Regulation Of Survivin Expression Research Articles

Opposing Interplay between Nuclear Factor Erythroid 2-Related Factor 2 and Forkhead BoxO 1/3 is Responsible for Sepantronium Bromide's Poor Efficacy and Resistance in Cancer cells: Opportunity for Combination Therapy in Triple Negative Breast Cancer.

Survivin, a cancer-cell-specific multifunctional protein, is regulated by many oncogenic signaling pathways and an effective therapeutic target. Although, several types of survivin-targeting agents have been developed over the past few decades, none of them received clinical approval. This could be because survivin expression is tightly controlled by the feedback interaction between different signaling molecules. Of the several signaling pathways that are known to regulate survivin expression, the phosphatidylinositol 3-kinase/AKT serine-threonine kinase/forkhead boxO (PI3K/AKT/FoxO) pathway is well-known for feedback loops constructed by cross-talk among different molecules. Using sepantronium bromide (YM155), the first of its class of survivin-suppressant, we uncovered the existence of an interesting cross-talk between Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) and FoxO transcription factors that also contributes to YM155 resistance in triple negative breast cancer (TNBC) cells. Pharmacological manipulation to interrupt this interaction not only helped restore/enhance the drug-sensitivity but also prompted effective immune clearance of cancer cells. Because the YM155-induced reactive oxygen species (ROS) initiates this feedback, we believe that it will be occurring for many ROS-producing chemotherapeutic agents. Our work provides a rational explanation for the poor efficacy of YM155 compared to standard chemotherapy in clinical trials. Finally, the triple drug combination approach used herein might help reintroducing YM155 into the clinical pipeline, and given the high survivin expression in TNBC cells in general, it could be effective in treating this subtype of breast cancer.

Targeting Cyclin-Dependent Kinase 1 Induces Apoptosis and Cell Cycle Arrest of Activated Hepatic Stellate Cells.

Liver fibrosis is the integral process of chronic liver diseases caused by multiple etiologies and characterized by excessive deposition of extracellular matrix (ECM). During liver fibrosis, hepatic stellate cells (HSCs) transform into a highly proliferative, activated state, producing various cytokines, chemokines, and ECM. However, the precise mechanisms that license HSCs into the highly proliferative state remain unclear. Cyclin-dependent kinase 1 (CDK1) is a requisite event for the transition of the G1/S and G2/M phases in eukaryotic cells. In this study, it is demonstrated that CDK1 and its activating partners, Cyclin A2 and Cyclin B1, are upregulated in both liver fibrosis/cirrhosis patient specimens and the murine hepatic fibrosis models, especially in activated HSCs. In vitro, CDK1 is upregulated in spontaneously activated HSCs, and inhibiting CDK1 with specific small-molecule inhibitors (CGP74514A, RO-3306, or Purvalanol A) orshorthairpinRNAs(shRNAs) resulted in HSC apoptosis and cell cycle arrest by regulating Survivin expression. Above all, it is illustrated that increased CDK1 expression licenses the HSCs into a highly proliferative state and can serve as a potential therapeutic target in liver fibrosis.

Claudin-2 protects against colitis-associated cancer by promoting colitis-associated mucosal healing.

Patients with inflammatory bowel disease (IBD) are susceptible to colitis-associated cancer (CAC). Chronic inflammation promotes the risk for CAC. In contrast, mucosal healing predicts improved prognosis in IBD and reduced risk of CAC. However, the molecular integration among colitis, mucosal healing, and CAC remains poorly understood. Claudin-2 (CLDN2) expression is upregulated in IBD; however, its role in CAC is not known. The current study was undertaken to examine the role for CLDN2 in CAC. The AOM/DSS-induced CAC model was used with WT and CLDN2-modified mice. High-throughput expression analyses, murine models of colitis/recovery, chronic colitis, ex vivo crypt culture, and pharmacological manipulations were employed in order to increase our mechanistic understanding. The Cldn2KO mice showed significant inhibition of CAC despite severe colitis compared with WT littermates. Cldn2 loss also resulted in impaired recovery from colitis and increased injury when mice were subjected to intestinal injury by other methods. Mechanistic studies demonstrated a possibly novel role of CLDN2 in promotion of mucosal healing downstream of EGFR signaling and by regulation of Survivin expression. An upregulated CLDN2 expression protected from CAC and associated positively with crypt regeneration and Survivin expression in patients with IBD. We demonstrate a potentially novel role of CLDN2 in promotion of mucosal healing in patients with IBD and thus regulation of vulnerability to colitis severity and CAC, which can be exploited for improved clinical management.

Survivin as a mediator of stiffness-induced cell cycle progression and proliferation of vascular smooth muscle cells.

Stiffened arteries are a pathology of atherosclerosis, hypertension, and coronary artery disease and a key risk factor for cardiovascular disease events. The increased stiffness of arteries triggers a phenotypic switch, hypermigration, and hyperproliferation of vascular smooth muscle cells (VSMCs), leading to neointimal hyperplasia and accelerated neointima formation. However, the mechanism underlying this trigger remains unknown. Our analyses of whole-transcriptome microarray data from mouse VSMCs cultured on stiff hydrogels simulating arterial pathology identified 623 genes that were significantly and differentially expressed (360 upregulated and 263 downregulated) relative to expression in VSMCs cultured on soft hydrogels. Functional enrichment and gene network analyses revealed that these stiffness-sensitive genes are linked to cell cycle progression and proliferation. Importantly, we found that survivin, an inhibitor of apoptosis protein, mediates stiffness-dependent cell cycle progression and proliferation as determined by gene network and pathway analyses, RT-qPCR, immunoblotting, and cell proliferation assays. Furthermore, we found that inhibition of cell cycle progression did not reduce survivin expression, suggesting that survivin functions as an upstream regulator of cell cycle progression and proliferation in response to ECM stiffness. Mechanistically, we found that the stiffness signal is mechanotransduced via the FAK-E2F1 signaling axis to regulate survivin expression, establishing a regulatory pathway for how the stiffness of the cellular microenvironment affects VSMC behaviors. Overall, our findings indicate that survivin is necessary for VSMC cycling and proliferation and plays a role in regulating stiffness-responsive phenotypes.

Dihydromyricetin suppresses tumor growth via downregulation of the EGFR/Akt/survivin signaling pathway.

Deregulation of epidermal growth factor receptor (EGFR) signaling is frequently observed in non-small cell lung cancer (NSCLC). The present study aimed to determine the impact of dihydromyricetin (DHM) on NSCLC, a natural compound extracted from Ampelopsis grossedentata with various pharmacological activities. Results of the present study demonstrated that DHM may act as a promising antitumor agent for NSCLC therapy, inhibiting the growth of cancer cells in vitro and in vivo. Mechanistically, results of the present study demonstrated that exposure to DHM downregulated the activity of wild-type (WT) and mutant EGFRs (mutations, exon 19 deletion, and L858R/T790M mutation). Moreover, western blot analysis indicated that DHM induced cell apoptosis via suppression of the antiapoptotic protein, survivin. Results of the present study further demonstrated that depletion or activation of EGFR/Akt signaling may regulate survivin expression though modulating ubiquitination. Collectively, these results suggested that DHM may act as a potential EGFR inhibitor, and may provide a novel choice of treatment strategy for patients with NSCLC.

Dual role of deubiquitinating enzyme USP19 regulates mitotic progression and tumorigenesis by stabilizing survivin

Survivin is a component of the chromosomal passenger complex, which includes Aurora B, INCENP, and Borealin, and is required for chromosome segregation and cytokinesis. We performed a genome-wide screen of deubiquitinating enzymes for survivin. For the first time, we report that USP19 has a dual role in the modulation of mitosis and tumorigenesis by regulating survivin expression. Our results found that USP19 stabilizes and interacts with survivin in HCT116 cells. USP19 deubiquitinates survivin protein and extends its half-life. We also found that USP19 functions as a mitotic regulator by controlling the downstream signaling of survivin protein. Targeted genome knockout verified that USP19 depletion leads to several mitotic defects, including cytokinesis failure. In addition, USP19 depletion results in significant enrichment of apoptosis and reduces the growth of tumors in the mouse xenograft. We envision that simultaneous targeting of USP19 and survivin in oncologic drug development would increase therapeutic value and minimize redundancy.

Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Protect Cardiomyocytes from Doxorubicin-Induced Cardiomyopathy by Upregulating Survivin Expression via the miR-199a-3p-Akt-Sp1/p53 Signaling Pathway.

Cardiotoxicity is associated with the long-term clinical application of doxorubicin (DOX) in cancer patients. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) including exosomes have been suggested for the treatment of various diseases, including ischemic diseases. However, the effects and functional mechanism of MSC-sEVs in DOX-induced cardiomyopathy have not been clarified. Here, MSC-sEVs were isolated from murine embryonic mesenchymal progenitor cell (C3H/10T1/2) culture media, using ultrafiltration. H9c2 cardiac myoblast cells were pretreated with MSC-sEVs and then exposed to DOX. For in vivo studies, male C57BL/6 mice were administered MSC-sEVs intravenously, prior to a single dose of DOX (15 mg/kg, intraperitoneal). The mice were sacrificed 14 days after DOX treatment. The results showed that MSC-sEVs protected cardiomyocytes from DOX-induced cell death. H9c2 cells treated with DOX showed downregulation of both phosphorylated Akt and survivin, whereas the treatment of MSC-sEVs recovered expression, indicating their anti-apoptotic effects. Three microRNAs (miRNAs) (miR 199a-3p, miR 424-5p, and miR 21-5p) in MSC-sEVs regulated the Akt-Sp1/p53 signaling pathway in cardiomyocytes. Among them, miR 199a-3p was involved in regulating survivin expression, which correlated with the anti-apoptotic effects of MSC-sEVs. In in vivo studies, the echocardiographic results showed that the group treated with MSC-sEVs recovered from DOX-induced cardiomyopathy, showing improvement of both the left ventricle fraction and ejection fraction. MSC-sEVs treatment also increased both survivin and B-cell lymphoma 2 expression in heart tissue compared to the DOX group. Our results demonstrate that MSC-sEVs have protective effects against DOX-induced cardiomyopathy by upregulating survivin expression, which is mediated by the regulation of Akt activation by miRNAs in MSC-sEVs. Thus, MSC-sEVs may be a novel therapy for the prevention of DOX-induced cardiomyopathy.

Abstract 4687: Oncogenic role of the transcription factor YY1 and its target Survivin in non-Hodgkin's lymphoma

Abstract Objective Yin Yang 1 (YY1) is a transcription factor with a dual role in cancer genesis. Hematological malignancies, including non-Hodgkin lymphomas (NHL), are often characterized by a frequent development of resistance to therapy and a high rate of tumor recurrence. Our previous observations showed that YY1 is overexpressed in hematological tumors. The aim of this study was to investigate the functional role of YY1 in anti-cancer therapy induced cell death and to identify YY1 downstream resistant factors. Experimental Designs The NHL Raji cell line was used as a cellular model. The expression of YY1 was assessed by q-PCR and WB. YY1 knock-down (KD) was achieved using retroviral shRNA. Viability was measured by both the MTT assay and the Trypan Blue live/dead cell count. Cell death was induced following Vincristine and Doxorubicin treatments. The following analyses were used in this study: the JASPAR database, the Encyclopedia of DNA Elements (ENCODE) and the Cancer Genome Atlas (TCGA) datasets relative to NHL patients have been analyzed to assess the expression levels of YY1 and apoptotic genes, according to tumor stages and patients' clinical-pathological features. Results Silencing of YY1 in Raji cells resulted in increased sensitivity to both Vincristine and Doxorubicin treatments and augmented apoptosis. Jaspar analysis identified several potential YY1 transcriptional targets involved in cellular death regulation. As a result of q-PCR screening and validation, Survivin has been identified as a potential YY1 target, as it was significantly downregulated following YY1 KD. Bioinformatic analysis of deposited Chip-Seq databases in ENCODE revealed that YY1 is able to target Survivin transcriptional regulatory regions in different cancer cell lines, as well as in normal B lymphocytes. The TCGA-deposited datasets identified specific clusters of YY1 expression significantly correlated with Survivin. Conclusions Our findings demonstrated that (1) YY1 silencing resulted in the sensitization of Raji tumor cells to drug-induced apoptosis (2) q-PCR-mediated screening of putative YY1 targets led to the identification of Survivin as a potential YY1 target, as when YY1 was KD, Survivin levels were strongly downregulated (3) YY1 might be positively regulating Survivin expression (4) Survivin could be a potential target of YY1 in NHL and (5) YY1 and Survivin in NHL may represent two novel diagnostic and prognostic biomarkers to assess patients' response to chemotherapy. Citation Format: Silvia Vivarelli, Luca Falzone, Saverio Candido, Benjamin Bonavida, Massimo Libra. Oncogenic role of the transcription factor YY1 and its target Survivin in non-Hodgkin's lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4687.

Pathological Mechanistic Studies of Osimertinib Resistance in Non-Small-Cell Lung Cancer Cells Using an Integrative Metabolomics-Proteomics Analysis

Background Osimertinib is the first-line therapeutic option for the T790M-mutant non-small-cell lung cancer and the acquired resistance obstructs its application. It is an urgent challenge to identify the potential mechanisms of osimertinib resistance for uncovering some novel therapeutic approaches. Methods In the current study, the cell metabolomics based on ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry and the qualitative and tandem mass tags quantitative proteomics were performed. Results 54 differential metabolites and 195 differentially expressed proteins were, respectively, identified. The amino acids metabolisms were significantly altered. HIF-1 signaling pathway modulating P-glycoproteins expression, PI3K-Akt pathway regulating survivin expression, and oxidative phosphorylation were upregulated, while arginine and proline metabolism regulating NO production and glycolysis/gluconeogenesis were downregulated during osimertinib resistance. Conclusion The regulation of HIF-1 and PI3K-Akt signaling pathway, energy supply process, and amino acids metabolism are the promising therapeutic tactics for osimertinib resistance.

MicroRNA-140-5p inhibits salivary adenoid cystic carcinoma progression and metastasis via targeting survivin

BackgroundSalivary adenoid cystic carcinoma (SACC) is one of the most frequent carcinomas derived from the salivary gland. Growing evidence implied the involvement of microRNAs (miRNAs) in SACC progression and metastasis. This study aimed to determine the regulatory role of miR-140-5p in SACC progression and metastasis and to explore the underlying mechanisms.Materials and methodsMiR-140-5p and survivin mRNA expression levels were determined by quantitative real-time PCR; protein levels were evaluated by western blot assay; cell proliferation, growth, invasion, apoptosis and caspase-3 activity were evaluated by respective in vitro functional assays; xenograft nude mice model was used to assess the in vivo tumor growth; a luciferase reporter assay determined the interaction between miR-140-5p and survivin.ResultsMiR-140-5p overexpression suppressed SACC cell proliferation and invasion, induced cell apoptosis and inhibited in vivo tumor growth of SACC cells. The loss-of-function studies showed that miR-140-5p knockdown enhanced SACC cell proliferation and invasion, inhibited cell apoptosis and led to an accelerated in vivo tumor growth. The bioinformatics prediction and luciferase reporter assay revealed that miR-140-5p directly targeted survivin 3′ untranslated region, and survivin was inversely regulated by miR-140-5p. Knockdown of survivin exerted tumor-suppressive effects on SACC cells, while enforced expression of survivin counteracted the tumor-suppressive actions of miR-140-5p overexpression in SACC cells. Mechanistically, miR-140-5p modulated the protein expression levels of apoptosis- and epithelial-mesenchymal transition-related mediators as well as matrix metallopeptidase-2/-9 via targeting survivin. More importantly, the down-regulation of miR-140-5p and the up-regulation of survivin were detected in the SACC clinical tissues, and miR-140-5 expression was inversely correlated with survivin mRNA expression level in SACC tissues.ConclusionOur data indicated that miR-140-5p suppressed SACC cell proliferation and invasion, induced cell apoptosis via regulating survivin expression. The present study provide evidence that that miR-140-5p could be a promising target for treating SACC, which requires further investigations.

LncRNA FENDRR represses proliferation, migration and invasion through suppression of survivin in cholangiocarcinoma cells

ABSTRACTThis study was to investigate the biological function and underlying mechanisms of FENDRR in cholangiocarcinoma (CCA) cell proliferation, migration and invasion. FENDRR and survivin expression in CCA tissues or cell lines were measured by qRT-PCR. In QBC939 and HuCCTl cells, cell proliferation was detected by CCK-8, cell migration and invasion were using transwell assay. RNA pull-down and RIP assay were performed to determine whether FENDRR can combine with SETDB1 in CCA cell. The effect of SETDB1 on survivin and H3K9me1 expression in CCA cells were determined by western blotting. ChIP analysis was performed to analyze the combination of SETDB1 with survivin promoter in CCA cell. The effect of SETDB1 knockdown on survivin and H3K9me1 expression in CCA cells after transfection with FENDRR were determined by western blotting. The results showed that lncRNA FENDRR was downregulated in CCA tissues and cells, and was negatively correlated with survivin expression. Further investigation demonstrated that FENDRR represses CCA cell proliferation, migration and invasion through regulating survivin expression. FENDRR associated with SETDB1 and H3K9 to epigenetically silence survivin and then regulated cell proliferation, migration and invasion. These findings indicate an important role for FENDRR–survivin axis in CCA cell proliferation, migration and invasion, and reveal a novel epigenetic mechanism for survivin silencing. Our data indicated that FENDRR silences survivin via SETDB1-mediated H3K9 methylation, thereby represses CCA cell proliferation, migration and invasion.

Survivin in Insulin-Like Growth Factor-Induced Resistance to Lapatinib in Head and Neck Squamous Carcinoma Cells

Epidermal growth factor receptor (EGFR) inhibitors have limited efficacy in head and neck squamous cell carcinoma (HNSCC) due to various resistance mechanisms, such as activation of the insulin-like growth factor-1 receptor (IGF1R), which initiates pro-survival signaling. Survivin, a member of the inhibitor of apoptosis proteins family, is expressed at relatively high levels in malignant tissues and plays a role in cell division. Expression of survivin in tumors has been shown to correlate with poor prognosis due to chemotherapy resistance and anti-apoptotic behavior. We previously demonstrated that activation of the IGF1R reduces sensitivity to EGFR-tyrosine kinase inhibitors (TKIs) via reduced apoptosis suggesting a role of survivin in this process. This study evaluates the role of survivin in IGF1R-mediated lapatinib resistance. Using HNSCC cell lines FaDu and SCC25, survivin expression increased and lapatinib sensitivity decreased with IGF1R activation. Further, these effects were reversed by the survivin inhibitor YM-155. Conversely, survivin expression and lapatinib sensitivity were unchanged with IGF1R activation in UNC10 cells. YM-155 enhanced the inhibitory effect of lapatinib on UNC10 cells, regardless of activation of the IGF1R. These results demonstrate that enhanced survivin expression correlates with IGF1R-mediated lapatinib resistance in HNSCC cells and suggest that regulation of survivin expression may be a key mechanistic element in IGF1R-based therapeutic resistance. Combinatorial treatment with survivin antagonists and EGFR-TKIs warrants further investigation.

Excision repair cross-complementing group 1 (ERCC1) overexpression inhibits cell apoptosis and is associated with unfavorable prognosis of esophageal squamous cell carcinoma.

Excision repair cross-complementing group 1 (ERCC1) functions as a nucleotide excision repair (NER) enzyme. Altered ERCC1 expression or function is closely associated with cancer development and progression. This study determined the association of ERCC1 expression with survivin expression, clinicopathological characteristics, and survival of esophageal squamous cell carcinoma (ESCC) patients after postoperative concurrent chemoradiotherapy.Tissue specimens from 102 resected ESCC patients were acquired for immunohistochemical analysis of ERCC1 and survivin protein expression.ERCC1 expression was detected in 62.7% of ESCC tissues and in 9.8% of normal squamous epithelium tissues (P < .01), while survivin expression was detected in 60.8% of ESCC tissues and in 19.6% of normal squamous epithelia (P < .01). ERCC1 overexpression associated with advanced tumor clinical stage and lymph node metastasis (P < .05), but not with tumor size, depth of invasion, or differentiation (P > .05). ERCC1 overexpression was also associated with survivin levels (r = 0.42, P < .01) and worse progression-free survival of ESCC patients after concurrent chemoradiotherapy. Multivariate analysis data revealed that ERCC1 and survivin protein expression were independent predictors of overall survival of ESCC patients after chemotherapy and/or radiotherapy (P < .05).ERCC1 overexpression is an important phenotype that is associated with ESCC lymph node metastasis and advanced tumor clinical stages. ERCC1 expression may also inhibit ESCC cell apoptosis via regulating survivin expression, and ERCC1 and survivin overexpression are independent predictors of prognosis for ESCC patients who receive chemotherapy and/or radiotherapy.

Smac combined with DDP can inhibit drug resistance of ovarian cancer through regulation of Survivin expression.

Ovarian cancer has the highest mortality rate among gynecological malignancies, presenting a major threat to women's life and health. It is essential to study the mechanisms of drug resistance to chemotherapy to identify ways to enhance drug-sensitivity. In recent years, many studies have shown that Smac/DIABLO is closely related to tumor drug resistance. Smac/DIABLO expression is markedly different between drug-resistant and chemo sensitive tumor cells. Up-regulation of Smac/DIABLO has been shown to increase tumor cell chemotherapy sensitivity. We found that Smac, combined with DDP greatly inhibited proliferation of subcutaneous xenografts of ovarian cancer cell line SKOV3/DDP without side effects. Mechanistic studies showed that Smac can inhibit the expression of Survivin, promote cell apoptosis of drug-resistant ovarian cancer cells and reverse the drug resistance.

Stomatin-like protein 2 regulates survivin expression in non-small cell lung cancer cells through \u03b2-catenin signaling pathway

The overexpression of stomatin-like protein-2 (SLP-2) is commonly observed in non-small cell lung cancer (NSCLC) cells. In the present study, we transfected a number of NSCLC cells with an SLP-2 shRNA-expressing vector (AdSLP2i) and examined its possible effects on cell growth and apoptosis. We found that suppression of SLP-2 expression inhibited cell growth, and that the apoptosis induced by SLP-2 suppression was correlated with decreased survivin protein expression. Moreover, the reduced survivin expression was found to be associated with reduced β-catenin nuclear localization and appeared not to be modulated through the AKT signaling pathway. By using immunoprecipitation and proteomics to analyze protein–protein interactions in A549 cells with SLP-2 overexpression, we found that annexin A2 interacted with SLP-2 and β-catenin directly. Our data further suggested that the knockdown of SLP-2 gene affected the SLP-2/Annexin A2/β-catenin cascade formation, reduced the translocation of cytoplasmic β-catenin into nucleus, and downregulated downstream target genes. The results presented in this study, together with our previous findings, suggest that SLP-2 promotes NSCLC cell proliferation by enhancing survivin expression mediated via β-catenin pathway.

Sublytic C5b-9 Induces Glomerular Mesangial Cell Apoptosis Through miR-3546/SOX4/Survivin Axis in Rat Thy-1 Nephritis

Background/Aims: The activation of complement system and the formation of C5b-9 complex have been confirmed in the glomeruli of patients with mesangioproliferative glomerulonephritis (MsPGN). However, the role and mechanism of C5b-9-induced injury in glomerular mesangial cell (GMC) are poorly understood. Rat Thy-1N is an animal model for studying MsPGN. It has been revealed that the attack of C5b-9 to the GMC in rat Thy-1N is sublytic, and sublytic C5b-9 can cause GMC apoptosis, but the underlying mechanism is not fully elucidated. To explore the role and regulatory mechanism of C5b-9 in MsPGN lesion, we used rat Thy-1N model and first detected the change of microRNA (miRNA) profiles both in Thy-1N rat renal tissues (in vivo) and in the cultured GMCs with sublytic C5b-9 stimulation (in vitro). Then we determined the effect of miR-3546, which increased both in vivo and in vitro, on GMC apoptosis upon sublytic C5b-9 as well as the involved mechanism. Methods: Rat Thy-1N model was established and GMCs were treated with sublytic C5b-9. The rat renal cortex and the stimulated GMCs were obtained for miRNA microarray detection. Subsequently, the increased miRNAs were verified by real-time PCR. Meanwhile, to ascertain the ability of some miRNAs to upregulate cleaved caspase 3 and induce GMC apoptosis, the corresponding miRNA mimics were transfected into GMCs, followed by western blotting (WB) and flow cytometry mesurement. Thereafter, the miR-3546-targeted gene (SOX4) was predicted using bioinformatics approaches, and SOX4 expression in Thy-1N tissues and in the GMCs upon sublytic C5b-9 stimulation or miR-3546 mimic/inhibitor transfection were detected using real-time PCR and WB. To prove that miR-3546 can affect SOX4 gene transcription and SOX4 can regulate survivin expression, dual luciferase reporter assay, real-time PCR, WB and chromatin immunoprecipitation (ChIP) assays were performed. Furthermore, the role of miR-3546/SOX4/survivin axis in the GMC apoptosis induced by sublytic C5b-9 was examined using WB and flow cytometry. Results: Compared with normal renal tissues and untreated GMCs, there were 43 and 62 upregulated miRNAs (> 2-fold) in Thy-1N tissues and sublytic C5b-9-stimulated GMCs respectively. A total of 17 miRNAs were increased both in vivo and in vitro, 11 of which were validated by real-time PCR. Among them, miR-3546 could markedly promote GMC apoptosis and inhibit SOX4 or survivin expression in response to sublytic C5b-9, and either SOX4 or survivin overexpression markedly rescued the GMC apoptosis mediated by miR-3546 mimic. Additionally, SOX4 overexpression could reverse the survivin suppression by miR-3546 mimic, and SOX4 could bind to survivin promoter (-1,278 to -853 nt) and activate survivin gene transcription. Conclusion: MiR-3546/ SOX4/survivin axis has a promoting role in the GMC apoptosis triggered by sublytic C5b-9, and our findings may provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN.

Survivin expression pattern in the intestine of normoxic and ischemic rats

BackgroundSurvivin, a member of the inhibitor of apoptosis protein (IAP) family, regulates mitosis and chromosome segregation. The expression of survivin proceeds during embryonic development and in addition has already been demonstrated in cancer cells. However, there is also evidence of survivin expression in differentiated tissues, including the gastro-intestinal tract of adult rats. A study with human colon specimens exhibited survivin in most basal crypt epithelial cells of normal mucosa. There is rather limited information on survivin expression in the small intestine. In order to paint a more detailed and thus complete picture of survivin expression patterns in the gastrointestinal tract, we used an immunohistochemical approach in normal adult rat small intestinal and ascending colonic tissue. Moreover, to get deeper insights in the regulation of survivin expression after tissue damage, we also studied its expression in mesenteric ischemia-reperfusion (I/R) injury.MethodsMesenteric ischemia-reperfusion injury was induced in male Wistar rats (six animals/group) by occlusion of the superior mesenteric artery for 90 min and subsequent reperfusion for 120 min. Paraffin sections of untreated or ischemically treated tissue were assessed immunohistochemically by survivin and Ki-67 staining.ResultsSurvivin could be detected in the small intestine and ascending colon of the normoxia group. It was expressed mainly in the epithelial cells of the crypts and only marginally in the villi. The individual small intestinal segments studied revealed comparable staining intensities. Likewise, expression of survivin was detected in the ischemically damaged small intestine and ascending colon. The expression pattern corresponded to the normoxic animals, as far as verifiable due to the existing tissue damage. Comparison of the expression pattern of Ki-67, a protein that acts as a cellular marker for proliferation, and survivin demonstrated a coincidental localization of the two proteins in the small intestinal and ascending colonic tissue.ConclusionsSurvivin was expressed strongly in epithelial cells of small intestinal as well as ascending colonic tissue. Its expression was located in cells with a high proliferation rate and regenerative capacity. This further supports the decisive role of survivin in cell division. Surprisingly, the ischemically damaged small intestinal and ascending colonic tissue showed a comparably high expression level. These results suggest that there is already a maximal survivin expression under normal conditions. However, the intestine is able to maintain the regenerative capacity even in spite of an ischemic injury. These findings reflect the important relevance of an intact intestinal barrier.

HIF-2α dictates the susceptibility of pancreatic cancer cells to TRAIL by regulating survivin expression.

Cancer cells develop resistance to therapy by adapting to hypoxic microenvironments, and hypoxia-inducible factors (HIFs) play crucial roles in this process. We investigated the roles of HIF-1α and HIF-2α in cancer cell death induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) using human pancreatic cancer cell lines. siRNA-mediated knockdown of HIF-2α, but not HIF-1α, increased susceptibility of two pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL in vitro under normoxic and hypoxic conditions. The enhanced sensitivity to TRAIL was also observed in vivo. This in vitro increased TRAIL sensitivity was observed in other three pancreatic cancer cell lines. An array assay of apoptosis-related proteins showed that knockdown of HIF-2α decreased survivin expression. Additionally, survivin promoter activity was decreased in HIF-2α knockdown Panc-1 cells and HIF-2α bound to the hypoxia-responsive element in the survivin promoter region. Conversely, forced expression of the survivin gene in HIF-2α shRNA-expressing Panc-1 cells increased resistance to TRAIL. In a xenograft mouse model, the survivin suppressant YM155 sensitized Panc-1 cells to TRAIL. Collectively, our results indicate that HIF-2α dictates the susceptibility of human pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL by regulating survivin expression transcriptionally, and that survivin could be a promising target to augment the therapeutic efficacy of death receptor-targeting anti-cancer therapy.

839 - Long noncoding RNA H19 regulates survivin expression in bladder cancer as sponge of miR-138-5p

839 - Long noncoding RNA H19 regulates survivin expression in bladder cancer as sponge of miR-138-5p

MiR-138-5p contributes to cell proliferation and invasion by targeting Survivin in bladder cancer cells.

BackgroundSurvivin (encoded by the gene BIRC5) plays an important role in the carcinogenesis of bladder cancer. Identifying miRNAs that target Survivin in the setting of bladder cancer will help to develop Survivin-based therapies for bladder cancer.MethodsThe expression levels of miR-138-5p and Survivin protein were measured in 12 resected bladder cancer specimens. The correlation between miR-138-5p and Survivin was further examined by evaluating Survivin expression in human bladder cancer cell lines that either overexpressed or knocked down miR-138-5p. A luciferase reporter assay was performed to test the direct binding of miR-138-5p to the target gene BIRC5. We also investigated the biological role of miR-138-5p targeting to Survivin in bladder cancer cell lines both in vivo and in vitro.ResultsIn this study, we found that the Survivin protein was either absent or weakly expressed in normal adjacent tissues and consistently up-regulated in bladder cancer tissues; however, the mRNA levels did not vary as much, suggesting that a post-transcriptional mechanism was involved. Because microRNAs are powerful post-transcriptional regulators of gene expression, we used bioinformatic analyses to search for microRNAs that could potentially target BIRC5 in the setting of bladder cancer. We identified 2 specific targeting sites for miR-138-5p in the 3′ untranslated region (3′-UTR) of BIRC5. We further identified an inverse correlation between miR-138-5p and Survivin protein levels in bladder cancer tissue samples. By overexpressing or knocking down miR-138-5p in bladder cancer cells, we experimentally confirmed that miR-138-5p directly recognizes the 3′-UTR of the BIRC5 transcript and regulates Survivin expression. Furthermore, the biological consequences of the targeting of BIRC5 by miR-138-5p were examined in vitro via cell proliferation and invasion assays and in vivo using a mouse xenograft tumor model. We demonstrated that BIRC5 repression by miR-138-5p suppressed the proliferative and invasive characteristics of bladder cancer cells and that miR-138-5p exerted an anti-tumor effect by negatively regulating BIRC5 in a xenograft mouse model.ConclusionsTaken together, our findings provide the first clues regarding the role of miR-138-5p as a tumor suppressor in bladder cancer by inhibiting BIRC5 translation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0569-4) contains supplementary material, which is available to authorized users.