstrate that miR-30 regulates the WNT pathway by targeting Bcl9 in MM cells in the BM milieu and represents a promising novel therapeutic target in MM. Disclosures:Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.