Lentiviral shRNA Expression Research Articles

Knockdown of ZNF280A inhibits cell proliferation and promotes cell apoptosis of bladder cancer.

ZNF280A is a member of the zinc finger protein family, whose role in human cancers is little known and rarely reported. This study aimed to investigate the role of ZNF280A in bladder cancer. Immunohistochemical analysis was performed to detect the expression of ZNF280A in clinical samples. ZNF280A knockdown cell models were constructed by transfection of shRNA-expressing lentivirus. MTT assay and flow cytometry were performed for detecting cell proliferation, apoptosis and cycle. Wound healing and Transwell assays were operated to detect cell migration. Western blotting and Human Apoptosis Antibody Microarray were used to measure expression of related proteins. A mouse xenograft model was constructed for in vivo study. Our study demonstrated that ZNF280A was up-regulated in bladder cancer tissues compared with normal tissues, whose high expression was significantly correlated with advanced malignant grade. Knockdown of ZNF280A inhibited cell proliferation and cell migration, promoted cell apoptosis and G1/G2 phase arrest. The tumor growth in vivo was also proved to be inhibited by ZNF280A. Moreover, ZNF280A may promote bladder cancer through regulation of MAPK9, Cyclin D1 and the Akt pathway. In this study, ZNF280A was shown as a potential tumor promoter and prognosis indicator for bladder cancer. Targeting ZNF280A may be a promising strategy for the development of novel bladder cancer treatment.

Agrin Inhibition in Enteric Neural Stem Cells Enhances Their Migration Following Colonic Transplantation.

Regenerative cell therapy to replenish the missing neurons and glia in the aganglionic segment of Hirschsprung disease represents a promising treatment option. However, the success of cell therapies for this condition are hindered by poor migration of the transplanted cells. This limitation is in part due to a markedly less permissive extracellular environment in the postnatal gut than that of the embryo. Coordinated interactions between enteric neural crest-derived cells (ENCDCs) and their local environment drive migration along the embryonic gut during development of the enteric nervous system. Modifying transplanted cells, or the postnatal extracellular environment, to better recapitulate embryonic ENCDC migration could be leveraged to improve the engraftment and coverage of stem cell transplants. We compared the transcriptomes of ENCDCs from the embryonic intestine to that of postnatal-derived neurospheres and identified 89 extracellular matrix (ECM)-associated genes that are differentially expressed. Agrin, a heparin sulfate proteoglycan with a known inhibitory effect on ENCDC migration, was highly over-expressed by postnatal-derived neurospheres. Using a function-blocking antibody and a shRNA-expressing lentivirus, we show that inhibiting agrin promotes ENCDC migration in vitro and following cell transplantation ex vivo and in vivo. This enhanced migration is associated with an increased proportion of GFAP + cells, whose migration is especially enhanced.

Development of short hairpin RNA expression vectors targeting the internal ribosomal entry site of the classical swine fever virus genomic RNA

BackgroundClassical swine fever (CSF) is a fatal contagious disease affecting pigs caused by classical swine fever virus (CSFV). The disease can be transmitted by pigs and wild boars, and it is difficult to prevent and control. To obtain necessary information to establish the CSFV resistant animals in a future study, we designed lentiviral vector-delivered short hairpin RNAs (shRNAs) targeting the conserved domain III of the internal ribosomal entry site (IRES) of the CSFV genomic RNA.ResultsFirst, we confirmed the effects of siRNAs on CSFV-IRES activity. We observed significant inhibition of CSFV-IRES activity by si42 (domain IIIa), si107 (domain IIIc), and si198 (domain IIIf) in SK-L cells and si56 (domain IIIb), si142 (domain IIId1) and si198 in HEK293 cells without affecting the amount of luciferase RNA. Next, we constructed lentiviral vectors expressing shRNA based on siRNA sequences. Treatment with shRNA-expressing lentivirus was examined at 7 and 14 days post infection in SK-L cells and HEK293 cells, and CSFV-IRES was significantly suppressed at 14 days (sh42) post infection in HEK293 cells without significant cytotoxicity. Next, we examined the silencing effect of siRNA on CSFV replicon RNA and observed a significant effect by si198 after 2 days of treatment and by shRNA-expressing lentivirus (sh56, sh142, and sh198) infection after 14 days of treatment. Treatment of sh198-expressing lentivirus significantly suppressed CSFV infection at 3 days after infection.ConclusionThe IRES targeting sh198 expressing lentivirus vector can be a candidate tool for CSFV infection control.

RSAD2 Is an Effective Target for High-Yield Vaccine Production in MDCK Cells.

Increasingly, attention has focused on improving vaccine production in cells using gene editing technology to specifically modify key virus regulation-related genes to promote virus replication. In this study, we used DIA proteomics analysis technology to compare protein expression differences between two groups of MDCK cells: uninfected and influenza A virus (IAV) H1N1-infected cells 16 h post infection (MOI = 0.01). Initially, 266 differentially expressed proteins were detected after infection, 157 of which were upregulated and 109 were downregulated. We screened these proteins to 23 genes related to antiviral innate immunity regulation based on functional annotation database analysis and verified the mRNA expression of these genes using qPCR. Combining our results with published literature, we focused on the proteins RSAD2, KCNN4, IDO1, and ISG20; we verified their expression using western blot, which was consistent with our proteomics results. Finally, we knocked down RSAD2 using lentiviral shRNA expression vectors and found that RSAD2 inhibition significantly increased IAV NP gene expression, effectively promoting influenza virus replication with no significant effect on cell proliferation. These results indicate that RSAD2 is potentially an effective target for establishing high-yield vaccine MDCK cell lines and will help to fully understand the interaction mechanism between host cells and influenza viruses.

Selected Articles from This Issue

Ceramide kinase (CERK) and its enzymatic product, ceramide-1-phosphate (C1P), have been implicated in oncogenesis, but molecular mechanisms underlying CERK/C1P-mediated oncogenesis have not been elucidated. While investigating CERK functionality in non–small cell lung cancer (NSCLC), Vu and colleagues discovered that CERK is more abundant in KRASG12 mutant NSCLC tumor samples. Further interrogation revealed that CERK inhibitor NVP-231 increases reactive oxygen species levels and ferroptotic cell death uniquely in KRASG12 mutant NSCLC cells. Accordingly, an in vivo model in which the authors used CRISPR/Cas9 to generate NSCLC cells with single CERK alleles demonstrated that limiting CERK abundance sensitizes KRASG12 mutant NSCLC cells to cisplatin. Voltage-dependent anion channel 1 (VDAC1) abrogation using siRNA reverses CERK inhibition-driven cytotoxicity and cisplatin sensitivity in vitro. Mechanistically, the authors found that CERK inhibition prevents AKT mitochondrial localization, reducing AKT-mediated VDAC1 phosphorylation and subsequent β-tubulin/VDAC1 interactions on mitochondrial outer membranes. Overall, this study demonstrates that KRASG12 mutant NSCLC survival is reliant on CERK, and that CERK dependence may be therapeutically targetable using existing chemotherapeutics.While increased Ecdysoneless (ECD) mRNA and protein abundance has been observed in breast cancer and other tumor types, molecular mechanisms by which ECD might promote breast cancer tumorigenesis have not been fully clarified. To query how ECD functions in breast cancer, Mohapatra and colleagues generated a mouse model in which an ECD transgene is overexpressed specifically in mammary epithelium (ECDTg). Mammary ECD overexpression produces mammary tumors with heterogeneous histological characteristics that occasionally metastasize to the lungs. Transcriptomic analyses of ECDTg and control tumors revealed that ECD overexpression enhances c-MYC transcriptional programming, particularly augmenting metabolic c-MYC target gene transcription. The authors demonstrated that ECD increases c-MYC abundance by facilitating c-MYC mRNA splicing and maturation. Furthermore, doxycycline disrupts ECD and c-MYC expression, unveiling a potential therapeutic vulnerability in ECD-overexpressing tumors. Taken together, this study defines novel, physiologically relevant ECD functions in mammary cancer and presents potential ECD-driven therapeutic targets.Small cell lung cancer (SCLC) is an aggressive disease for which current therapeutic options provide limited benefits, necessitating therapeutic target discovery. While it is known that MUC1-C facilitates tumorigenesis in several cancers, it has not been studied in SCLC. To assess the role of MUC1-C in SCLC, Fushimi and colleagues abrogated MUC1-C expression in classic, variant, and non-neuroendocrine SCLC cell lines using doxycycline-inducible shRNA and performed transcriptomic analyses. The authors found that MUC1-C enhances MYC transcriptional programming in each SCLC subtype. Further studies revealed that MUC1-C is required for MYC localization to the NOTCH2 promoter and enhancer, and that ablating MUC1-C or MYC expression decreases expression of NOTCH2 and master transcription factors that dictate SCLC subtypes. Genetic and pharmacological disruption of MUC1-C, as well as CRISPR-Cas9-mediated NOTCH2 elimination, inhibit SCLC cell tumorsphere formation in vitro and tumor growth in vivo, suggesting the MUC1-C/MYC/NOTCH2 axis drives stemness and tumor progression in SCLC. In sum, this study defines a novel, potentially therapeutically targetable molecular mechanism underlying SCLC tumor progression.Proteosome inhibitor bortezomib demonstrates effectiveness in many multiple myeloma patients upon initial treatment. However, patients often relapse after initial therapy and develop drug resistance. Previously, Shigeki Miyamoto's group discovered that bone marrow stromal cells (BMSC) produce cleaved forms of hyaluronan and proteoglycan link protein 1 (HAPLN1) that activate NF-κB to engender bortezomib resistance. How cleaved HAPLN1 matrikines are generated in multiple myeloma remains unknown. To test if BMSC-derived HAPLN matrikines might derive from proteolytic cleavage, Miyamoto and co-authors performed a qRT-PCR–based matrix metalloproteinase (MMP) screen in BMSC derived from healthy donors and multiple myeloma patients. The screen revealed heightened MMP2 expression in multiple myeloma patient BMSC, and gelatin zymography activity assays demonstrated increased MMP2 activity in multiple myeloma patient BMSC. Subsequent in vitro assays showed that MMP2 cleaves HAPLN1 into products similar in size to the previously discovered matrikines. Abrogating HAPLN1 and MMP2 expression via shRNA-expressing lentivirus transduction in multiple myeloma patient BMSC decreases NF-κB activation and fosters bortezomib sensitivity in multiple myeloma cells. Altogether, this study unveils a novel molecular mechanism by which BMSC support multiple myeloma cell bortezomib resistance.

CtBP1 Mediates Hypoxia-Induced Sarcomatoid Transformation in Hepatocellular Carcinoma.

BackgroundSarcomatoid hepatocellular carcinoma (sHCC), a highly aggressive subtype of hepatocellular carcinoma (HCC), mostly transforms from classical hepatocellular carcinoma (cHCC). The study intended to explore the role of C-terminal binding protein 1 (CtBP1) in sarcomatoid transformation of hepatocellular carcinoma.MethodsWestern blotting and/or immunohistochemistry were used to confirm the expression of CtBP1 and other proteins in HCC cells, xenografts and clinical tissue samples. CtBP1 shRNA-expressing lentivirus was used to infect HepG2 cells to construct CtBP1 knockdown cells. Cell migration was determined by scratch wound assays and Transwell assays. Immunofluorescence was used to label the a-tubulin cytoskeleton to evaluate cell morphology. HepG2 cells were inoculated subcutaneously in nude mice to construct xenografts and beneath the liver capsule to evaluate in vivo metastasis.ResultsCompared to that in the cHCC area, CtBP1 expression was significantly upregulated in the sHCC area, as shown by immunohistochemistry. HE staining showed that cells in the sHCC area were spindle-shaped, while those in the cHCC area were polygonal. Immunohistochemically, the epithelial markers pancytokeratin (CK) and E-cadherin were partially or completely lost, while the expression of the mesenchymal marker vimentin was upregulated in the sHCC area. Moreover, HepG2, an HCC cell line with high expression of CtBP1, autonomously underwent sarcomatoid transformation, showing a sarcomatoid morphology and phenotype. HIF1a expression was upregulated in epithelial cells adjacent to the sHCC area. Hypoxia upregulated CtBP1 protein expression and induced an EMT phenotype with increased migration and a spindle-shaped morphology in HepG2 cells. Knockdown of CtBP1 partially reversed the EMT phenotype induced by hypoxia. Silencing CtBP1 completely blocked the sarcomatoid transformation of subcutaneous xenografts and decreased lung metastasis in subcapsular xenografts of the liver in nude mice.ConclusionCtBP1 plays a key role in hypoxia-induced EMT and sarcomatoid transformation in HCC and could be a candidate target for the management of sHCC.

MAFA Expression Promotes Proliferation of Myeloma Cells and Its Protein Stability Is Regulated By Proteasome Inhibitors

IntroductionMultiple myeloma (MM) patients with a t(14;16) or t(14;20) which result in high expression of MAF and MAFB, respectively, have a poor prognosis, and have not benefitted from the recent advances in MM therapy. Our previous work showed that post-translational modifications (PTM) further up-regulated MAF (Blood, 2016) and MAFB proteins (ASH Abstract #2091, 2014) conferring innate resistance to proteasome inhibitors (PI). The large MAF family of proteins contains a third member, MAFA. Little is known about its biological role in MM pathogenesis, as a probeset for MAFA is not present in gene expression profiling (GEP) analysis of molecular subgroups, and MAFA probes are not routinely used in fluorescence in situ hybridization (FISH) analysis. In the present study we therefore investigated the biological role of MAFA in MM and its effect on PI resistance.MethodsThe MAFA translocation in primary MM cells was determined by FISH analysis in 19 newly diagnosed MM patients. MAFA protein expression and protein level in response to PIs were assessed by immunoblotting and immunofluorescence staining (IFS). The effect of MAFA on proliferation of MM cells was measured by MTT assay. Annexin V staining and BrdU incorporation, analyzed by FACS analysis, was performed to determine the molecular action of MAFA on the survival of MM cells. MAFA expression was down-regulated in human MM cell lines (HMCLs). HMCLs using a lentiviral shRNA expression system. qRT-PCR analysis was used to determine MAFA mRNA expression in 32 HMCL and primary CD138+ plasma cells from 14 MM patients, and MAFA levels after shRNA treatment.ResultsFour levels of MAFA mRNA expression (relative to GAPDH) by RT-PCR analysis were determined: high levels were detected in 5 HMCLs, intermediate levels (100 to 500 ) in 10, low levels (10<100) in 7 and negative (<10) in 10 HMCLs. Among primary CD138+ patient cells (n=14), high levels were found in one patient, intermediate levels in 3, low levels in 4 and negative in 6 patients. FISH analysis found MAFA /t(8;14)(q24;q32) translocation in one out of 19 patients, which was also in the MF group by GEP analysis suggesting common downstream targets of MAFA with MAF and MAFB . MAFA protein was detected in the 6 HMCLs which expressed the highest MAFA mRNA levels by RT-PCR but not in the others.To determine if MAFA is required for MM cell survival, we knocked-down MAFA expression in three HMCLs that expressed high levels of MAFA . sh MAFA lentiviral supernatant infected HMCLs had 75% lower levels of MAFA mRNA and protein level compared with the cells infected with scrambled shRNA. Additionally, bioinformatically defined MAFA target genes were significantly decreased, including TLR4 (2.4-fold, P<0.01), NUAK1 (2.3-fold, p<0.01) and SPP1 (osteopontin, 2.2-fold, P<0.01), compared with HMCLs infected with control viral vector. Silencing MAFA expression significantly decreased the proliferation of HMCLs (62.5% decrease, P=2.1x10-4). Among 6 high MAFA expressing HMCLs, 3 co-expressed MAF and 2 co-expressed MAFB . To determine if MAFA synergized with MAF in promoting MM proliferation, we silenced MAFA and MAFB genes in cells co-expressing these genes. Simultaneously silencing MAFA and MAFB did not further decrease proliferation compared to either silencing MAFA or MAFB alone. These results indicate that MAFA promotes MM cell proliferation and that there is no synergetic effect between MAFA and MAFB in regulating MM proliferation.To determine if MAFA protein is regulated by PTM, we treated MM cells with PIs including bortezomib (Bzb) and carfilzomib (CFZ). Bzb induced stabilization of MAFA protein in a dose-dependent manner. Similar effects were seen with CFZ indicating that MAFA protein may be degraded by the ubiquitin proteasome system.ConclusionTaken together, our results indicate that increased levels of MAFA are seen in MM including the MAFA /t(8;14) translocation. High expression of MAFA mRNA and protein occurs in 18.7 % of MM cell lines. MAFA promotes myeloma proliferation and regulates TLR4, NUAK1 and SPP1 gene expression. The stability of MAF protein is regulated by PIs. These results provide new insights in understanding the role of the MAF family and, in particular, MAFA in MM pathogenesis. Experiments are ongoing to determine the function of MAFA in PI-resistance and to confirm MAFA targets in order to identify potential alternate approaches for the treatment of this poor performing molecular subgroup. DisclosuresMorgan:Bristol Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Davies:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria.

Silencing of long non-coding RNA CRNDE promotes autophagy and alleviates neonatal hypoxic-ischemic brain damage in rats.

Hypoxic-ischemic (HI) brain damage (HIBD) leads to high neonatal mortality and severe neurologic morbidity. Autophagy is involved in the pathogenesis of HIBD. This study aims to investigate the effect of long non-coding RNA colorectal neoplasia differentially expressed (CRNDE) on HIBD and to validate whether autophagy is involved in this process. A HIBD model in rat pups and a HI model in rat primary cerebrocortical neurons were established. Autophagy was evaluated by western blot. The HIBD in rats was evaluated by hematoxylin and eosin staining, TUNEL staining, triphenyl tetrazolium chloride staining, and morris water maze test. The HI injury in vitro was evaluated by determining cell viability and apoptosis. The results showed that CRNDE expression was time-dependently increased in the brain after HIBD. Administration with CRNDE shRNA-expressing lentiviruses alleviated pathological injury and apoptosis in rat hippocampus, decreased infarct volume, and improved behavior performance of rats subjected to HIBD. Furthermore, CRNDE silencing promoted cell viability and inhibited cell apoptosis in neurons exposed to HI. Moreover, CRNDE silencing promoted autophagy and the autophagy inhibitor 3-methyladenine counteracted the neuroprotective effect of CRNDE silencing on HI-induced neuronal injury both in vivo and in vitro. Collectively, CRNDE silencing alleviates HIBD, at least partially, through promoting autophagy.

Effect of histone demethylase KDM5A on the odontogenic differentiation of human dental pulp cells

ABSTRACT Human dental pulp cells (hDPCs) possess the capacity to differentiate into odontoblast-like cells in response to exogenous stimuli. Histone methylation is one of the most robust epigenetic marks and is essential for the regulation of multiple cellular processes. Previous studies have shown that histone methyltransferases (HMTs) and histone demethylases (HDMs) are crucial for the osteogenic differentiation of human bone marrow, adipose tissue, and tooth tissue. However, little is known about the role of histone methylation in hDPC differentiation. Here, the expression levels of HMTs and HDMs were profiled in hDPCs undergoing odontogenic induction. Among several differentially expressed enzymes, HDM KDM5A demonstrated significantly enhanced expression during cytodifferentiation. Furthermore, KDM5A expression increased during early passages and in a time-dependent manner during odontogenic induction. Using a shRNA-expressing lentivirus, KDM5A was knocked down in hDPCs. KDM5A depletion resulted in greater alkaline phosphatase activity and more mineral deposition formation. Meanwhile, the expression levels of the odontogenic markers DMP1, DSPP, OSX, and OCN were increased by KDM5A knockdown. As a histone demethylase specific for tri- and dimethylated histone H3 at lysine 4 (H3K4me3/me2), KDM5A deficiency led to a significant increment in total H3K4me3 levels, whereas no significant difference was found for H3K4 me2. H3K4me3 levels on the promoters of the odontogenic markers increased after KDM5A knockdown in hDPCs. These results demonstrated that KDM5A is present in hDPCs and inhibits the odontogenic differentiation potentiality of hDPCs by removing H3K4me3 from specific gene promoters, suggesting that KDM5A-dependent histone demethylation may play an important role in reparative dentinogenesis.

EZH2 regulates PD-L1 expression via HIF-1α in non-small cell lung cancer cells

Lung cancer is the most commonly diagnosed cancer and accounts for most cancer-related mortalities worldwide. The high expression of programmed death ligand 1 (PD-L1) is an important factor that promotes immune escape of lung cancer, thus aggravates chemotherapy resistance and poor prognosis. Therefore, understanding the regulatory mechanism of PD-L1 in lung cancer is critical for tumor immunotherapy. Enhancer of Zeste homolog2 (EZH2), an epigenetic regulatory molecule with histone methyltransferase activity, promotes the formation of an immunosuppressive microenvironment. This study aimed to investigate the role of EZH2 in PD-L1 expression and in the progression of lung tumors. We found that EZH2 was upregulated in lung cancer tissues and positively correlated with PD-L1 levels and poor prognosis. Further, shRNA-expressing lentivirus mediated EZH2 knockdown suppressed both the mRNA and protein expression level of PD-L1, thus delaying lung cancer progression in vivo by enhancing anti-tumor immune responses. Moreover, the regulatory effect of EZH2 on PD-L1 depended on HIF-1α. The present results indicate that EZH2 regulates the immunosuppressive molecule PD-L1 expression via HIF-1α in non-small cell lung cancer cells.

Pridopidine stabilizes mushroom spines in mouse models of Alzheimer's disease by acting on the sigma-1 receptor

There is evidence that cognitive decline in Alzheimer's disease (AD) results from deficiencies in synaptic communication (e.g., loss of mushroom-shaped ‘memory spines’) and neurodegenerative processes. This might be treated with sigma-1 receptor (S1R) agonists, which are broadly neuroprotective and modulate synaptic plasticity. For example, we previously found that the mixed muscarinic/S1R agonist AF710B prevents mushroom spine loss in hippocampal cultures from APP knock-in (APP-KI) and presenilin-1-M146 V knock-in (PS1-KI) mice. We also found that the “dopaminergic stabilizer” pridopidine (structurally similar to the S1R agonist R(+)-3-PPP), is a high-affinity S1R agonist and is synaptoprotective in a mouse model of Huntington disease. Here we tested whether pridopidine and R(+)-3-PPP are synaptoprotective in models of AD and whether this requires S1R. We also examined the effects of pridopidine on long-term potentiation (LTP), endoplasmic reticulum calcium and neuronal store-operated calcium entry (nSOC) in spines, all of which are dysregulated in AD, contributing to synaptic pathology. We report here that pridopidine and 3-PPP protect mushroom spines from Aβ42 oligomer toxicity in primary WT hippocampal cultures from mice. Pridopidine also reversed LTP defects in hippocampal slices resulting from application of Aβ42 oligomers. Pridopidine and 3-PPP rescued mushroom spines in hippocampal cultures from APP-KI and PS1-KI mice. S1R knockdown from lenti-viral shRNA expression destabilized WT mushroom spines and prevented the synaptoprotective effects of pridopidine in PS1-KI cultures. Knockout of PS1/2 destabilized mushroom spines and pridopidine was unable to prevent this. Pridopidine lowered endoplasmic reticulum calcium levels in WT, PS1-KO, PS1-KI and PS2 KO neurons, but not in PS1/2 KO neurons. S1R was required for pridopidine to enhance spine nSOC in PS1-KI neurons. Pridopidine was unable to rescue PS1-KI mushroom spines during pharmacological or genetic inhibition of nSOC. Oral pridopidine treatment rescued mushroom spines in vivo in aged PS1-KI-GFP mice. Pridopidine stabilizes mushroom spines in mouse models of AD and this requires S1R, endoplasmic reticulum calcium leakage through PS1/2 and nSOC. Thus, pridopidine may be useful to explore for the treatment of AD.

The presumed MTH1-inhibitor TH588 sensitizes colorectal carcinoma cells to ionizing radiation in hypoxia

BackgroundThe nudix family member enzyme MutT homologue-1 (MTH1) hydrolyses the oxidized nucleotides 8-oxo-dGTP and 2-hydroxy-dATP and thus prevents the incorporation of damaged nucleotides into nuclear and mitochondrial DNA. Therefore MTH1 was proposed to protect cancer cells from oxidative DNA lesions and subsequent cell death. We investigated whether the bona fide MTH1 inhibitor TH588 affects responses of cultured colorectal tumor cells to ionizing radiation (IR) in normoxia and in moderate or severe hypoxia.MethodsTH588 was tested in cell viability and survival assays (tetrazolium dye (MTT), propidium iodide staining, caspase-3 activity, and colony formation assays (CFA)) in colorectal carcinoma cells (HCT116 and SW480) in combination with IR in normoxia and in hypoxia. Additionally, MTH1 was targeted by lentiviral shRNA expression. Human umbilical vein endothelial cells (HUVEC) were assessed in MTT assays.ResultsIn all cell lines tested, TH588 dose-dependently impaired cell survival. In CFAs, TH588 and IR effects on carcinoma cells were additive in normoxia and in hypoxia. Using 3 different shRNAs, the lentiviral approach was detrimental to SW480, but not to HCT116.ConclusionsTH588 has cytotoxic effects on transformed and untransformed cells and synergizes with IR in normoxia and in hypoxia. TH588 toxicity is not fully explained by MTH1 inhibition as HCT116 were unaffected by lentiviral suppression of MTH1 expression. TH588 should be explored further because it has radiosensitizing effects in hypoxia.

LGR6 promotes the progression of gastric cancer through PI3K/AKT/mTOR pathway.

BackgroundIn the present study, we aimed to investigate the role of LGR6 in the progression of gastric cancer (GC) and explore the intrinsic molecular mechanisms.Materials and methodsThe lentiviral LGR6 shRNA (sh-LGR6) and lentiviral expression vector of LGR6 gene (OE-LGR6) were used to regulate the LGR6 expression. Furthermore, we performed in vitro experiments to observe whether PI3K/AKT/mTOR pathway was affected by LGR6 and assess the role of LGR6 in the proliferation, apoptosis, migration, and invasion of GC cells.ResultsOur data showed that phosphorylated AKT and mTOR were downregulated by sh-LGR6 (P<0.05). The expressions of proapoptotic proteins Bax and Caspase-3 were upregulated by sh-LGR6 (P<0.05); the expression of antiapoptotic protein Bcl2 was downregulated by sh-LGR6 (P<0.001). Besides, the functional experiments proved that sh-LGR6 could promote the apoptosis of GC cells and inhibit the proliferation, invasion, and migration of GC cells (P<0.001). Compared with sh-LGR6, OE-LGR6 led to the opposite results.ConclusionLGR6 is an antiapoptosis protein which controls the progression of GC through PI3K/AKT/mTOR pathway. More in vivo experiments and clinical trials are necessary to confirm the possibility of LGR6 in tumor therapy.

Construction and identification of a model for HJURP gene defect expression in human embryo villus cells

To construct a lentiviral vector for RNA interference (RNAi) of the HJURP gene and to identify the silencing efficiency in the human embryo villus cells and to provide a human embryo villus cells multiplication and chromosome segregation. In accordance with the study, three specific sequences of siRNA targeting HJURP gene were designed, synthesized, then the complementary DNA containing both sense and antisense oligonucleotides of the targeting sequences were annealed and inserted into the lentiviral vector.The correct clonings were confirmed by PCR and sequencing. The most effective recombinant lentivirus vector was screened, and the recombinant plasmids with the lentivirus packaging mixes were co-transfected into 293T cells to obtain packaged lentivirus particles. Then viral titer was determined. The silencing efficiency of target gene in human embryo villus cells was detected by Real-Time PCR. DNA sequencing showed that the shRNA sequence was successfully inserted into the lentivirus vector. The recombinant lentiviral vector was successfully transfected into 293T cells. The recombinant lentivirus had a titer of 108 PFU/ml. After silencing HJURP gene in human embryo villus cells, the expression level of HJURP mRNA decreased significantly and the RNAi efficiency was greater than 70%. A lentiviral shRNA expression vector targeting the HJURP gene was successfully constructed and may effectively silence the target gene at a cellular level, which provides a experimental model for the influence of HJURP gene expressing inhibition on human embryo villus cells multiplication and chromosome segregation.

Abstract 522: Role and regulation of Aurora A in radiation resistance mechanisms in glioblastoma

Abstract Glioblastoma (GBM) is the most aggressive tumor of the human brain, which inevitably escapes the standard treatment modalities that include surgery, radiotherapy and chemotherapy. Consequently, the median survival of GBM patients remains 12-15 months despite these aggressive treatments. Therefore, to develop effective therapies, it is important to understand how GBM cells develop therapeutic resistance. Aurora A is a ubiquitously expressed serine/threonine kinase, which plays essential roles in mitotic regulation. This protein kinase, which phosphorylates p53, PP1, CEBP, and histone H3 among other regulators of cell division, is overexpressed in various neoplasms including GBM and invariably associated with poor prognosis. Recent studies have demonstrated that Aurora A contributes to radio-resistance in various solid neoplasm including GBM. Importantly, Aurora A inhibitors are currently in phase I/II clinical trials for solid cancer. Notably, Aurora A inhibitor MLN8237 that crosses the blood brain barrier specifically inhibits Aurora A at concentrations ≤ maximally tolerated dose in animal models and in phase I clinical trials. We found that Aurora A protein level is upregulated by ionizing radiation in GBM cells. This was, in part, mediated by radiation-induced inhibition of 26S-proteosomal degradation of Aurora A. Aurora A was silenced using both lentiviral mediated shRNA expression and a specific Aurora A inhibitor in three different established cell lines, and were treated with 6 gy radiation. We found that pharmacological inhibition and RNAi-mediated knockdown of Aurora A sensitized the GBM cells to radiation therapy resulting in reduced cell proliferation and increased apoptosis. Interestingly, we observed that RNAi-mediated down-regulation of Aurora A was associated with a decrease in X-linked inhibitor of apoptosis (XIAP) expression in GBM cells. We also found that Aurora A and XIAP were co-localized in GBM cells. Taken, together our results reveal a mechanism by which Aurora A confers radio-resistance in GBM cells and further rationalize the use of Aurora A inhibitors as radio-sensitizer in solid neoplasms including GBM. Funding sources: This work was supported by R01CA108633 (To AC), 1RC2CA148190 (To AC) U10CA180850-01 (To AC), 1R01CA169368 (To AC) from the National Cancer Institute (NCI), Brain Tumor Funders Collaborative Grant (To AC), Ohio State University Comprehensive Cancer Center Award (To AC) Citation Format: Brinda Ramasubramanian, Kamalakannan Palanichamy, Disha Patel, Saikh Jaharul Haque, Arnab Chakravarti. Role and regulation of Aurora A in radiation resistance mechanisms in glioblastoma. [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 522.

Knockdown of COUP-TFII inhibits cell proliferation and induces apoptosis through upregulating BRCA1 in renal cell carcinoma cells.

COUP-TFII belongs to the nuclear receptor family, which is highly expressed in many kinds of tumors. Previous studies have shown that COUP-TFII can promote tumor progression through regulating tumor angiogenesis and cell proliferation and migration of certain cancer cells. However, the function of COUP-TFII in renal cell carcinoma (RCC) is not clear. Here, we showed that clinical RCC tumor tissues showed much higher COUP-TFII expression level than adjacent normal tissues. When COUP-TFII was knocked down in RCC 769-P and 786-O cells by siRNA or shRNA-expressing lentivirus, the cell proliferation was markedly inhibited, and apoptosis increased. Moreover, the tumor growth of COUP-TFII knockdown 769-P and 786-O xenografts in nude mice was also obviously inhibited. Using qRT-PCR and Western blot, we showed that the expression of the tumor suppressor gene BRCA1 was upregulated in COUP-TFII knockdown cells. Simultaneously knockdown of BRCA1 and COUP-TFII partially rescued the inhibited cell proliferation and increased apoptosis in COUP-TFII single knockdown cells. These results indicate that COUP-TFII may play an oncogenic role in RCC, and COUP-TFII may promote tumor progression through inhibiting BRCA1.

Knockdown of ZFR suppresses cell proliferation and invasion of human pancreatic cancer.

BackgroundZinc finger RNA binding protein (ZFR) is involved in the regulation of growth and cancer development. However, little is known about ZFR function in pancreatic cancer.MethodsHerein, to investigate whether ZFR is involved in tumor growth, Oncomine microarray data was firstly used to evaluate ZFR gene expression in human pancreatic tumors. Then short hairpin RNA (shRNA) targeting ZFR was designed and delivered into PANC-1 pancreatic cancer cells to knock down ZFR expression. Cell viability, cell proliferation and cell cycle analysis after ZFR knockdown were determined by MTT, colony forming and FACS, respectively. In addition, cell migration and invasion were assessed using the Transwell system.ResultsThe expression of ZFR was significantly higher in pancreatic tumors than normal pancreas tissues by Oncomine database analysis. Knockdown of ZFR by shRNA-expressing lentivirus significantly decreased the viability and invasion ability of pancreatic cancer cells. Moreover, FACS analysis showed that knockdown of ZFR in PANC-1 cells caused a significant cell cycle arrest at G0/G1 phase. Furthermore, knockdown of ZFR decreased the levels of CDK2, CDK4, CyclinA and CyclinD1 and enhanced the expression of p27, which has evidenced by qRT-PCR and Western blot analysis.ConclusionsKnockdown of ZFR might provide a novel alternative to targeted therapy of pancreatic cancer and deserves further investigation.

Construction and identification of a lentiviral vector for RNA interference of human GLUT3 gene

To construct an effective lentiviral vector for RNA interference (RNAi) with human glucose transporter 3 (GLUT3)gene. Four pairs of shRNA sequences against different parts of GLUT3-mRNA were separately cloned into the RNAi plasmid vector pLV-shRNA by recombinant DNA technology to construct shRNA expression vectors pLV-shRNA-GLUT3-1, pLV-shRNA-GLUT3-2, pLV-shRNA-GLUT3-3, and pLV-shRNA-GLUT3-4. The vectors were transfected into HeLa cells to detect the effectiveness of GLUT3 gene silencing. One of effective vectors was selected and co-transfected into 293T cells with lentivirus packaging plasmids to obtain packaged lentivirus particles LV-GLUT3. After viral titer determination, U251 glioblastoma cells were infected with LV-GLUT3 at a multiplicity of infection (MOI) of 10. Finally, the expression of GLUT3 protein was detected by Western blot. DNA sequencing demonstrated that the shRNA sequences were successfully inserted into the pLV-shRNA vectors. In HeLa cells, the expression of GLUT3-mRNA was significantly down-regulated by the recombinant vectors compared with negative control. The recombinant lentivirus LV-GLUT3 harvested from 293T cells had a titer of 1.5×10(9) TU/mL. After infection with LV-GLUT3, the expression of GLUT3 protein in U251 glioblastoma cells was down-regulated. An effective lentiviral shRNA expression vector targeting the GLUT3 gene is successfully constructed and can be used for further study on the functions of GLUT3 gene.

Abstract B40: Autocrine and paracrine IL-6 and IL-8 drive osteosarcoma lung tropism and facilitate metastasis

Abstract Osteosarcoma originates in bone, but metastasizes predictably to the lungs. Patients who develop lung metastases become much more difficult to treat. Metastatic lung disease responds poorly to traditional chemotherapy. We know very little of the mechanisms that mediate this lung tropism, or predilection for spread to lung tissue. A better understanding of this process could facilitate disruption of pathways which drive the metastatic process, potentially preventing or even treating lung metastasis. We thus sought to identify tumor-lung interactions that facilitate extravasation, survival, and growth of tumor cells specifically within the lung environment. Drawing on published signatures of osteosarcoma metastasis, we curated a list of 59 candidate genes that might facilitate lung metastasis. By comparing expression in metastatic lung lesions relative to paired primary tumors, we found that metastatic tumors reliably express two cytokines, IL-6 and IL-8, at much higher levels than matched primary bone tumors. In a mouse model of osteosarcoma, we found that tumors expressing high levels of autocrine IL-6 and IL-8 established lung metastases much more readily than tumors which expressed low or no IL-6 or IL-8. Using immunohistochemistry to identify sources of these cytokines within human metastatic lesions, we found higher levels of IL-6 and IL-8 coming from non-malignant cells within the tumor, especially mononuclear infiltrates and peri-bronchiolar cells. Modelling tumor-lung interactions in vitro using a co-culture system, much higher levels of IL-6 and IL-8 were produced when osteosarcoma cells were cultured with bronchial epithelial cells, smooth muscle cells, or macrophages suggesting that paracrine interactions drive production of these cytokines upon interaction of tumor cells with resident lung cells. In murine models of osteosarcoma metastasis, blocking IL-6 and/or IL-8 production within tumor cells using inducible shRNA-expressing lentiviruses dramatically reduced metastatic efficiency. Our data suggest that IL-6 and IL-8 play a primary role in the development of osteosarcoma lung metastasis. The readily targetable nature of these pathways makes them attractive candidates for therapies designed to prevent and possibly treat osteosarcoma lung metastasis. Citation Format: Ryan D. Roberts, Amy C. Gross, Hemant K. Bid, Doris Phelps, Mary F. Wedekind, Peter J. Houghton. Autocrine and paracrine IL-6 and IL-8 drive osteosarcoma lung tropism and facilitate metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B40.

Double transduction of a Cre/LoxP lentiviral vector: a simple method to generate kidney cell-specific knockdown mice.

In a lentivirus-based gene delivery system, the incorporated gene is continuously expressed for a long time. In this study, we devised a simple way to knock down a specific gene in a kidney cell-specific pattern in adult mice by lentivirus-assisted transfer of short hairpin RNA (shRNA). Kidney collecting duct (CD)-specific aquaporin-3 (AQP3)-knockdown mice were generated by consecutive injection of Hoxb7-Cre-expressing lentivirus (LV-Hoxb7 Cre) and loxP-AQP3 shRNA-expressing lentivirus (LV-loxP shAQP3) in adult C57BL6/J mice. LV-Hoxb7 Cre was designed to express mCherry, while LV-loxP shAQP3 was designed with a floxed enhanced green fluorescent protein (EGFP)-tagged stop sequence, and thus EGFP would be expressed only in the absence of Cre recombination. In mice treated with LV-Hoxb7 Cre alone, mCherry protein expression, which indicates the presence of Cre recombinase, occurred only in CD cells. However, LV-loxP shAQP3 injection alone resulted in an increase in EGFP expression in all kidney cells, indicating the transcription of the floxed region. When LV-Hoxb7 Cre and LV-loxP shAQP3 were sequentially transduced, EGFP expression was attenuated while mCherry expression was sustained in CD cells, demonstrating a CD cell-specific recombination of the floxed region. AQP3 expression in mice injected with LV-Hoxb7 Cre or LV-loxP shAQP3 alone did not differ, but consecutive injection of LV-Hoxb7 Cre and LV-loxP shAQP3 significantly reduced AQP3 expression in CD cells. However, the expression levels of AQP3 were not altered in other cell types. Double transduction of Cre- and loxP-based lentivirus can easily generate kidney cell-specific knockdown mice, and this method might be applicable to other species.