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shRNA screen

Sijie Lin, Kuancan Liu, Yongchun Zhang, Ming Jiang, Rong Lu, Christopher J Folts, Xia Gao, Mark D Noble, Tingting Zhao, Zhongren Zhou, Xiaopeng Lan, Jianwen Que
Drug repurposing with a better understanding of the underlying mechanism has provided new avenues to find treatment for malignancies. Esophageal adenocarcinoma (EAC) is a rapidly increasing cancer with a dismal 5-year survival rate of <15%. Lack of efficient treatment options contributes to the high mortality rate of EAC. To find new therapy against EAC we performed unbiased drug screening of an FDA-approved drug library and identified that the cardiac glycosides including Ouabain, Digoxin and Digitoxin efficiently inhibit the proliferation of EAC cell lines (OE33 and OE19) both in vitro and in vivo...
August 10, 2018: Cellular Signalling
Chongyang Shen, Qingli Quan, Chuan Yang, Yueqiang Wen, Hong Li
BACKGROUND: Adipose-derived mesenchymal stem cells (ADSCs) have been extensively explored as a promising therapeutic agent due to their differentiation, proliferation and migration abilities. The epigenetic mechanisms that regulate the fate of mesenchymal stem cells (MSCs) have been described in detail. However, the epigenetic modulation of ADSCs proliferation and migration is poorly understood. METHODS: The present study examined histone demethylases roles and expression by RT-PCR, as well as through siRNA screening and ChIP-qPCR assay...
August 9, 2018: Stem Cell Research & Therapy
Biswajit Das, Curtis Dobrowolski, Benjamin Luttge, Saba Valadkhan, Nicolas Chomont, Rowena Johnston, Peter Bacchetti, Rebecca Hoh, Monica Gandhi, Steven G Deeks, Eileen Scully, Jonathan Karn
Unbiased shRNA library screens revealed that the estrogen receptor-1 (ESR-1) is a key factor regulating HIV-1 latency. In both Jurkat T cells and a Th17 primary cell model for HIV-1 latency, selective estrogen receptor modulators (SERMs, i.e., fulvestrant, raloxifene, and tamoxifen) are weak proviral activators and sensitize cells to latency-reversing agents (LRAs) including low doses of TNF-α (an NF-κB inducer), the histone deacetylase inhibitor vorinostat (soruberoylanilide hydroxamic acid, SAHA), and IL-15...
July 30, 2018: Proceedings of the National Academy of Sciences of the United States of America
Shaoli Das, Xiang Deng, Kevin Camphausen, Uma Shankavaram
Summary: Synthetic lethality is a state when simultaneous loss of two genes is lethal to a cancer cell, while the loss of the individual genes is not. We developed an R package DiscoverSL to predict and visualize synthetic lethality in cancers using multi-omic cancer data. Mutation, copy number alteration, and gene expression data from The Cancer Genome Atlas (TCGA) project were combined to develop a multi-parametric Random Forest classifier. The effects of selectively targeting the predicted synthetic lethal genes is tested in silico using shRNA and drug screening data from cancer cell line databases...
July 28, 2018: Bioinformatics
Nathalie Falk, Kristin Kessler, Sinja-Fee Schramm, Karsten Boldt, Elvir Becirovic, Stylianos Michalakis, Hanna Regus-Leidig, Angelika A Noegel, Marius Ueffing, Christian T Thiel, Ronald Roepman, Johann Helmut Brandstätter, Andreas Gießl
Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human PCNT gene are associated with diseases including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptor connecting cilium is unknown. We downregulated Pcnt in the retina ex vivo and in vivo via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the photoreceptor connecting cilium and outer segment and showed a nuclear migration defect...
July 27, 2018: Journal of Cell Science
Yi Pan, Joanna Hung Man Tong, Raymond Wai Ming Lung, Wei Kang, Johnny Sheung Him Kwan, Wing Po Chak, Ka Yee Tin, Lau Ying Chung, Feng Wu, Simon Siu Man Ng, Tony Wing Chung Mak, Jun Yu, Kwok Wai Lo, Anthony Wing Hung Chan, Ka Fai To
BACKGROUND: Patients with colorectal cancer (CRC) have a high incidence of regional and distant metastases. Although metastasis is the main cause of CRC-related death, its molecular mechanisms remain largely unknown. METHODS: Using array-CGH and expression microarray analyses, changes in DNA copy number and mRNA expression levels were investigated in human CRC samples. The mRNA expression level of RASAL2 was validated by qRT-PCR, and the protein expression was evaluated by western blot as well as immunohistochemistry in CRC cell lines and primary tumors...
July 23, 2018: Molecular Cancer
T Fang, X X Cui, N Shen, Y H Li, P Q Xi, Y Zhang, Y Xie, G W Li, F S Tian
Objective: To investigate the mechanisms of telmisartan on delaying the course of type 2 diabetes. Methods: 3T3-L1 preadipocytes were induced to 80% mature adipocytes (control group) and stimulated with 50 ng/ml tumor necrosis factor (TNF-α) for 1 hour (TNF-α group). Then 0.1, 5, 10 μmol/L telmisartan was added to the culture medium for 24 h, respectively(T(0.1, )T(5) and T(10) group). The cells from each group was collected to detect peroxisome proliferator-activated receptors γ (PPARγ) and its phosphorylation level, as well as upstream kinase cell cycle dependent kinase 5 (CDK5) by Western blot...
July 10, 2018: Zhonghua Yi Xue za Zhi [Chinese medical journal]
Tingyue Tu, Mao Yu, Yanping Zhang, Xiafei Shi, Jinhao Xu, Junqing Hu, Jingjing Gan, Wei He, Lei Dong, Jianlin Han, Zhen Huang, Yi Pan, Junfeng Zhang
Hyperactivated macrophages play a key role in the initiation and perpetuation of mucosal inflammation in Crohn's disease (CD). Increasing evidence suggests that the basic helix-loop-helix (bHLH) repressor Twist1 can suppress activation of nuclear factor-κB (NF-κB) and the subsequent production of TNF-α, which are both essential elements of macrophage activation. Thus, developing novel therapeutic strategies to enhance Twist1 expression and to inhibit macrophage activation may be beneficial for CD treatment...
July 17, 2018: Biochemical Pharmacology
Ting Yan, Wentong Liang, Enli Jiang, Aizhu Ye, Qian Wu, Mingrong Xi
Go-Ichi-Ni-San 2 (GINS2), also known as partner of Sld five 2, is involved in the initiation of DNA replication and cell cycle progression. GINS2 is abundantly expressed in a number of malignant solid tumors, including breast cancer, melanoma and hepatic carcinoma. However, the functions of GINS2 in epithelial ovarian cancer (EOC) remain unclear. The aim of the present study was to investigate these functions. GINS2 expression was detected in EOC and normal ovarian tissues using immunohistochemistry. To investigate the functions of GINS2 in EOC, GINS2 expression was stably knocked down in SKOV-3 cells using lentiviral short hairpin RNA (shRNA)...
August 2018: Oncology Letters
Taomei Yang, Haoran Cui, Mingxin Wen, Johannes Zuber, Scott C Kogan, Guangwei Wei
Leukemia is a malignance with complex pathogenesis and poor prognosis. Discovery of noval regulators amenable to leukemia could be of value to gain insight into the pathogenesis, diagnosis and prognosis of leukemia. Here, we conducted a large-scale shRNA library screening for functional regulators in the development of myeloid cells in primary cells. We identified eighteen candidate regulators in the primary screening. Those genes cover a wide range of cellular functions, including gene expression regulation, intracellular signaling transduction, nucleotide excision repair, cell cycle control and transcription regulation...
July 25, 2018: Experimental Cell Research
He Huang, Chongyang Zhang, Bei Wang, Fei Wang, Bin Pei, Chaofei Cheng, Wei Yang, Zhendong Zhao
RNA interference (RNAi) is widely used in gene-knockdown analysis and as a tool to screen host genes involved in viral infection. Owing to the limitations of transducing cells with synthetic small interfering RNAs (siRNA), lentiviral short-hairpin RNA (shRNA) vectors are more widely used. However, we found that stable transduction with lentiviral shRNA vectors inhibited hepatitis C virus (HCV) propagation in human hepatoma cells. We found by miRNA microarray analysis that this inhibition was induced by the alteration of host microRNA (miRNA) expression...
July 11, 2018: Journal of Virology
Sara E Meyer, David E Muench, Andrew M Rogers, Tess J Newkold, Emily Orr, Eric O'Brien, John P Perentesis, John G Doench, Ashish Lal, Patrick J Morris, Craig J Thomas, Judy Lieberman, Edwina McGlinn, Bruce J Aronow, Nathan Salomonis, H Leighton Grimes
We have shown that antagomiR inhibition of miRNA miR-21 and miR-196b activity is sufficient to ablate MLL-AF9 leukemia stem cells (LSC) in vivo. Here, we used an shRNA screening approach to mimic miRNA activity on experimentally verified miR-196b targets to identify functionally important and therapeutically relevant pathways downstream of oncogenic miRNA in MLL-r AML. We found Cdkn1b (p27Kip1 ) is a direct miR-196b target whose repression enhanced an embryonic stem cell-like signature associated with decreased leukemia latency and increased numbers of leukemia stem cells in vivo...
July 11, 2018: Journal of Experimental Medicine
F Liu, W B Xie, L Y Zhou, Y H Liu, W Y Fang, K T Yao
Objective: To observe the effect of knockdown A20 gene expression on the proliferation, invasion and metastasis of human nasopharyngeal carcinoma cell in vivo and in vivo . Methods: Human nasopharyngeal carcinoma cell 5-8F-H3 was transfected with A20-specific shRNA Tet-on inducible plasmid vectors, and A20 silenced cells were screened by Puromycin. Quantitative RT-PCR and Western blot analysis were used to detect the mRNA level and protein of A20. The cell proliferation was detected by cell counting kit-8 (CCK8) and plate colony formation assays...
June 26, 2018: Zhonghua Yi Xue za Zhi [Chinese medical journal]
Shili Xu, Arthur Catapang, Daniel Braas, Linsey Stiles, Hanna M Doh, Jason T Lee, Thomas G Graeber, Robert Damoiseaux, Orian Shirihai, Harvey R Herschman
Background: Precision medicine therapies require identification of unique molecular cancer characteristics. Hexokinase (HK) activity has been proposed as a therapeutic target; however, different hexokinase isoforms have not been well characterized as alternative targets. While HK2 is highly expressed in the majority of cancers, cancer subtypes with differential HK1 and HK2 expression have not been characterized for their sensitivities to HK2 silencing. Methods: HK1 and HK2 expression in the Cancer Cell Line Encyclopedia dataset was analyzed...
2018: Cancer & Metabolism
Alexander Ling, Robert F Gruener, Jessica Fessler, R Stephanie Huang
High-throughput screens in cancer cell lines (CCLs) have been used for decades to help researchers identify compounds with the potential to improve the treatment of cancer and, more recently, to identify genomic susceptibilities in cancer via genome-wide shRNA and CRISPR/Cas9 screens. Additionally, rich genomic and transcriptomic data of these CCLs has allowed researchers to pair this screening data with biological features, enabling efforts to identify biomarkers of treatment response and gene dependencies...
June 25, 2018: Pharmacology & Therapeutics
Xiaorong Li, Jin Zhang, Duan Ma
PPP2R2A is one of the regulatory subunits of the PP2A phosphatase complexes, and previous studies showed that its upregulation promotes cancer cell survival and growth. In this research, we used the tandem affinity purification and the HPLC-Chip-ESI/MS/MS mass spectrometry to screen the PPP2R2A-binding proteins and the results indicated that the GFPT-1/-2 were the potential partners of PPP2R2A. We further validated the interaction between PPP2R2A and GFPT-1/-2 through GST Pull-down, co-immunoprecipitation and immunofluorescence assays...
June 25, 2018: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Konstantin Stoletov, Lian Willetts, Robert J Paproski, David J Bond, Srijan Raha, Juan Jovel, Benjamin Adam, Amy E Robertson, Francis Wong, Emma Woolner, Deborah L Sosnowski, Tarek A Bismar, Gane Ka-Shu Wong, Andries Zijlstra, John D Lewis
Metastasis is the most lethal aspect of cancer, yet current therapeutic strategies do not target its key rate-limiting steps. We have previously shown that the entry of cancer cells into the blood stream, or intravasation, is highly dependent upon in vivo cancer cell motility, making it an attractive therapeutic target. To systemically identify genes required for tumor cell motility in an in vivo tumor microenvironment, we established a novel quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos...
June 14, 2018: Nature Communications
Haixia Wu, Cheng Qian, Chungang Liu, Junyu Xiang, Di Ye, Zhenfang Zhang, Xianquan Zhang
This study was aimed to investigate the effect of Forkhead Box G1 (FOXG1) on the epithelial-mesenchymal transition (EMT) of colorectal cancer (CRC) cells and the underlying mechanism. For this purpose, FOXG1 lentiviral interference (shRNA) plasmid and expression plasmid were constructed. Western blotting was used to analyze the expression of FOXG1 protein in five CRC cells, namely RKO, SW480, SW620, LoVo and DLD-1. The shRNA fragment of FOXG1 (shFOXG1) was designed and synthesized. Recombinant plasmids were obtained with the aid of DNA recombination technique...
May 25, 2018: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Ana Janic, Liz J Valente, Matthew J Wakefield, Leon Di Stefano, Liz Milla, Stephen Wilcox, Haoyu Yang, Lin Tai, Cassandra J Vandenberg, Andrew J Kueh, Shinsuke Mizutani, Margs S Brennan, Robyn L Schenk, Lisa M Lindqvist, Anthony T Papenfuss, Liam O'Connor, Andreas Strasser, Marco J Herold
It has long been assumed that p53 suppresses tumor development through induction of apoptosis, possibly with contributions by cell cycle arrest and cell senescence1,2 . However, combined deficiency in these three processes does not result in spontaneous tumor formation as observed upon loss of p53, suggesting the existence of additional mechanisms that are critical mediators of p53-dependent tumor suppression function3-5 . To define such mechanisms, we performed in vivo shRNA screens targeting p53-regulated genes in sensitized genetic backgrounds...
June 11, 2018: Nature Medicine
Ji-Chao Chen, Xu Wu
OBJECTIVE: To explore the role of fatty acid binding protein 4 (FABP4) in regulating lung cancer cell metastasis and identify miRNAs that target FABP4. METHODS: The expression of FABP4 in lung cancer cells with different metastatic potentials was detected using enzyme-linked immunosorbent assay (ELISA) and Western blotting. The effects of FABP4 knockdown or overexpression by shRNA or a recombinant lentivirus, respectively, on lung cancer cells metastasis were assessed...
May 20, 2018: Nan Fang Yi Ke da Xue Xue Bao, Journal of Southern Medical University
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