shRNA Plasmid Research Articles

More than mitochondrial biogenesis: alternative roles of PGC-1α in exercise adaptation.

More than mitochondrial biogenesis: alternative roles of PGC-1α in exercise adaptation.

Suppression of lentiviral vector-mediated mTOR gene silencing on growth of human lens epithelial cell in vitro

Background Mammalian target of raparrmycin (mTOR) acts as a critical molecular link between growth signals and the processes underlying cell growth. The overexpression of mTOR in human lens epithelial cells (HLECs) is associated with posterior capsular opacification (PCO). Applying the mTOR shRNA transfection technique to silence mTOR in HLECs is significant for the targeting treatment of PCO. Objective This study was to investigate the function and morphology of HLECs infected by mTOR shRNA lentiviral vector. Methods 293T cells were transfected by recombinant plasmid containing mTOR shRNA, blank plasmid and PBS to prepare different lentiviral vectors. HLECs were routinely cultured and were divided into the mTOR shRNA transfected 1, 2, 3 groups, blank plasmid group and PBS group, and different lentiviral vectors were added in the medium to infect the cells. The expression of mTOR protein in HLECs was detected using Western blot to determine mTOR gene silence, and the group with the highest efficiency of silence was screened as the sequent experiment group. The proportion of HLECs in the G1 phase was analyzed by flow cotymetry. The expressions of α-smooth muscle actin (α-SMA) and E-Cadherin proteins in the cells were assayed by Western blot. The viability of HLECs was assessed by cell counting kit-8 (CCK8) test. Results Successfully packaged lentivirus were found in the supernatant after infection of mTOR shRNA in the mTOR shRNA transfected 1, 2, 3 groups with the green fluorescence in >80% cells, while green fluorescence protein (GFP) response was absent in the blank plasmid group and the PBS group. The HLECs of mTOR shRNA transfected groups and blank plasmid group grew well due to the resistance for puromycin of plasmid, however, more dead cells were found in the PBS group. Western blot revealed the weakest expression of mTOR mRNA in the cells in the mTOR shRNA transfected 3 group. The expressing bland of α-SMA in the HLECs was weaker in the mTOR shRNA transfectd 3 group than that in the blank plasmid group or PBS group, and the expression of E-Cadherin was stronger in in the mTOR shRNA transfectd 3 group than that in the blank plasmid group or PBS group. The absorbance of the mTOR shRNA transfectd 3 group was significantly declined in comparison with the blank plasmid group and the PBS group, with significant difference among the three groups in both 1 hour and 2 hours after experiment (all at P<0.05). The proportion of cells in G1 phase was (60.00±1.78)% in the mTOR shRNA transfected 3 group, which was significantly increased in comparison with (53.48±1.86)% in the blank plasmid group and (53.02±1.49)% in the PBS group (F=18.910, P=0.002). Conclusions mTOR gene is silenced after mTOR shRNA plasmid transfected into HLECs. This procedure can invert the epithelial-mesenchymal transition (EMT), inhibit cell growth and put off cellular cycle in HLECs in vitro. Key words: Lentiviral vector; Short hairpin RNA; Posterior capsule opacification; Mammalian target of raparrmycin; Epithelial-mesenchymal transition; Lens, crystal; Epithelial cells; Gene silence

Lentivirus-mediated RNA interference suppresses DNA binding protein A expression and inhibits proliferation of colorectal cancer cells

Objective To investigate the effects of lentivirus-mediated RNA interference(RNAi)targeting DNA binding protein A(dbpA)on the proliferation of colorectal cancer cell line SW620. Methods The lentiviral vector shRNA-dbp A was constructed and verified by polymerase chain reaction(PCR)and DNA sequencing. SW620 cells were transfected with shRNA- dbp A plasmid, nontargeting shRNA plasmid, or empty plasmid.Forty- eight hours after the transfection, the cells were examined for dbp A expression using Western blotting. Seventy- two hours after transfection, flow cytometry was used to detect the cell apoptosis and cell cycle changes. The cell growth inhibition rate was detected by methyl thiazol tetrazolium(MTT)assay,and then clone formation was detected. Results PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting dbpA gene was successfully inserted into the lentiviral vector.shRNA- dbpA transfection resulted in obviously reduced expression of dbpA in SW620 cells. After transfection,the apoptosis rate of shRNA- dbpA- transfected cells were increased to(26.60±0.38)%,significantly higher than that in cells transfected with the nontargeting plasmid or the empty plasmid[(12.54±0.25)% and(4.46±0.19)%, respectively,P<0.01]; the cell number in G0/G1 phase increased while that in G2/M phase decreased in shRNA- dbpA- transfected cells. The growth inhibition test indicate that the OD value of the fifth day in shRNA- dbpA group was 1.183±0.226, significantly lower than that in the other two groups(5.295± 0.282 and 10.207±0.383, respectively,P<0.01).The colony formation number is(37±3)、(64±5)and (175±10) respectively,shRNA-dbpA is significantly higher than that in the other two groups(P<0.01). Conclusion Lentivirus-mediated RNAi targeting dbpA can effectively suppress the expression of dbpA in SW620 cells and decrease the proliferation of the colorectal cells. Key words: DNA binding protein A; RNA interference; SW620 cells; Proliferation

NRP-1/shRNA silencing suppresses nasopharyngeal carcinoma cell growth in vitro and in vivo

To investigate the effect of RNA interference (RNAi) targeting NRP-1 gene on the growth and proliferation of nasopharyngeal carcinoma (NPC) CNE-2Z cells. Short hairpin RNA (shRNA) plasmids targeting NRP-1 were designed and synthesized. These plasmids were respectively transfected into human NPC CNE-2Z cells and xenograft tumors in nude mice. The expressions of Fluorescein-labeled plasmids in NPC CNE-2Z cells and xenograft tumors were observed by fluorescence microscopy. Cell proliferation was detected by MTT assay. The inhibitory effects on target genes were evaluated with RT-PCR and Western blotting, respectively. The inhibitory effect of plasmids with the most effective sequence on xenograft tumors in nude mice was observed. CNE-2Z cell proliferation was significantly inhibited by NRP-1/shRNA silencing. RT-PCR showed NRP-1 mRNA expression was significantly decreased. Western blotting demonstrated the NRP-1/shRNA silencing can effectively inhibit the expression of target proteins in CNE-2Z cells. After six weeks, there were significant differences in the mean tumor volumes in nude mice between plasmid group and negative control group [(0.599±0.002) vs (1.141±0.013) cm(3), P<0.05] or blank control group [(0.599±0.002) vs (1.165±0.308) cm(3), P<0.05], and the inhibitory rate of tumor growth was 48.6%. RNA interference targeting NRP-1 can remarkably inhibit the growth of CNE-2Z cells in vitro and in vivo.

Endoglin silencing has significant antitumor effect on murine mammary adenocarcinoma mediated by vascular targeted effect.

New targets and therapeutic approaches for vascular targeted strategies in oncology are continuously explored. Endoglin, a co-receptor of TGF-β, is a known target, however, its silencing with vector-based RNA interference technology has not been evaluated yet. Therefore, in our study, we assembled plasmid DNA coding for shRNA against endoglin, and used gene electrotransfer as a delivery method to determine its antitumor and vascular targeted effects. In vitro and in vivo data provide evidence of vascular targeted effects of endoglin silencing. The vascular targeted action of endoglin silencing could be described as a result of two separated effect; antiangiogenic and vascular disrupting effect. This was first supported by in vitro data; predominantly by reduction of proliferation and tube formation of endothelial cells. In the TS/A murine mammary carcinoma model, in which the tumor cells do not express endoglin, reduced tumor growth and number of vessels were observed. Quick destruction of existing activated blood vessels at the site of tumor cells' injection and sustained growth of tumors afterwards was observed in tumors that were growing in dorsal window chamber by intravital microscopy. This observation supports both vascular disrupting and antiangiogenic action. In conclusion, the results of our study provide evidence of endoglin as a valid target for cancer therapy and support further development of plasmid shRNA delivery, which have prolonged antitumor effect, especially in combined schedules.

β-catenin knockdown inhibits pituitary adenoma cell proliferation and invasion via interfering with AKT and gelatinases expression

Pituitary adenomas are among the most prevalent forms of intrinsic brain tumors. Although most pituitary adenomas are benign, some of them may become invasive and cause significant mass effect and hormonal dysfunction. We have previously shown that β-catenin is overexpressed in human pituitary adenomas and its level correlates to tumor grades. In the present study, we further investigated the role of β-catenin in pituitary adenoma cell proliferation and invasion in vitro. Stable β-catenin knockdown pituitary adenoma cell line was created by transfecting mouse growth hormone pituitary adenoma GT1.1 cells with β-catenin shRNA plasmid. Cell proliferation and invasion were assessed using CCK-8 kit and transwell assay, respectively. Our data demonstrated that knockdown of β-catenin with shRNA significantly inhibited the proliferation and invasion of GT1.1 cells. In β-catenin shRNA transfected cells, the expression of AKT, STAT3, cyclin D1 and CDK4 were significantly suppressed, which accounted for the observed growth retardation following β-catenin shRNA transfection. Moreover, β-catenin shRNA transfection led to a drastic reduction in MMP-2/9 secretion into the conditioned media, which might be responsible for the reduced invasiveness of β-catenin shRNA-transfected pituitary adenoma cells. These results indicate that β-catenin may regulate the expression of AKT, STAT3, cyclin D1, CDK4 and MMP-2/9 to promote pituitary adenoma cell proliferation and invasion.

HGPAEs vector-based RNAi inhibits the expression of gene encoding MyD88 in rat liver tissues

Objective To investigate the inhibitory effects of histidine grafted poly (β-amino ester) (HGPAEs) vector-based RNA interference (RNAi) on the expression of gene encoding myeloid differentiation factor 88 (MyD88) in rat liver tissues. Methods The sequence of small hairpin RNA (shRNA) was designed based on the genetic information of MyD88. HGPAEs vector was constructed and coupled with shRNA plasmid targeting MyD88 to construct pMyD88-HGPAEs vector. Rats were divided into five groups including control group, HGPAEs treatment group, pHK-HGPAEs treatment group, shRNA treatment group and pMyD88-HGPAEs treatment group. The rats in each group were transfected with the corresponding interventions through portal vein injection. Real-time PCR and Western blot assay were performed to detect the expression of MyD88 in liver tissues 3 days after transfection. Results The pMyD88-HGPAEs vector was successfully constructed. The expression of gene encoding MyD88 was inhibited in rats from shRNA treatment group and pMyD88-HGPAEs treatment group (P<0.05). Significantly decreased expression of gene encoding MyD88 at mRNA and protein levels were observed in rats from pMyD88-HGPAEs treatment group as compared with those from other groups (P<0.01). Conclusion HGPAEs vector might be used as a potential gene carrier. The expression of gene encoding MyD88 in rat liver tissues could be significantly inhibited through portal vein injection of pMyD88-HGPAEs vector. This study provided evidences for further research on pMyD88-HGPAEs vector in a high responder model of rat orthotopic liver transplantation. Key words: Histidine grafted poly (β-amino ester); Myeloid differentiation factor 88; RNA interference

Expression of TLR5 in different types of non-small cell lung cancer cell lines and its activation mechanism

背景与目的已有的研究表明：Toll样受体5（toll-like receptor 5, TLR5）在肿瘤起始和发展中发挥重要作用。我们前期研究发现，TLR5在非小细胞肺癌（non-small cell lung cancer, NSCLC）组织中高表达，但其在NSCLC高表达后的信号通路活化情况的研究并不多见。本研究旨在探讨TLR5在不同NSCLC细胞株上的表达，及其在NSCLC细胞中活化的机制。方法用免疫荧光、RT-PCR和Western blot方法检测TLR5在三种不同NSCLC细胞株中的表达。分别用0 μg/mL、0.01 μg/mL、0.1 μg/mL、1 μg/mL、5 μg/mL、10 μg/mL的鞭毛蛋白刺激，用NF-κB荧光素酶报告基因质粒瞬时转染后，检测细胞内NF-κB荧光素酶的活性。选择TLR5表达最高的SPC-A-1细胞株为实验对象，选择0.1 μg/mL的鞭毛蛋白，分别用0 μg/mL、0.01 μg/mL、0.1 μg/mL、1 μg/mL、10 μg/mL的TLR5抗体抑制通路活化，检测细胞内NF-κB荧光素酶的活性，验证TLR5活化通路。构建TLR5-shRNA，转染SPC-A-1细胞48 h后，以0.1 μg/mL浓度鞭毛蛋白分别刺激SPC-A-1细胞及转染的SPC-A-1细胞，在刺激0 min、10 min、30 min、60 min，用Western blot方法比较TLR5信号通路因子p-IKBα、p-ERK1/2、p-JNK、IKBα、ERK1/2的变化。结果TLR5在肺腺癌细胞株SPC-A-1中呈高表达，且主要表达在细胞膜上。三种细胞株中SPC-A-1细胞NF-κB荧光素酶的活性最高，呈浓度依赖性，0.1 μg/mL鞭毛蛋白即可明显增强NF-κB荧光素酶的活性（P < 0.05）；而SPC-A-1细胞内NF-κB荧光素酶的活性可被TLR5抗体抑制，与TLR5抗体浓度负相关（P < 0.05）。与0 min相比较，SPC-A-1细胞内p-IKBα、p-ERK1/2、p-JNK水平在鞭毛蛋白刺激10 min即明显增高，30 min达到高峰，60 min开始下降（P < 0.05），且与10 min和60 min组相比，p-IKBα、p-ERK1/2、p-JNK水平在30 min增高（P < 0.05）；而IKBα、ERK1/2的水平无明显变化（P > 0.05）。以适合浓度鞭毛蛋白刺激转染的SPC-A-1细胞，p-IKBα、p-JNK蛋白均未检出，IKBα、ERK1/2蛋白的水平无明显变化（P > 0.05），p-ERK1/2蛋白水平随着时间延长明显增高（P < 0.05）。结论外源性配体鞭毛蛋白可激活NSCLC细胞株TLR5蛋白，启动下游信号通路，可能与NSCLC的发生发展有关。

Potential roles of EZH2, Bmi-1 and miR-203 in cell proliferation and invasion in hepatocellular carcinoma cell line Hep3B

To investigate the potential roles of enhancer of zeste homolog2 (EZH2), Bmi-1 and miR-203 in cell proliferation and invasion in hepatocellular carcinoma (HCC) cell line Hep3B. A total of 73 patients who underwent surgical resection at Fuzong Clinical Medical College of Fujian Medical University were enrolled in this study. Hep3B cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C. Vectors that containing cDNA of the EZH2 gene or miR-203 targeted shRNA plasmid were constructed, and then transfected into Hep3B cells. The mRNA expression of miR-203, EZH2, and Bmi-1 was analyzed using quantitative real-time polymerase chain reaction analysis, and the protein levels of EZH2 and Bmi-1 were detected by Western blot analysis. Effect of EZH2 or miR-203 on cell proliferation was observed by methyl thiazolyl tetrazolium assay, and cell apoptosis was assessed using flow cytometry. Besides, effect of EZH2 or miR-203 on tumor cell invasion was detected using Transwell assay. The mRNA levels of EZH2 and Bmi-1 in HCC tissues and in Hep3B cells were significantly higher compared with those in normal samples (P < 0.01), while miR-203 level was significantly lower in HCC tissues (P < 0.01). Hep3B cells transfected with EZH2-shRNA or miR-203-shRNA showed lower expression levels of EZH2 and Bmi-1 (P < 0.05). Compared with controls, Hep3B cells transfected with EZH2-shRNA had relative slow cell proliferation, indicating that low expression of EZH2 and Bmi-1 and overexpression of miR-203 could inhibit Hep3B cell proliferation (P < 0.05). The average apoptosis rate of Hep3B cells transfected with EZH2-shRNA vector was about 18.631%, while that of Hep3B cells transfected with shRNA vector was about 5.33%, suggesting that EZH2 was down-regulated by transfecting with EZH2-shRNA, and the down-regulated EZH2 contributed to the cell apoptosis. Low expression of EZH2 and Bmi-1 and overexpression of miR-203 could reduce Hep3B cell invasion (P < 0.05). Our study suggests that EZH2 and Bmi-1 are up-regulated while miR-203 is down-regulated in Hep3B cells. MiR-203 may contribute to the metastasis and enhance apoptosis of HCC cells by regulating EZH2 and Bmi-1. Our study may provide a theoretical basis for metastasis of HCC and targeted therapy of HCC.

Nuclear SMAD2 Restrains Proliferation of Glioblastoma

Aims: Although TGFβ receptor signaling has been shown to play a role in regulation of the growth and metastasis of glioblastoma multiforme (GBM), the downstream pathway through either SMAD2 or SMAD3 has not been elucidated. In this study, we investigate whether nuclear SMAD2 can restrain the proliferation of glioblastoma. Methods: A total of 23 resected specimens from GBM patients were collected for SMAD2 detection. Human GBM cell line A172, U87mg, D341m and Hs683 were maintained in Dulbecco's modified Eagle's medium and transfected with SMAD2 and SMAD3 shRNA plasmids. Gene expression was detected by RT-qPCR and Western and cell growth were detected by MTT assay. Results: Our results showed that the phosphorylated SMAD2 (pSMAD2, the nuclear and functional form of SMAD2) levels in GBM were significantly lower than the paired normal brain tissue in patients. Depletion of SMAD2, but not SMAD3, significantly abolished the inhibitory effects of TGFβ1 on the growth of GBM cells, possibly through pSMAD2-mediated increases in cell-cycle inhibitor, p27. Conclusion: Our data suggest that TGFβ/SMAD2 signaling cascades restrains growth of GBM.

New technology and resources for cryptococcal research

Rapid advances in molecular biology and genome sequencing have enabled the generation of new technology and resources for cryptococcal research. RNAi-mediated specific gene knock down has become routine and more efficient by utilizing modified shRNA plasmids and convergent promoter RNAi constructs. This system was recently applied in a high-throughput screen to identify genes involved in host-pathogen interactions. Gene deletion efficiencies have also been improved by increasing rates of homologous recombination through a number of approaches, including a combination of double-joint PCR with split-marker transformation, the use of dominant selectable markers and the introduction of Cre-Loxp systems into Cryptococcus. Moreover, visualization of cryptococcal proteins has become more facile using fusions with codon-optimized fluorescent tags, such as green or red fluorescent proteins or, mCherry. Using recent genome-wide analytical tools, new transcriptional factors and regulatory proteins have been identified in novel virulence-related signaling pathways by employing microarray analysis, RNA-sequencing and proteomic analysis.

Aberrant levels of histone H3 acetylation induce spermatid anomaly in mouse testis

Histone acetylation is involved in the regulation of chromatin structure and gene function. We reported previously that histone H3 acetylation pattern is subject to dynamic changes and limited to certain stages of germ cell differentiation during murine spermatogenesis, suggesting a crucial role for acetylation in the process. In the present study, we investigated the effects of hyper- and hypo-acetylation on spermatogenesis. Changes in acetylation level were induced by either in vivo administration of sodium phenylbutyrate, a histone deacetylase inhibitor, or by knockdown of histone acetyltransferases using short hairpin RNA plasmids transfection. Administration of sodium phenylbutyrate induced accumulation of acetylated histone H3 at lysine 9 and lysine 18 in round spermatids, together with spermatid morphological abnormalities and induction of apoptosis through a Bax-related pathway. Knockdown of steroid receptor coactivator 1, a member of histone acetyltransferases, but not general control of amino acid synthesis 5 nor elongator protein 3 by in vivo electroporation of shRNA plasmids, reduced acetylated histone H3 at lysine 9 in round spermatids, and induced morphological abnormalities. We concluded that the proper regulation of histone H3 acetylation levels is important for spermatid differentiation and complex chromatin remodeling during spermiogenesis.

Short hairpin RNA targeting Notch2 inhibits U87 human glioma cell proliferation by inducing cell cycle arrest and apoptosis in vitro and in vivo.

Notch signaling has been reported to be oncogenic or tumor suppressive, depending on the tissue context. To investigate the effects of Notch2 knockdown on U87 human glioma cell proliferation in vitro and in vivo, and the associated mechanisms, U87 cells were stably transfected with p green fluorescent protein (GFP)-V-RS Notch2 short hairpin (sh) RNA plasmid and pGFP-V-RS scramble-shRNA plasmid. The former was referred to as the Notch2-shRNA group and the latter as the negative-shRNA group. mRNA and protein expression, cell proliferation, cell cycle and apoptosis were measured by reverse transcription-polymerase chain reaction, western blot analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis and flow cytometry using propidium iodide, respectively. Tumor volume, tumor weight and cumulative survival rate were determined in a nude mouse xenograft tumor model. Notch2 mRNA and protein expression in the Notch2-shRNA group were reduced by 87.6 and 94.5% compared with the negative-shRNA group (P<0.001). Notch2 knockdown significantly inhibited U87 cell proliferation after three days of culture (P<0.05). Notch2 silencing induced cell cycle arrest at G0/G1 phase by upregulation of p21 protein expression and downregulation of mini chromosome maintenance complex 2 and cyclin-D1 protein expression. Furthermore, knockdown of Notch2 also induced U87 cell apoptosis. On day 50 after inoculation, tumor weight in the Notch2-shRNA group was significantly lower than that in the negative-shRNA group (0.55±0.10 vs. 1.23±0.52 g; P<0.01). The cumulative survival rate was significantly longer in the Notch2-shRNA group compared with the negative-shRNA group (log rank test P=0.01). In conclusion, Notch2 silencing inhibited U87 glioma cell proliferation by inducing cell cycle arrest and apoptosis in vitro and in vivo. Thus, Notch2 may be a key therapeutic target for the treatment of glioma.

Abstract 4393: miR-138 overexpression most effectively enhanced efficacy of apigenin to impair cell cycle progression and induce apoptosis in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cells in vivo

Abstract Malignant neuroblastoma, which mostly occurs in children, remains incurable with conventional chemotherapeutic agents. Innovative therapeutic strategies must be developed to control the growth of this childhood malignancy. Inhibition of cyclin D3 may induce cell cycle arrest and apoptosis in human malignant neuroblastoma. Also, inhibition of the catalytic component of human telomerase reverse transcriptase (hTERT) may induce apoptosis in many human cancers including malignant neuroblastoma. Decrease in expression of the tumor suppressor microRNA-138 (miR-138) is often associated with increases in expression of hTERT and cyclin D3. We hypothesized that miR-138 overexpression could be a better therapeutic strategy than hTERT knockdown to potentiate pro-apoptotic effect of apigenin (APG), a plant-derived flavonoid, in human malignant neuroblastoma. We found that transfection of miR-138 mimics was more powerful than transfection of hTERT shRNA plasmid in potentiating efficacy of APG for decreasing cell viability in human neuroblastoma SK-N-DZ and SK-N-BE2 cells. We then examined comparative effectiveness of different treatment strategies in reducing tumor growth and inducing apoptosis in SK-N-DZ and SK-N-BE2 xenografts in athymic nude mice. Combination of miR-138 overexpression and APG treatment showed better efficacy than combination of hTERT knockdown and APG treatment in reducing tumor growth and inducing cell death in both neuroblastoma xenograft models. According to the microRNA database (miRDB), miR-138 may target and inhibit expression of cyclin D3 so as to impair cell cylce progression. Combination of miR-138 overexpression and APG treatment caused the highest inhibition of expression of cyclin D3, CDK4, and CDK6, which were required for G1/S transition in cell cycle. Down regulation of cyclin D3 could inhibit phosphorylation of the tumor suppressor Rb and impair cell cycle progression. We delineated that apoptosis occurred with induction of molecular components of extrinsic and intrinsic pathways in the xenografts. Combination of miR-138 overexpression and APG treatment most effectively activated caspase-8 and cleaved Bid to tBid to induce extrinsic pathway of apoptosis and also raised Bax:Bcl-2 ratio to trigger intrinsic pathway of apoptosis with mitochondrial release of Smac into the cytosol to neutralize a set of endogenous inhibitor-of-apoptosis proteins such as Survivin so as to facilitate activation of caspase-3. Activation of both calpain and caspase-3 caused proteolysis of α-spectrin to generate calpain-specific 145 kD SBDP and caspase-3-specific 120 kD SBDP, respectively, in course of apoptosis in the xenografts. In conclusion, miR-138 overexpression was more powerful than hTERT knockdown in enhancing pro-apoptotic effect of APG for controlling growth of human malignant neuroblastomas in vivo. Citation Format: Mrinmay Chakrabarti, Walden Ai, Swapan K. Ray. miR-138 overexpression most effectively enhanced efficacy of apigenin to impair cell cycle progression and induce apoptosis in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cells in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4393. doi:10.1158/1538-7445.AM2014-4393

Abstract 1409: The BP1 homeobox gene is dysregulated in triple negative breast cancer

Abstract Background: Triple negative breast cancers (TNBC) lack the expression of ER, PR and HER2 receptors, making targeted treatments against these receptors ineffective. We are studying BETA PROTEIN 1 (BP1), a member of homeobox gene family, which we cloned and showed is activated in 80% of invasive breast cancers; its expression correlates with breast cancer progression and invasion. The BP1 gene is located on 17q21, a region that we showed is altered in 30% of breast tumors. BP1 protein has been shown to regulate genes including BCL2, BRCA1, VEGF and c-MYC. Also BP1 mRNA and protein are highly expressed in ER and PR negative and clinically aggressive tumors. Thus we hypothesize that BP1 may play a role in TNBC. Methods: To delineate pathways in TNBC cells that are dysregulated by BP1 expression, we studied the 5 TNBC parental cell lines, 2 non-TNBC cell lines and a stably overexpressed BP1 TNBC cell line. Microarray analysis was performed in all these cell lines and the data obtained was verified using qPCR and ELISA. A BP1 shRNA plasmid was stably transfected into MDA-MB-231 cell lines to generate sh-MDA-MB-231 cell lines with about 80% knockdown of BP1 protein. ELISA was performed to verify the effects of BP1 expression on target genes. Formalin-Fixed, Paraffin-Embedded (FFPE) tissues of TNBC were analyzed by array-CGH and confirmed by Taqman copy number (using a BP1-TAM specific probe) and FISH (using our constructed BP1-BAC probe) assays. Results: All of the TNBC cell lines had statistically more BP1 protein (pBP1) than non-TNBC cell lines, suggesting that TNBC tumors may have higher levels of pBP1 than non-TNBC tumors. Gene expression microarrays in the TNBC cell lines revealed up/down regulation of several genes in comparison to the normal breast cell lines, including the IL-6 and IL-8 genes, which were significantly upregulated in TNBC cells overexpressing BP1. ELISA assays confirmed a direct correlation between elevated levels of IL-6 in conditioned media and overexpression of BP1. Knockdown of BP1 led to a decrease in secreted IL-6. Studies are underway to establish the mechanism by which these pathways are regulated by BP1. Increased BP1 copy number was observed in 37% (25/67) of the clinical cases and all (8/8) of TNBC cell lines by array-CGH. Confirmation of these changes by FISH and TaqMan copy number assays revealed increased copy number in 22% (18/82) and 23% (9/38) of the clinical cases, respectively. Protein expression analysis by IHC is currently being conducted to determine if BP1 increased DNA copy number may be one of the mechanisms leading to pBP1 overexpression. Conclusions: Both BP1 copy number and protein expression are dysregulated in TNBC. Microarray analysis in TNBC cell lines have revealed important cancer related genes and oncogenic pathways that may be upregulated by BP1, suggesting that BP1 could be a valuable molecular marker and therapeutic target in this aggressive form of breast cancer. Citation Format: Saurabh Kirolikar, Mandeep Gill, Silma Pereira, Svetlana Ghinbouschi, Luciane Cavalli, Patricia Berg. The BP1 homeobox gene is dysregulated in triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1409. doi:10.1158/1538-7445.AM2014-1409

CAPN5 gene silencing by short hairpin RNA interference.

BackgroundThe purpose of this project was to identify short hairpin RNA (shRNA) sequences that can suppress expression of human CAPN5 in which gain-of-function mutants cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). We created HEK293T cells that stably express an ADNIV disease allele, CAPN5-p.R243L. Transfection protocols were optimized for neuroblastoma SHSY5Y cells. The gene silencing effect of four different shRNA plasmids that target CAPN5 was tested. RNA and protein expression was determined using quantitative RT-PCR and immunoblot analysis.FindingsTwo of four shRNA plasmids reduced mutant CAPN5 RNA in a stable cell line. Similar knockdown was observed in SH-SY5Y cells that natively express CAPN5. Lactose dehydrogenase assays showed that down-regulation of CAPN5 was not cytotoxic.ConclusionsCAPN5 expression can be suppressed by shRNA-based RNA interference. Further testing in ADNIV models will determine the potential of gene silencing as a strategy to treat, delay, or prevent blindness in ADNIV patients.

908P - Nuclear Expression of Bcl10 Has a Role in the Regulation of Cell Growth of Ovarian Cancer Through the Activation of Nf-&Kgr;B‐Dependent Cyclin D1 Signaling

ABSTRACT Aim: We previously characterized the molecular linkage that directs both BCL10 overexpression and nuclear translocation in response to inflammation-related NF-kB signaling pathway in breast cancer cell line, lymphoma cell line, and cervical cancer line (J Biol Chem 2006;281:167-75, Blood 2008;112:2927-34; Gynecol Oncol 2012;126:245-51). Since NF-kB activation has been shown to occur in the pathogenesis of ovarian cancer, we sought to investigate whether BCL10 possesses clinical significance in relation to ovarian cancer. Methods: Two ovarian cancer cell lines (SK-OV3, and CAOV-3) with overexpressing BCL10 were used in this study. The proliferation effect of BCL10 and its mechanism were evaluated in vitro and in vivo. The DNA binding activity of NF-kB was determined by the luciferase assay. The expression of BCL10, NF-kB, and cyclin D1 in tumor cells from an array of 100 tissue samples was examined using immunohistochemical staining. Results: We found that inhibition of BCL10 with BCL10 small interfering RNA (siRNA) significantly reduced the cell growth rate of these two ovarian cancer cell lines through the G1 arrest. BCL10 siRNA treatment inhibited the expression of p-IKKb and p-IkB, and also down-regulated NF-kB activation and its downstream cell cycle protein, cyclin D1. Furthermore, we used BCL10 short hairpin RNA (shRNA) plasmids to transfect two ovarian cancer cell lines, and found that the down-regulation of BCL10 inhibited the tumor growth rate of ovarian cancer and prolonged the survival rate in xenograft model of immunodeficient mice (SK-OV3: 38.7 ± 4.4 in WT; 59.2 ± 6.2 in BCL10 knockdown, and CAOV-3: 37.2 ± 7.8 in WT; 76.7 ± 11.6 in BCL10 knockdown). In tissue samples, we detected that serous cystadenocarcinoma had a higher rate of BCL10 nuclear expression (35/49 [71.4%]) than mucinous cystadenocarcinoma (4/18 [22.2%]) and endometrioid adenocarcinoma (16/28 [57.1%]) (P = 0.001). BCL10 nuclear expression was also closely associated with tumor grading (P = 0.017). Furthermore, nuclear expression of BCL10 was closely associated with nuclear NF-kB expression (P Conclusions: Our findings indicate that nuclear BCL10 plays an important role in controlling the growth and progression of ovarian cancer cells through NF-kB dependent cyclin D1 regulation. Disclosure: All authors have declared no conflicts of interest.

Expression of netrin-1 receptors in retina of oxygen-induced retinopathy in mice.

BackgroundNetrin-1 has been reported to promote retinal neovascularization in oxygen-induced retinopathy (OIR). However, netrin-1 receptors, which may mediate netrin-1 action during retinal neovascularization, have not been characterized. In this study, we investigated netrin-1 receptor subtype expression and associated changes in the retinas of mice with OIR.MethodsC57BL/6J mice were exposed to 75±2% oxygen for 5 days and then returned to normal air to induce retinal neovascularization. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used to examine the expression of netrin-1 receptor subtypes in the mouse retinas. Double staining of netrin-1 receptor subtypes and isolectin B4 was used to determine the location of the netrin-1 receptor subtypes in the retinas. Inhibition of retinal neovascularization was achieved by UNC5B shRNA plasmid intravitreal injection. Retinal neovascularization was examined by fluorescein angiography and quantification of preretinal neovascular nuclei in retinal sections.ResultsRT-PCR results showed that, except for UNC5A, netrin-1 receptor subtypes UNC5B, UNC5C, UNC5D, DCC, neogenin, and A2b were all expressed in the retinas of OIR mice 17 days after birth. Western blots showed that only UNC5B expression was significantly increased on that day, and immunofluorescence results showed that only UNC5B and neogenin were expressed in retinal vessels. Treatment of OIR mice with the UNC5B shRNA plasmid dramatically reduced neovascular tufts and neovascular outgrowth into the inner limiting membrane.ConclusionsUNC5B may promote retinal neovascularization in OIR mice.

Effects of interfering RNA of α-1,3-galactosyltransferase and nuclear factor-kappa B on cardiac xenotransplantation

BackgroundBoth α1,3-galactosyltransferase (α1,3GT) and nuclear factor kappa B (NF-κB) play an important role in the immune response of xenotransplantation. The purpose of this study is to investigate the effect of RNAi of α1,3GT and NF-κB on xenotransplantation. MethodsLentiviral vectors with shRNA focusing on α1, 3GT and RelA were constructed. The effect of RNAi on α1, 3GT and RelA was examined in vitro and in vivo. Additionally, we established a mouse-to-rat heterotopic cardiac xenotransplantatic model (donor hearts transplanted to the right side of the neck in rat) using a modified cuff technique. The survival time of donor hearts in each group was monitored. The expressions of α1, 3GT and RelA mRNA, Galα1,3Gal antigen, and RelA protein were detected by RT-PCR, immunofluorescence, and Western blot respectively. The expressions of C3, IgM, IgG, NK, macrophages, ICAM-1 on donor hearts were examined by immunohistochemistry. ResultsHigh titer lentiviral vectors carrying α1, 3GT and RelA shRNA plasmids had a high and stable transfection rate on EOMA in vitro. In vivo, heart tissue showed a much stronger GFP expression and significant decrease in target gene mRNA expression and protein expression in shRNA interfering groups (p<0.01). The survival time of α1,3GTi-3 and dual lentiviral vector groups was significantly longer than other groups. The mRNA expression levels of α1,3GT and RelA, as well as Galα(1,3)Gal and RelA proteins, in α1,3GTi-3, RelAi-3, and dual lentiviral vector groups were downregulated and compared to other groups (p<0.01). The depositions of C3, IgM, IgG in α1,3GTi-3 group and dual lentiviral vector group were less than other groups (p<0.01). The infiltration of NK, macrophages and ICAM-1 in α1,3GTi-3 group and dual lentiviral vector group was more than other groups (p<0.01), but the infiltration of NK, macrophages and ICAM-1 in dual lentiviral vector group was less than α1,3GTi-3 group (p<0.01). ConclusionsOur results indicate that RNAi technology with lentiviral vectors is an effective method to transmit exogenous genes into living bodies and stably inhibit the expression of target genes. Moreover, siRNA targeting the α1,3GT gene was found to control the immune process and obviously prolong the survival time of donors, whereas knocking down NF-κB alone showed no differences. However, the RNAi of NF-κB can make the infiltration of macrophages and natural killer cells decrease, and the expression of ICAM-1 in the xenografts also decreases, contributing to the restraining of AVR.

Effects of immune inhibitory receptor LILRB2 on proliferation and apoptosis of leukemia cell lines

Objective To explore the expression, function, and relevant mechanism of the immune inhibitory receptor of leukocyte immunoglobulin-like receptors subfamily B member 2( LILRB2) in human leukemia cell lines and to provide new clues for the targeting treatment of leukemia. Methods T he expressions of LILRB2 in leukemia cell lines, such as T HP1, U937, and K562 were detected by the flow cytometry and W estern blotting. T he shRNA plasmids specific for the knockdown of LILRB2( with GFP tag) was constructed and by which the virus was packaged. T HP1 cells with high expression of LILRB2 were then infected by the virus. C ell lines with steady expression of GFP+were obtained by the flow cell sorting. T he knockdown efficiency of shRNA at both mRNA level and protein level was detected. T he changes of cell proliferation and apoptosis at different time points were observed and the relevant molecular mechanisms, such as changes of downstream regulatory signals, were analyzed after the knockdown of LILRB2. Results All three cell lines expressed LILRB2 and the expression level of LILRB2 of T HP1 cells was the highest. T he Real-T ime PC R and W estern blotting findings showed that among four constructed shRNAs plasmids, shLILRB2-717 and shLILRB2-1312 significantly downregulated the expression of LILRB2. T he inhibition of LILRB2 expression in T HP1 cells dramatically decreased the proliferation and significantly increased the apoptosis. T he results of the flow cytometry indicated that the apoptotic rates of cells of the shLILRB2-717 group and shLILRB2-1312 group at the early stage were( 7. 90 ±1. 61) % and( 24. 80 ± 1. 32) %, much higher than( 3. 96 ± 0. 48) % of the control group( Scramble group)( P 0. 05). T he apoptotic rates of cells of the two groups at the late stage were( 13. 80 ± 0. 98) % and( 23. 60 ±1. 03) %, much higher than( 10. 80 ± 0. 48) % of the Scramble group( P 0. 05). T he knockdown of LILRB2 led to significant down-regulation of expressions of SHP1 and p-SHP1, which indicated that LILRB2 might regulate the proliferation and apoptosis of leukemia cell lines through SHP1. Conclusion Immune inhibitory receptor LILRB2 can be expressed in a variety of leukemia cell lines. T he inhibition of LILRB2 expression can lead to significant down-regulation of SHP1 and p-SHP1 in T HP1 cells and mass apoptosis, which suggests that SHP1 may positively regulate the development of leukemia.