HIF2α Upregulates the Migration Factor ODZ1 under Hypoxia in Glioblastoma Stem Cells

Background: Glioblastoma (GBM) remains a major clinical challenge due to its invasive capacity, resistance to treatment, and recurrence. We have previously shown that ODZ1 contributes to glioblastoma invasion and that ODZ1 mRNA levels can be upregulated by epigenetic mechanisms in response to hypoxia. Herein, we have further studied the transcriptional regulation of ODZ1 in GBM stem cells (GSCs) under hypoxic conditions and analyzed whether HIF2α has any role in this regulation. Methods: We performed the experiments in three primary GSC cell lines established from tumor specimens. GSCs were cultured under hypoxia, treated with HIF regulators (DMOG, chetomin), or transfected with specific siRNAs, and the expression levels of ODZ1 and HIF2α were analyzed. In addition, the response of the ODZ1 promoter cloned into a luciferase reporter plasmid to the activation of HIF was also studied. Results: The upregulation of both mRNA and protein levels of HIF2α under hypoxia conditions correlated with the expression of ODZ1 mRNA. Moreover, the knockdown of HIF2α by siRNAs downregulated the expression of ODZ1. We found, in the ODZ1 promoter, a HIF consensus binding site (GCGTG) 1358 bp from the transcription start site (TSS) and a HIF-like site (CCGTG) 826 bp from the TSS. Luciferase assays revealed that the stabilization of HIF by DMOG resulted in the increased activity of the ODZ1 promoter. Conclusions: Our data indicate that the HIF2α-mediated upregulation of ODZ1 helps strengthen the transcriptional control of this migration factor under hypoxia in glioblastoma stem cells. The discovery of this novel transcriptional pathway identifies new targets to develop strategies that may avoid GBM tumor invasion and recurrence.

Qi Ling Inhibits Progression of Androgen-Independent Prostate Cancer via Negative Regulation of TRIM66/HP1γ/AR Axis

<b><i>Aim:</i></b> This study aimed to understand the molecular mechanism underlying the therapeutic effect of Qi Ling (QL) against androgen-independent prostate cancer. <b><i>Methods:</i></b> The relative expression of TRIM66 in prostate tumor was interrogated by microarray. Real-time polymerase chain reaction and Western blotting were performed to determine the transcript abundances and protein expressions of TRIM66, HP1γ, AR, c-Myc, and GAPDH. Cell proliferation and apoptosis were analyzed by cell counting kit-8 method and flow cytometry. The regulatory action of c-Myc on TRIM66 was interrogated with luciferase reporter plasmid and the direct binding was demonstrated by chromatin immunoprecipitation. The secretory prostate-specific antigen was quantified by enzyme-linked immunosorbent assay. <b><i>Results:</i></b> TRIM66 was aberrantly overexpressed in prostate cancer and associated with unfavorable prognosis. TRIM66/HP1γ/AR was upregulated during the androgen-independent transition in hormone-deprived medium. The TRIM66 level positively linked to cell proliferation and negatively linked to cell apoptosis in androgen-independent prostate cancer cells. QL treatment specifically inhibited c-Myc and therefore directly downregulated TRIM66 via binding to its promoter. Ectopic introduction of TRIM66 significantly reversed the anti-tumor effects of QL against androgen-independent prostate cancer. <b><i>Conclusion:</i></b> Our study uncovered the importance of downregulated TRIM66/HP1γ/AR signaling in mediating the anti-tumor properties of QL.

Targeting MYC and HDAC8 with a Combination of siRNAs Inhibits Neuroblastoma Cells Proliferation In Vitro and In Vivo Xenograft Tumor Growth

HDAC8, c MYC and MYCN are involved in the tumorigenesis of neuroblastoma. A mouse Neuroblastoma (NB) tumor model was used to understand the role of miRNA, miR-665 in NB tumorigenesis and cellular differentiation. During cellular differentiation of NB cells there is an up regulated miRNA-665. We found that HDAC 8, c MYC and MYCN are the direct targets of mimic miR-665 which was validated by luciferase reporter plasmid with 3’ UTR and ELISA. Mimic miR-665 inhibited cell proliferation, arrested cells in G1 stage and decreased S Phase in cell cycle. miR-665 increased the acetylation of histones and activated Caspase 3. This is the first report to recognize miRNA 665 as a suppressor miRNA of NB. The effects of miR-665 were confirmed with the transfection of siRNA for HDAC8 and siRNA for MYC. Individual siRNA- HDAC8 or siRNA-MYC inhibited 40–50% of cell proliferation in vitro, however, the treatment with the combination of both siRNA-MYC + siRNA- HDAC8 inhibited 86% of cell proliferation. Indicating that both the targets c MYC and HDAC 8 should be reduced to obtain a significant inhibition of cell proliferation. Intratumoral treatment of xenograft tumors in mice with the combination of siRNA-MYC + siRNA- HDAC8 reduced the levels of target c-MYC protein by 64% and target HDAC 8 protein by 85% and the average tumor growth reduced by 80% compared to control tumors treated with NC-siRNA. Our results suggest the potential therapeutic effect of suppressor miR-665 and the combination of siRNA-MYC + siRNA-HDAC8 for neuroblastoma treatment.

Potentials and pitfalls of transient in vitro reporter bioassays: interference by vector geometry and cytotoxicity in recombinant zebrafish cell lines

The water framework directive re-evaluation proposes the integration of effect-based tools, increasing the need for alternative methods. Especially within aquatic toxicology, coverage of specific toxicity pathways is scarce, and most applications are based on mammalian or bacterial models, not reflecting realistic exposure scenarios. The use of transient reporter gene assays in cells from organisms of interest could be a quick and inexpensive solution. However, interference with cellular homeostasis may impact the system beyond the function of the manipulated gene and thus lead to non-specific results. We describe how varying vector geometry and different regulatory gene elements on plasmids used for transfection in zebrafish hepatocytes and embryonic fibroblasts may lead up to a tenfold difference in potency. Cells were transiently co-transfected with an Nrf2-responsive Firefly luciferase reporter plasmid and eight different Renilla luciferase normalization plasmids. Transfected cells were exposed to two different regimes (0.1–100 µM and 7.8–250 µM) of the oxidative stress-inducing compounds, sulforaphane, tertbutylhydroquinone, and metazachlor. Nrf2 activity was measured in dual-luciferase assays. In parallel, cytotoxicity was assessed for different endpoints (energy metabolism, protein amount, membrane stability, and cell proliferation) in non-transfected cells and cells co-transfected with constructs of increasing size, to be used for normalization. Transfected cells were more susceptible to cytotoxicity in a vector size-dependent manner. Conclusively, we report that vector geometries (size, backbones, gene-regulatory units), cell line (tissue origin), applied transfection methods, and signal normalization may alter the sensitivity of reporter bioassays in a synergistic manner. Further, we propose that thorough bioassay design is needed to ensure reliability and regulatory acceptance.

miR-182 facilitates invasion and EMT by targeting Numb in Malignant Pleural Mesothelioma

Background Malignant Pleural Mesothelioma (MPM) is a highly aggressive tumor which need effective therapeutic methods to improve the prognosis. We carried out this study to explore the role of miR-182 in MPM development, its correlation with Numb expression and EMT. Methods First, we investigated the level of miR-182 and Numb-mRNA by qRT-PCR. Furthermore, we introduced the putative miR-182 binding site into a luciferase reporter plasmid to illustrate the impact of miR-182 on Numb. Then, we down-regulated the expression of miR-182 with/without Numb knocked down in NCI-H2452 cells to investigated their effect. Data were presented as mean ± SD of three independent experiments. Student’s test, correlation analysis and analysis of variance (ANOVA) were used. Results Our results revealed that miR-182 has a high level of expression in MPM, it has a negative correlation with Numb and targeted Numb in MPM cells. miR-182 facilitated the invasion of MPM cells while down-regulation of miR-182 restrained the progression of EMT and made MPM cells more susceptible to pemetrexed. Conclusions miR-182 and Numb can serve as potential therapeutic targets for MPM.

Identification of Serum miRNA-423-5p Expression Signature in Somatotroph Adenomas

Circulating miRNAs are novel disease biomarkers that are valuable for diagnosis and prognosis. But the circulating miRNAs profile in somatotroph adenomas is still unknown. Therefore, serum exosomal miRNAs expression profiling in somatotroph adenomas was performed on 6 somatotroph adenomas and 6 normal controls. From the exosomal miRNAs expression profiling, we found 169 miRNAs differently expressed between somatotroph adenomas and healthy pituitary samples (p< 0.05, FC > 2). Among the 169 miRNAs, miR-423-5p was expressed lower in somatotroph adenomas than in healthy pituitary samples, which was proved by miRSCan Panel Chip™ qPCR. PTTG1 and SYT1 were the target mRNAs of miR-423-5p, and transcriptomics and proteomics profile both indicated the high expression of PTTG1 and SYT1 in somatotroph adenomas. H-scores were 223.1 ± 34.7 for PTTG1 and 163.4 ± 42.3 for SYT1 in 62 somatotroph adenomas specimens and 84.2 ± 21.3 for PTTG1 and 47.4 ± 17.2 for SYT1 in 6 healthy pituitary specimens by IHC. miR-423-5p inhibited the expression of SYT1 and PTTG1 at the mRNA and protein levels. Dual luciferase reporter gene assay shown was significantly reduced in the presence of miR-423-5p in GH3 cells transfected with wild-type PTTG1 3'UTR luciferase reporter plasmid but not reduced when transfected with the mutation PTTG1 3'UTR luciferase reporter plasmid (p<0.01). In vitro experiments showed that miR-423-5p induced cell apoptosis, inhibited cell proliferation, and reduced growth hormone release and migration of GH3 cells. The activity of miR-423-5p in GH3 cell was nearly blocked by its inhibitor. These results verified the central role of low miR-423-5p in promoting tumorigenesis in somatotroph adenomas. PTTG1 may act as biomarkers for clinical treatment of somatotroph adenomas.

MKL1 and STAT3 activate the activity of the luciferase reporter plasmid containing the CAAP1 gene promoter

Breast cancer is the leading cause of cancer death in women worldwide. The etiology of the disease is not yet clear. We know that MKL1 and STAT3 play an important part in the development and progression of breast cancer. CAAP1 is a ubiquitous and highly conserved protein that is closely related to the apoptotic process of tumors. However, the definitive transcriptional mechanism of the CAAP1 gene is still unclear. In our study, we constructed a luciferase reporter plasmid for the human CAAP1 gene promoter. Then one or both of the two overexpression vectors of MKL-1 and STAT3 were co-transfected into MCF-7 cells with CAAP1 promoter plasmid, and we then tested activation of the CAAP1 promoter by luciferase reporter assay. The results show that compared with the transfected pcDNA3.1 group, MKL1 can evidently increase the transcription activity of the CAAP1 gene promoter, while the STAT3 group can slightly upregulate the transcription activity of the CAAP1 gene promoter. Our research will further reveal the relationship between CAAP1 and the occurrence and development of breast cancer cells, and provide a new idea and direction for the cures of breast cancer.

Cyclosporine-A induces apoptosis in human prostate cancer cells PC3 and DU145 via downregulation of COX-2 and upregulation of TGFβ

Background Potential targets for prostate cancer therapy are urgently needed for curative of patients. Cyclosporine-A (CsA), an immunosuppressive and a selective cyclooxygenase-2 (COX-2) inhibitor, exerts antitumor activity. However, the molecular effects of CsA is not fully understood in prostate cancer. In this research, we sought to determine role and mechanism of CsA in prostate cancer. Materials and methods PC3 and DU145 cells were treated with CsA time (12, 24, 48 h) and dose dependent (2.5, 10, 25 μM) and cell survival, migration, colony formation, expression of apoptosis related proteins/genes using MTT assay, scratch assay, Western blotting/qPCR. At the same time, cells treated with CsA to test on the effects of COX-2 promoter activity using luciferase reporter plasmid. Lastly, functional role in the CsA treatment prostate cancer cells were interrogated for relationship of TGFβ, Akt, caspases and COX-2. Results These study findings provided direct evidences that the CsA induced apoptosis and downregulated migration. Conclusions CsA downregulated Akt as well as COX-2 and upregulated TGFβ, resulting in the suppression of cell migration which was augmented a potential therapeutic of CsA in prostate cancer cells.