shRNA Vector Research Articles

Differential expression of APE1 in hepatocellular carcinoma and the effects on proliferation and apoptosis of cancer cells.

This research aimed to investigate the differential expression of apurinic-apyrimidinic endonuclease 1 (APE1) in hepatocellular carcinoma (HCC) tissues and cells and the effects on proliferation and apoptosis of cancer cells. Immunohistochemical techniques were used to detect the expression of APE1 in 80 cases of HCC and the corresponding paracancerous tissue microarrays; meanwhile, Western blots were used to detect the expression of APE1 in both human HCC BEL-7402, BEL-7405, HCC-9204, Hep3B, HepG2, SMMC-7721 and Huh-7 cells, and normal hepatocyte L-02 cells. The relationship between APE1 expression and clinical pathological characteristics of HCC was statistically analyzed. APE1 shRNA vector was constructed in Hep 3B cells to establish a stably transfected cell line, using Western blots to determine the interference efficiency. Cell proliferation activity was detected with MTT assays, while apoptosis was detected with the Annexin V-FITC/PI double-labeling technique. The expression of APE1 in HCC tissues and cells was significantly up-regulated, and its expression was significantly different from TNM staging and histopathological grading. Down-regulation of APE1 expression significantly reduced the proliferative activity and increased the apoptosis rate of Hep 3B cells. In conclusion, APE1 demonstrates cancer progression potential at the clinical, tissue and cell level. It provides a new idea and theoretical basis for APE1-based clinical diagnosis, prognosis determination and molecular targeted therapy in treatment of HCC.

A6645 Sirtuin 3-mediated glycolysis enhancing during Angiotension II-induced endothelial-to-mesenchymal transition

Objectives: Enhanced glycolysis has been recognized as a critical role in initiating endothelial to mesenchymal transition (EndMT) progression. The underlying regulators of the metabolic program in Angiotension II (Ang II)-induced EndMT remain unclear. Sirtuin 3 (SIRT3), an NAD-dependent deacetylase, is a major cellular sensor of energy metabolism and mediates adaptation responses. The present study is designed to investigate the role of SIRT3-medated glycolysis enhancing in regulating the processes of EndMT. Methods: Experiments were preformed with murine aortic endothelial cells (MAECs) which were infected with lentivirus SIRT3 shRNA, ATG5 shRNA or GFP-expressing vector. EndMT was induced by Ang II (1 μM for 24 h) and the expression of endothelial and mesenchymal markers (CD31, FSP1 and α-SMA) were detected by Western blot analysis. Results: AngII leads to impaired mitophagy flux in SIRT3-deficient ECs, accompanying marked changes in EC architecture, endothelial loss and gain of mesenchymal markers. The accumulation of damaged mitochondria during EndMT is coupled to a metabolic shift with increased lactate production and elevated expression of glycolytic enzymes, which correlates with the upregulation of acetylation of endogenous ATG5. ATG5 acetylation inhibits autophagosome maturation andinduces metabolic reprogramming. Consistent with this, en face analyses displays a marked increase in α-SMC-positive endothelial cells in SIRT3−/− mice challenged with Ang II. Further, we find that SIRT3 overexpression represses glycolysis and transition in endothelial cells. Pharmacological or genetic inhibition of glycolysis significantly attenuates α-SMA expression. Conclusion: Taken together, these findings reveal that SIRT3-deficient ECs display impaired autophagy and accelerate glycolysis and the processes of EndMT.

Effect of shRNA-mediated Xist knockdown on the quality of porcine parthenogenetic embryos.

Parthenogenetically activated oocytes exhibit poor embryo development and lower total numbers of cells per blastocyst accompanied by abnormally increased expression of Xist, a long noncoding RNA that plays an important role in triggering X chromosome inactivation during embryogenesis. To investigate whether knockdown of Xist influences parthenogenetic development in pigs. We developed an anti-Xist short hairpin RNA (shRNA) vector, which can significantly inhibit Xist expression for at least seven days when injected at 12-13 hr after parthenogenetic activation. Embryonic cleavage, blastocyst formation, and total blastocyst cell numbers were compared during the blastocyst stage, as well as the expression of an X-linked gene and three pluripotent transcription factors. Knockdown of Xist significantly increases the total blastocyst cell number, but does not influence the rate of embryo cleavage and blastocyst formation. The expressions of Sox2, Nanog, and Oct4 were also significantly improved in the injected embryos compared with the control at the blastocyst stage, but the Foxp3 expression level was not increased significantly. The present study provides valuable information for understanding the role of Xist in parthenogenesis and presents a new approach for improving the quality of porcine parthenogenetic embryos. Developmental Dynamics 248:140-148, 2019. © 2018 Wiley Periodicals, Inc.

Targeted Suppression and Knockout of ASCT2 or LAT1 in Epithelial and Mesenchymal Human Liver Cancer Cells Fail to Inhibit Growth.

Amino acid transporters alanine-serine-cysteine transporter 2 (ASCT2) and L-Type Amino Acid Transporter 1 (LAT1) are coordinately enhanced in human cancers where among other roles, they are thought to drive mechanistic target-of-rapamycin (mTOR) growth signaling. To assess ASCT2 and LAT1 as therapeutic targets, nine unique short hairpin RNA (shRNA) vectors were used to stably suppress transporter expression in human epithelial (Hep3B) and mesenchymal (SK-Hep1) hepatocellular carcinoma (HCC) cell lines. In addition, six unique CRISPR-Cas9 vectors were used to edit the ASCT2 (SLC1A5) and LAT1 (SLC7A5) genes in epithelial (HUH7) and mesenchymal (SK-Hep1) HCC cells. Both approaches successfully diminished glutamine (ASCT2) and leucine (LAT1) initial-rate transport proportional to transporter protein suppression. In spite of profoundly reduced glutamine or leucine transport (up to 90%), transporter suppression or knockout failed to substantially affect cellular proliferation or basal and amino acid-stimulated mTORC1 growth signaling in either HCC cell type. Only LAT1 knockout in HUH7 slightly reduced growth rate. However, intracellular accumulation of radiolabeled glutamine and leucine over longer time periods largely recovered to control levels in ASCT2 and LAT1 knockout cells, respectively, which partially explains the lack of an impaired growth phenotype. These data collectively establish that in an in vitro context, human epithelial and mesenchymal HCC cell lines adapt to ASCT2 or LAT1 knockout. These results comport with an emerging model of amino acid exchangers like ASCT2 and LAT1 as “harmonizers”, not drivers, of amino acid accumulation and signaling, despite their long-established dominant role in initial-rate amino acid transport.

Abstract 3460: p38-p14ARF-CtBP2 axis as a novel regulator of CSC phenotype and tumor cell dormancy

Abstract Introduction: Cancer cell dormancy and the cancer stem cell (CSC) phenotype are interlinked and play a crucial role in disease relapse. The pp38/pERK signaling ratio positively regulates cellular dormancy. We observed that heparin hexasaccharide (HS06) inhibited colon CSC self-renewal through isoform specific induction of pp38α/β, increasing the pp38/pERK ratio, and downregulation of C-terminal binding protein-2 (CtBP2) levels which is known to play a critical role in CSC growth. Hence, we hypothesized that HS06 inhibits CSCs self-renewal and prevents dormancy escape through regulation of CtBP2 in a manner dependent on p38 activation. Method: Spheroid culture of HT29 and HCT116 colon cancer cells was used for CSC self-renewal monitoring. Transient transfection with p38α, p14ARF, CtBP2 shRNA and/or expression vector with appropriate scrambled or vector controls was used to modulate cell signaling through this pathway. Standard western blotting, Q-PCR, flow cytometry, and immunoprecipitation were used to examine protein levels, their phosphorylation status and protein-protein interactions. Short term exposure of colon cancer cells (HT29) to chemotherapy (5-Fluorouracil (125μM) and oxaliplatin (5μM) (5-FUOX)) was used to study a transient dormancy-like state followed by growth escape. HT29 CSC (1x105 CD133+/CXCR4+ cells) induced xenografts were established s.c. in NCr nude mice were treated with HS06 (150mg/kg 3xwk., 3 weeks) and monitored for tumor growth, and xenografts were analyzed for CSC makers and p38-p14ARF-CtBP2 axis status. Results: HS06 inhibited CtBP2 levels but increased the expression of its negative regulator p14 ARF in vitro and in vivo in a manner dependent on p38 activation, as SB203580, a selective p38α/β inhibitor, as well as p38α shRNA, both near-completely reversed the effects of HS06 on p14ARF, CtBP2, and CSC self-renewal. This regulation of p14ARF and CtBP2 was post-transcriptional in nature. Moreover, reciprocal coimmunoprecipitation studies revealed a novel physical interaction between endogenous pp38 and p14ARF, which resulted in serine phosphorylation of p14ARF in a p38 activation-dependent manner that was critical for regulation of CtBP2 levels and CSC self-renewal. Using a chemotherapy induced cellular dormancy model, we observed that activation/upregulation of p38 and p14ARF prevented escape from dormancy in vitro, whereas increased CtBP2 had the opposite effect. Conclusion: We have discovered a novel p38-p14ARF-CtBP2 axis that plays a dual role in colon cancer CSC self-renewal and cellular dormancy. Agents targeting this pathway e.g., HS06 and CtBP2 inhibitors, represent novel therapeutic strategies to inhibit CSCs and prevent dormancy escape leading to reduced tumor relapse and/or cure. Citation Format: Rio S. Boothello, Nirmita J. Patel, Priyadarshan K. Damle, Chetna Sharon, Kranthi Kumar Chougoni, Umesh R. Desai, Steven R. Grossman, Bhaumik B. Patel. p38-p14ARF-CtBP2 axis as a novel regulator of CSC phenotype and tumor cell dormancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3460.

PO-345 The role of the deubiquitinase USP11 in endocrine-driven breast cancer

IntroductionApproximately 80% of breast cancers overexpress the oestrogen receptor α (ERα) and depend on this key transcriptional regulator for growth. The discovery of novel mechanisms controlling ERα function represent major advances in our understanding of breast cancer progression and potentially offer new therapeutic opportunities. Here, we investigated the role of deubiquitinating enzymes (DUBs), which remove ubiquitin moieties from proteins, in regulating ERα activity in breast cancer.Material and methodsTo identify DUBs involved in ERα transcriptional activity, we performed an RNAi loss-of-function screen using a library of shRNA vectors targeting all 108 known or putative human DUB genes. We found that suppression of the BRCA-associated DUB, USP11, downregulated ERα transcriptional activity in ZR-75–1 cells. Dual luciferase reporter assays and qRT-PCR were used to determine ERα activity in stable ZR-75–1 USP11 knockdown cell lines. PTMScan technology, which allows for enrichment of ubiquitinated proteins and quantitative profiling by mass spectrometry, was used to reveal the ubiquitinome in these cells and results are pending. RNA sequencing (RNA-seq) technology was carried out in estrogen-independent LCC1 USP11 knockdown cells, were USP11 was hypothesised to play a vital role. Finally, immunohistochemistry (IHC) was used to investigate the prognostic relevance of USP11 in breast cancer patients.Results and discussionsKnockdown of USP11 in ZR-75–1 cells decreased ERα transcriptional activity and mRNA expression of ERα target genes. These results were further validated in a HEK293T USP11 CRISPR knockout model with ectopic ERα expression. Interestingly, USP11 expression was upregulated in LCC1 cells, an isogenic, estrogen-independent model derived from MCF-7 cells. Knockdown of USP11 in LCC1 cells decreased the expression of multiple ERα target genes and cell cycle-associated genes, as determined by RNA-seq. IHC staining of a breast cancer tissue microarray (103 ER +patients) and subsequent Kaplan-Meier analysis of this cohort revealed a significant association between high USP11 expression and poor overall (p=0.030) and breast cancer-specific survival (p=0.041). In silico analysis of publically available breast cancer gene expression datasets further supported an association between high USP11 expression and poor prognosis.ConclusionThese results suggest a role for USP11 in ERα transcriptional activity and reveal a novel mechanism as to how this receptor is regulated in breast cancer.

Knockdown of Trnau1ap inhibits the proliferation and migration of NIH3T3, JEG-3 and Bewo cells via the PI3K/Akt signaling pathway

The tRNA selenocysteine 1 associated protein 1 (Trnau1ap, initially named SECp43) is involved in Selenocysteine (Sec) biosynthesis and incorporation into selenoproteins, which play a key role in biological processes, such as embryonic development. We previously reported that downregulation of Trnau1ap inhibited proliferation of cardiomyocyte-like H9c2 cells. However, the effects of Trnau1ap on cell proliferation and migration of embryonic development are not known, and the mechanisms remain elusive. Herein, lentiviral shRNA vectors were transfected in NIH3T3, JEG-3 and Bewo cells (embryonic, trophoblast and placental cells). We found that knockdown of Trnau1ap resulted in reduced expression levels of selenoproteins. The data of Cell Count Kit-8 (CCK-8) assay and wound scratch assay revealed the proliferation and migration rates were reduced in the Trnau1ap-shRNA groups. Furthermore, western blot analysis showed that the phosphorylation level of Akt in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was attenuated. These results indicate that Trnau1ap plays an important role in regulation of cell proliferation and migration through the PI3K/Akt signaling pathway, as well as being essential for embryonic development by regulating the expression of selenoproteins.

RNAi screening of subtracted transcriptomes reveals tumor suppression by taurine-activated GABAA receptors involved in volume regulation.

To identify coding and non-coding suppressor genes of anchorage-independent proliferation by efficient loss-of-function screening, we have developed a method for enzymatic production of low complexity shRNA libraries from subtracted transcriptomes. We produced and screened two LEGO (Low-complexity by Enrichment for Genes shut Off) shRNA libraries that were enriched for shRNA vectors targeting coding and non-coding polyadenylated transcripts that were reduced in transformed Mouse Embryonic Fibroblasts (MEFs). The LEGO shRNA libraries included ~25 shRNA vectors per transcript which limited off-target artifacts. Our method identified 79 coding and non-coding suppressor transcripts. We found that taurine-responsive GABAA receptor subunits, including GABRA5 and GABRB3, were induced during the arrest of non-transformed anchor-deprived MEFs and prevented anchorless proliferation. We show that taurine activates chloride currents through GABAA receptors on MEFs, causing seclusion of cell volume in large membrane protrusions. Volume seclusion from cells by taurine correlated with reduced proliferation and, conversely, suppression of this pathway allowed anchorage-independent proliferation. In human cholangiocarcinomas, we found that several proteins involved in taurine signaling via GABAA receptors were repressed. Low GABRA5 expression typified hyperproliferative tumors, and loss of taurine signaling correlated with reduced patient survival, suggesting this tumor suppressive mechanism operates in vivo.

Adenoviral shRNA vector targeting ribonucleotide reductase M1 (RRM1) has a strong antitumor activity and decrease the drug resistance to GEM in RRM1-overexpressing bladder cells.

e24237Background: Ribonucleotide reductase large subunit (RRM1) is the main enzyme responsible for synthesis of the deoxyribonucleotides and involved in cell proliferation. It is also the primary c...

Thrombotic characteristics of extracellular vesicles derived from prostate cancer cells.

Prostate cancer (PC) patients in advanced stages of the disease have high risk of blood coagulation complications. The procoagulant molecule Tissue factor (TF), and the fibrinolysis inhibitor plasminogen activator inhibitor-1 PAI-1 play important role in this complication. Extracellular vesicles (EV) shed from cancer cells may contribute to the regulation of TF and PAI-1. The procoagulant activity of EV can be associated with the oncogenic and metastatic characteristics of their cells. We have expressed EGFRvIII in DU145 cells to assess the role of this oncogene in the procoagulant activity of EV. The intercellular exchange of TF via EV was assessed by downregulating its expression in DU145 cells using shRNA vector, and determining the transfer of TF via EV enriched with the protein. Two PC cell lines with different metastatic potential were used to assess the correlation between the procoagulant activity of EV and the metastatic potential of PC cells. Photometric assays were used to determine FXa-activity and thrombin generation as indicators for the procoagulant activity of EV. Double-tagged proteinase-activated receptor 1(PAR-1) expressed in CHO cells to assess its activation by EV. The expression of EGFRvIII in DU145 cells led to increased mRNA levels for TF and PAI-1, but the increase in these proteins expression was detected mostly in the EV. EV with enhanced levels of TF protein conferred higher TF procoagulant activity on the acceptor cells by intercellular exchange of this protein. Procoagulant activity of EV, assessed by FXa activity, and thrombin generation, was correlated with the oncogenic and metastatic potential of PC cells. The ability of EV to generate thrombin led to the activation of PAR-1, which was evident by the truncation of tagged-PAR-1. The active oncogene EGFRvIII increases the concentration of TF and PAI-1 in EV. The procoagulant activity of EV is associated with the oncogenic and metastatic characteristics of their PC cells. Also, EV may contribute to the high procoagulant activity in the tumour microenvironment by the intercellular exchange of TF. Finally, through the generation of thrombin, EV can activate PAR-1, which evidently contributes to cancer progression, linking the coagulation system to tumor progression.

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.

Cystatin F involvement in adenosine A2A receptor-mediated neuroinflammation in BV2 microglial cells

Our previous studies have shown adenosine A2A R activation markedly promotes the expression of cystatin F (CF) and exacerbates the white matter lesions induced by hypoxic brain injuries. Thus, we hypothesized that CF was probably involved in neuroinflammation of activated microglia induced by A2A R activation. We transfected the BV2 cells with a CF shRNA vector and examined the production of pro-inflammatory cytokines in hypoxic-BV2 cells in which A2A R was activated or inactivated to confirm this hypothesis. Additionally, we also investigated the probable signaling pathways involved in modulation of A2A R activation on CF expression in hypoxia-activated BV2 cells. Activation of A2A R promoted CF expression, which was significantly increased after the low glucose and hypoxia treatments in BV2 cells. CF gene knockdown markedly inhibited the increase in the expression of pro-inflammatory cytokines induced by A2A R activation in hypoxic-BV2 cells. Furthermore, the increased expression of the CF induced by A2A R activation was remarkably inhibited in hypoxic-BV2 cells administrated with the PKA inhibitor H-89 and the PKC inhibitor staurosporine. Hence, these results indicate that hypoxia BV2 cells highly express CF, which is involved in A2A R activation-mediated neuroinflammation via the PKA/CREB and PKC/CREB or ERK1/2 signaling pathways.

Decreased expression of A20 is associated with ocular Behcet’s disease (BD) but not with Vogt-Koyanagi-Harada (VKH) disease

PurposeA20 is a ubiquitously expressed and inducible cytosolic protein, which plays an important role in the negative regulation of inflammation and immunity. In this study, we investigated the role of...

Abstract P2-05-02: Functional genomic screening identifies ubiquitin-specific protease 11 (USP11) as a novel regulator of ER-alpha transcription in breast cancer

Abstract Approximately 70% of breast cancers overexpress the estrogen receptor α (ERα) and depend on this key transcriptional regulator for growth and differentiation. The discovery of novel mechanisms controlling ERα function represent major advances in our understanding of breast cancer progression and potentially offer attractive new therapeutic opportunities. Here, we investigated the role of deubiquitinating enzymes (DUBs), which act to remove ubiquitin moieties from proteins, in regulating transcriptional activity of ERα in breast cancer. To identify DUBs involved in the regulation of ERα transcriptional activity, we performed an RNAi loss-of-function screen using a library of shRNA vectors targeting all human DUB genes. The DUB library consisted of pools of four non-overlapping shRNAs targeting all 108 known or putative DUBs (432 shRNAs in total). We found that suppression of a number of DUBs markedly repressed or enhanced the activity of an estrogen-response-element (ERE) luciferase reporter following estradiol (E2) stimulation. Of particular interest, suppression of the BRCA2-associated DUB, USP11, was found to down-regulate ERα transcriptional activity. Subsequent validation using two individual siRNAs targeted to USP11 revealed a notable reduction in expression of endogenous ERα target genes in the ZR-75-1 cell line, as quantified using qRT-PCR. Further validation was carried out in a HEK293T USP11 knockout cell line, where reduced activity of an ERE-luciferase reporter was detected when compared to wild-type cells. This phenotype was rescued with a USP11 overexpression vector, both in the presence and absence of E2. Furthermore, USP11 expression was found to be upregulated in the estrogen-independent cell line LCC1 when compared to their parental MCF7 cells. Knockdown of USP11 in LCC1 cells resulted in decreased mRNA expression of a panel of ERα target genes, while RNA-seq revealed a downregulation of several putative ERα target genes and a downregulation of many cell cycle-associated proteins. To support the prognostic relevance of USP11, immunohistochemical staining of a breast cancer tissue microarray (103 ER+ patients available for final analysis) was performed. Kaplan-Meier analysis of this cohort revealed a highly significant association between high USP11 expression and poor overall (p=0.030) and breast cancer-specific survival (p=0.041). In silico analysis of publically available breast cancer gene expression datasets further supported an association between high USP11 mRNA levels and poor prognosis. We observed a significant correlation between high expression of USP11 mRNA in ER-positive patients and poor distant metastasis-free survival (HR 2, CI 1.37-2.91, p=0.00023). This correlation was also significant in ER-positive patients who had received tamoxifen only (HR 2.9, CI 1.63-5.15, p=0.00015). These results suggest a role for USP11 in driving cellular growth and identify USP11 as novel therapeutic target in breast cancer. Citation Format: Dwane L, Das S, Moran B, O'Connor AE, Mulrane L, Dirac AM, Jirstrom K, Crown JP, Bernards R, Gallagher WM, Ní Chonghaile T, O'Connor DP. Functional genomic screening identifies ubiquitin-specific protease 11 (USP11) as a novel regulator of ER-alpha transcription in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-05-02.

The effects of caveolin1 on β cell proliferation.

Our study aims to access the influence of caveolin1 (CAV1) on β cell expression profiles. We knocked down the expression of CAV1 in both NIT-1 cells and islets isolated from C57BL/6J mice using an RNA interference technique, which was realized by the transfer of an shRNA vector targeting CAV1 mRNA into NIT-1 cells or islets through latent virus infection. First, we identified the change in gene expression profiles in islets, in which the CAV1 expression level was down-regulated, as ascertained by mouse gene expression microarray, and the results showed that pathways related to β cell proliferation and pancreatic secretion functions were significantly influenced. The results of MTT demonstrated that the knockdown of CAV1 expression in NIT-1 cells promoted proliferation. The protein array results showed that pro-apoptotic cytokines were down-regulated in the NIT-1 cell line with CAV1 knockdown. These findings suggest that CAV1 might be involved in apoptosis and proliferation regulation in β cells, and therefore could be a potential target for the development of novel therapies for diabetes mellitus.

Doxycycline modulates VEGF-A expression: Failure of doxycycline-inducible lentivirus shRNA vector to knockdown VEGF-A expression in transgenic mice.

Vascular endothelial growth factor-A (VEGF-A) is the master regulator of angiogenesis, vascular permeability and growth. However, its role in mature blood vessels is still not well understood. To better understand the role of VEGF-A in the adult vasculature, we generated a VEGF-A knockdown mouse model carrying a doxycycline (dox)-regulatable short hairpin RNA (shRNA) transgene, which silences VEGF-A. The aim was to find the critical level of VEGF-A reduction for vascular well-being in vivo. In vitro, the dox-inducible lentiviral shRNA vector decreased VEGF-A expression efficiently and dose-dependently in mouse endothelial cells and cardiomyocytes. In the generated transgenic mice plasma VEGF-A levels decreased shortly after the dox treatment but returned back to normal after two weeks. VEGF-A expression decreased shortly after the dox treatment only in some tissues. Surprisingly, increasing the dox exposure time and dose led to elevated VEGF-A expression in some tissues of both wildtype and knockdown mice, suggesting that dox itself has an effect on VEGF-A expression. When the effect of dox on VEGF-A levels was further tested in naïve/non-transduced cells, the dox administration led to a decreased VEGF-A expression in endothelial cells but to an increased expression in cardiomyocytes. In conclusion, the VEGF-A knockdown was achieved in a dox-regulatable fashion with a VEGF-A shRNA vector in vitro, but not in the knockdown mouse model in vivo. Dox itself was found to regulate VEGF-A expression explaining the unexpected results in mice. The effect of dox on VEGF-A levels might at least partly explain its previously reported beneficial effects on myocardial and brain ischemia. Also, this effect on VEGF-A should be taken into account in all studies using dox-regulated vectors.

Silencing of ENO1 by shRNA Inhibits the Proliferation of Gastric Cancer Cells

α-Enolase is a significant subunit of enolase and acts as a glycolytic enzyme responsible for catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate in the anaerobic glycolysis pathway. The research about their role is known little in tumor invasion and metastasis. This research analyzed the effect of α-enolase in proliferation and progression of human gastric cancer. The constructed PLKO.1-ENO1 shRNA vector was transfected into 293 T cells and used to infect gastric cancer cells, MKN45, by using lentivirus method. Negative controls were generated by infection with viruses containing empty vector PLKO.1-scramble-shRNA by the same protocol and using wild-type MKN45 cells as blank control. The silencing effect was confirmed by reverse transcription polymerase chain reaction and Western blotting at messenger RNA and protein levels, respectively. Cell proliferation and chemosensitivity were tested by methyl-thiazolyl-tetrazolium assay. Cell apoptosis was tested by flow cytometry. The cell line α-enolase short hairpin RNA stabling silence α-enolase was successfully constructed. In the α-enolase short hairpin RNA cell lines, messenger RNA and protein expression of α-enolase were significantly lower than those in negative control and blank control groups. The proliferation and clone formation ability were significantly inhibited, cell apoptosis was increased significantly, and the inhibition rate of chemotherapy drugs was increased (P < .05). Our data provide strong evidence that α-enolase short hairpin RNA interference vector can effectively suppress the proliferation and increase chemosensitivity of MKN45 cells, which may provide a novel gene therapy for gastric cancer.

IGF2BP2 Overexpression Indicates Poor Survival in Patients with Acute Myelocytic Leukemia

Background/Aims: IGF2BP2 has been reported to serve as an oncogene in various solid cancers. However, the role of IGF2BP2 in acute myelocytic leukemia (AML) is still unknown. Methods: Public databases Gene Omnibus was used to evaluate the expression of IGF2BP2 in AML patients and healthy controls. In addition, primary cells from these two populations were prepared by Ficoll density centrifugation. Rt-qPCR and western blot were used to detect IGF2BP2 expression in the primary cells from these two populations. Meta-analysis was performed to evaluate the association of IGF2BP2 and prognosis. Lentivirus-based shRNAs were used to knock down IGF2BP2 in AML cell lines KG-1a and Kasumi. Results: We searched the public database Gene Omnibus and analyzed IGF2BP2 expression in both AML and healthy populations. The results showed that IGF2BP2 was overexpressed in AML patients. To verify this phenomenon in fresh human samples, we compared the expression of IGF2BP2 in primary cells from 10 AML patients and 10 healthy controls and found that the expression of IGF2BP2 was upregulated in AML primary cells. More importantly, we observed that IGF2BP2 expression was negatively correlated with the CEBPA mutation status, which is an indicator of good prognosis (RR=0.648, p=0.0001). In addition, IGF2BP2 expression was positively associated with poor prognostic factors, such as the FLT3-ITD mutation (RR=1.198, p=0.0009) and IDH1 mutation (RR=1.354, p=0.0003), as well as intermediate and poor cytogenetic risk (RR=1.214, p=0.0026). To evaluate the prognostic value of IGF2BP2 in AML, we further performed a meta-analysis of 8 studies consisting of 1731 patients and found that IGF2BP2 overexpression was correlated with worse overall survival in AML patients [HR=1.31(1.16-1.49); p = 0.00]. Furthermore, we performed Gene Omnibus and Gene Set Enrichment analyses and found that the genes regulated by IGF2BP2 were mainly enriched in cell proliferation. IGF2BP2 knockdown by 4 different shRNA vectors significantly inhibited the growth of two AML cell lines, KG-1a and Kasumi. Conclusion: Thus, IGF2BP2 may serve as a biomarker to predict the prognosis of AML and as a potential target in AML.

Eyes absent gene (EYA1) is a pathogenic driver and a therapeutic target for melanoma.

EYA1 is a DNA repair enzyme that is induced after DNA damage and is upregulated in melanoma. However, its role in pathogenesis and therapeutic targeting of melanoma is unknown. Our objectives are (1) to study the relationship between EYA1 expression levels and melanoma patients’ clinical pathologic parameters including survival; (2) to investigate its impact on cultured melanoma cells in vitro; and (3) to evaluate EYA1 inhibitors’ potential as a treatment of melanoma.Melanoma tissue microarrays were used to assess EYA1 protein expression in 326 melanoma tissues, and to correlate the expression with patients’ clinical pathological parameters. In addition, retroviral ShRNA vectors were used to silence expression of EYA1 in A375 melanoma cells, and the resultant cells examined for changes in growth, DNA synthesis, and tumor formation in vitro. Lastly, melanoma cells were treated with benzbromarone with or without the BRAF inhibitor vemurafenib.Our results showed that EYA1 protein is low in benign nevi, but is significantly up-regulated in melanoma in situ, and remains high in invasive and metastatic melanoma. In addition, silencing of EYA1 gene expression resulted in decreased proliferation and colony formation. These were associated with decreased cyclin D1 and increased phosphorylated histone protein γH2AX. Finally, treatment with benzbromarone, a specific inhibitor of EYA1, caused significant inhibition of melanoma cell proliferation, and increased sensitivity to the BRAF inhibitor vemurafenib.In conclusion, EYA1 gene is a pathogenic driver in melanoma pathogenesis. Targeting EYA1 may be a valuable strategy for treatment of melanoma.

MiR-221/222 promote cancer stem-like cell properties and tumor growth of breast cancer via targeting PTEN and sustained Akt/NF-κB/COX-2 activation

MicroRNAs (miRNAs) play an important role in regulating cancer stem cell (CSC). Previous studies have shown that microRNA-221/222 (miR-221/222) cluster are involved in the propagation of breast cancer stem cell (BCSC), however, the underlying molecular mechanisms are still not fully understood. In this study, we found that miR-221/222 were overexpressed in highly aggressive breast cancer MDA-MB-231 cells, that are enriched in markers for epithelial-mesenchymal transition (EMT) and BCSCs, than in MCF-7 cells. Phosphatase and tensin homolog (PTEN) was confirmed to be the target of miR-221/222 in breast cancer cells. MiR-221/222 enhanced breast cancer cell growth, migration and invasion by downregulating PTEN. Importantly, both ectopic expression of miR-221/222 and PTEN knockdown increased the mammosphere formation capacity and the expression of the stemness marker ALDH1. MiR-221/222 lentivirus vector infected MCF-7 cells produced larger subcutaneous tumors, while shRNA vector of PTEN showed similar trend. Along with the downregulation of PTEN caused by miR-221/222 in the breast cancer cells and the xenograft tumor tissues, Akt phosphorylation (p-Akt), NF-κB p65 and phosphorylated p65 (p-p65), and cyclooxygenase-2 (COX-2) were all overexpressed compared to the negative control. Taken together, our findings indicate that miR-221/222 play a critical role in the propagation of BCSCs and tumor growth possibly through targeting PTEN, which in turn activating the Akt/NF-κB/COX-2 pathway. MiR-221/222 might represent the potential target of breast cancer therapy.