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shRNA screen

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https://www.readbyqxmd.com/read/30341149/shroom3-fyn-interaction-regulates-nephrin-phosphorylation-and-affects-albuminuria-in-allografts
#1
Chengguo Wei, Khadija Banu, Felipe Garzon, John M Basgen, Nimrod Philippe, Zhengzi Yi, Ruijie Liu, Jui Choudhuri, Miguel Fribourg, Tong Liu, Arun Cumpelik, Jenny Wong, Mubeen Khan, Bhaskar Das, Karen Keung, Fadi Salem, Kirk N Campbell, Lewis Kaufman, Paolo Cravedi, Weijia Zhang, Philip J O'Connell, John Cijiang He, Barbara Murphy, Madhav C Menon
BACKGROUND: We previously showed that the presence of a CKD-associated locus in SHROOM3 in a donor kidney results in increased expression of SHROOM3 (an F-actin-binding protein important for epithelial morphogenesis, via rho-kinase [ROCK] binding); this facilitates TGF-b signaling and allograft fibrosis. However, other evidence suggests Shroom3 may have a protective role in glomerular development. METHODS: We used human data, Shroom3 knockdown podocytes, and inducible shRNA-mediated knockdown mice to study the role of Shroom3 in adult glomeruli...
October 19, 2018: Journal of the American Society of Nephrology: JASN
https://www.readbyqxmd.com/read/30321816/adeno-associated-virus-mediated-rnai-against-mutant-alleles-attenuates-abnormal-calvarial-phenotypes-in-an-apert-syndrome-mouse-model
#2
Fengtao Luo, Yangli Xie, Zuqiang Wang, Junlan Huang, Qiaoyan Tan, Xianding Sun, Fangfang Li, Can Li, Mi Liu, Dali Zhang, Meng Xu, Nan Su, Zhenhong Ni, Wanling Jiang, Jinhong Chang, Hangang Chen, Shuai Chen, Xiaoling Xu, Chuxia Deng, Zhugang Wang, Xiaolan Du, Lin Chen
Apert syndrome (AS), the most severe form of craniosynostosis, is caused by missense mutations including Pro253Arg(P253R) of fibroblast growth factor receptor 2 (FGFR2), which leads to enhanced FGF/FGFR2-signaling activity. Surgical correction of the deformed skull is the typical treatment for AS. Because of constant maldevelopment of sutures, the corrective surgery is often executed several times, resulting in increased patient challenge and complications. Biological therapies targeting the signaling of mutant FGFR2 allele, in combination with surgery, may bring better outcome...
September 22, 2018: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/30320399/knockdown-of-the-hippo-transducer-yap-reduces-proliferation-and-promotes-apoptosis-in-the-jurkat-leukemia-cell
#3
Ran Wu, Hui Yang, Jiangbo Wan, Xiaohui Deng, Linjun Chen, Siguo Hao, Liyuan Ma
Leukemia and lymphoma are common hematological malignancies in children and young adults, which pose a tremendous threat to the survival of these young patients worldwide, despite availability of various effective treatments. The Hippo pathway is a novel‑signaling pathway that regulates organ size, cell proliferation, apoptosis and tumorigenesis. The chief component of this pathway is the transducer yes‑associated protein (YAP) which is over‑expressed in numerous categories of tumors. However, little is known about the effect of YAP in hematological malignancies...
October 15, 2018: Molecular Medicine Reports
https://www.readbyqxmd.com/read/30314410/effects-of-human-cyclooxygenase-2-gene-silencing-on-synovial-cells-of-rheumatoid-arthritis-mediated-by-lentivirus
#4
Ping Leng, Dawei Li, Yi Sun, Yingzhen Wang, Haining Zhang
The aim of the study is to screen the effective shRNA sequence which can silence the human COX-2 expression level in synovial cells of rheumatoid arthritis (RA) patient transfected by the lentivirus. Four pairs of hCOX-2 shRNA were designed and inserted into lentivirus to form pGPHI/GFP/Neo-shRNA vector. The reconstructed virus was transfected into synovial cells derived from RA patients, and then the expression level of hCOX-2 mRNA and the protein of the inflammatory factors including prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), interleukin-1β (IL-1β) and tumour necrosis factor alpha (TNF-α) in the supernatants were examined with real-time PCR and ELISA, respectively...
October 12, 2018: Artificial Cells, Nanomedicine, and Biotechnology
https://www.readbyqxmd.com/read/30292640/hiv-1-latency-is-maintained-by-the-estrogen-receptor
#5
Alexis J Bick, Janet P Hapgood
Persistence of the latent reservoir remains a challenge to curing HIV infection. Using shRNA screening, new insights into the molecular mechanisms underlying latency regulation indicate that the estrogen receptor is a potent repressor of proviral reactivation and may serve as a promising therapeutic target in combination with other latency-reversing agents.
October 3, 2018: Trends in Microbiology
https://www.readbyqxmd.com/read/30287689/er-stress-signalling-has-an-activating-transcription-factor-6%C3%AE-atf6-dependent-off-switch
#6
Franziska Walter, Aisling O'Brien, Caoimhín G Concannon, Heiko Düssmann, Jochen Hm Prehn
In response to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen, three ER transmembrane signalling proteins, inositol-requiring enzyme 1 (IRE1), PRKR-like ER kinase (PERK), and activating transcription factor 6α (ATF6α), are activated. These proteins initiate a signalling and transcriptional network termed the unfolded protein response (UPR), which re-establishes cellular proteostasis. When this restoration fails, however, cells undergo apoptosis. To investigate cross-talk between these different UPR enzymes, here we developed a high-content live cell screening platform to image fluorescent UPR reporter cell lines derived from human SH-SY5Y neuroblastoma cells in which different ER stress signalling proteins were silenced through lentivirus-delivered shRNA constructs...
October 4, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/30262461/cbp-modulates-sensitivity-to-dasatinib-in-pre-bcr-acute-lymphoblastic-leukemia
#7
Jesus Duque-Afonso, Chiou-Hong Lin, Kyuho Han, David W Morgens, Edwin E Jeng, Ziming Weng, Johan Jeong, Stephen H K Wong, Li Zhu, Michael C Wei, Hee-Don Chae, Martin Schrappe, Gunnar Cario, Justus Duyster, Kathleen M Sakamoto, Michael C Bassik, Michael L Cleary
Dasatinib is a multi-tyrosine kinase inhibitor approved for treatment of Ph+ acute lymphoblastic leukemia (ALL), but its efficacy is limited by resistance. Recent preclinical studies suggest that dasatinib may be a candidate therapy in additional ALL subtypes including pre-BCR+ ALL. Here we utilized shRNA library screening and global transcriptomic analysis to identify several novel genes and pathways that may enhance dasatinib efficacy or mitigate potential resistance in human pre-BCR+ ALL. Depletion of the transcriptional co-activator CBP increased dasatinib sensitivity by activating transcription of the pre-BCR signaling pathway previously associated with dasatinib sensitivity...
September 27, 2018: Cancer Research
https://www.readbyqxmd.com/read/30254182/cyclooxygenase-2-influences-response-to-co-targeting-of-mek-and-cdk4-6-in-a-subpopulation-of-pancreatic-cancers
#8
Joel D Maust, Christy L Frankowski-McGregor, Armand Bankhead, Diane M Simeone, Judith S Sebolt-Leopold
The ineffectiveness of chemotherapy in patients with pancreatic cancer highlights a critical unmet need in pancreatic cancer therapy. Two commonly mutated genes in pancreatic cancer, KRAS and CDKN2A, have an incidence exceeding 90%, supporting investigation of dual targeting of MEK and CDK4/6 as a potential therapeutic strategy for this patient population. An in vitro proliferation synergy screen was conducted to evaluate response of a panel of high passage and patient-derived pancreatic cancer models to the combination of trametinib and palbociclib to inhibit MEK and CDK4/6, respectively...
September 25, 2018: Molecular Cancer Therapeutics
https://www.readbyqxmd.com/read/30241742/nanoparticles-based-on-poly-%C3%AE-amino-ester-and-hpv16-targeting-crispr-shrna-as-potential-drugs-for-hpv16-related-cervical-malignancy
#9
Da Zhu, Hui Shen, Songwei Tan, Zheng Hu, Liming Wang, Lan Yu, Xun Tian, Wencheng Ding, Ci Ren, Chun Gao, Jing Cheng, Ming Deng, Rong Liu, Junbo Hu, Ling Xi, Peng Wu, Zhiping Zhang, Ding Ma, Hui Wang
Persistent high-risk HPV infection is the main cause of cervical cancer. The HPV oncogene E7 plays an important role in HPV carcinogenesis. Currently, HPV vaccines do not offer an effective treatment for women who already present with cervical disease, and recommended periodical cervical screenings are difficult to perform in countries and areas lacking medical resources. Our aim was to develop nanoparticles (NPs) based on poly (β-amino ester) (PBAE) and HPV16 E7-targeting CRISPR/short hairpin RNA (shRNA) to reduce the levels of HPV16 E7 as a preliminary form of a drug to treat HPV infection and its related cervical malignancy...
October 3, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/30229833/suppressed-ccl2-expression-inhibits-the-proliferation-of-leukemia-cells-via-the-cell-cycle-protein-cyclin-d1-preliminary-in-vitro-data
#10
S-Y Wu, J Yang, D Hong, P-F Xiao, J Lu, L Gao, Y-X Hu, M Wang, X-J Shao, C-Y Zhou, J-Q Li, J Pan, J Ling, W-Y Gu, R-H Chen, S-Y Hu
OBJECTIVE: Chemokine (C-C motif) ligand 2 (CCL2) is a member of the CC subfamily, which displays chemotactic activity for monocytes and basophils. This molecule plays a very important role in many solid tumors and shows changes in the bone marrow microenvironment. However, its role in acute myeloid leukaemia (AML) is still unclear. MATERIALS AND METHODS: In this study, we established a HL-60 cell line with CCL2 knockdown to explore its effect on leukemogenesis. Lentivirus with CCL2-knockdown was successfully constructed after screening effective CCL2 short hairpin RNA (shRNA) sequences and was transfected into HL-60 cells, which was further validated at the mRNA and protein levels by real-time polymerase chain reaction (PCR) and Western blotting, respectively...
September 2018: European Review for Medical and Pharmacological Sciences
https://www.readbyqxmd.com/read/30218356/unbiased-genetic-screen-to-identify-factors-involved-in-x-chromosome-inactivation-using-a-pooled-bar-coded-shrna-library
#11
Benoit Moindrot, Neil Brockdorff
In mammals, a dosage compensation mechanism exists to equalize gene expression levels between male and female. This process is initiated by Xist RNA, a long noncoding RNA that mediates the transcriptional silencing of a complete chromosome. The kinetics of events occurring on the future inactive X-chromosome has been described in detail over the last 20 years. More recently, parallel studies using advanced biochemical assays and genetic screens identified key factors critical for the silencing cascade. Here, we describe the procedure adopted in one of these studies, an shRNA-based loss-of-function screen in mouse embryonic stem cells (mESCs)...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/30191987/role-of-cxcl12-cxcr4-axis-in-ovarian-cancer-metastasis-and-cxcl12-cxcr4-blockade-with-amd3100-suppresses-tumor-cell-migration-and-invasion-in-vitro
#12
Yan Liu, Chen-Chen Ren, Li Yang, Yi-Ming Xu, Yan-Nan Chen
Ovarian cancer (OC) is a lethal gynecologic tumor, which brings its mortality to the head. CXCL12 and its receptor chemokine receptor 4 ( CXCR4) have been found to be highly expressed in OC and contribute to the disease progression by affecting tumor cell proliferation and invasion. Here, in this study, we aim to explore whether the blockade of CXCL12-CXCR4 axis with AMD3100 (a selective CXCR4 antagonist) has effects on the progression of OC. On the basis of the gene expression omnibus database of OC gene expression chips, the OC differentially expressed genes were screened by microarray analysis...
September 7, 2018: Journal of Cellular Physiology
https://www.readbyqxmd.com/read/30179066/inhibition-of-suv39h1-enhances-transgenic-ifn%C3%AE-2b-gene-expression-in-bcap-37-cells
#13
Liangliang Xie, Lang Lin, Shihai Huang, Ting Yang, Deshun Shi, Xiangping Li
The low expression of exogenous transferred gene limited the application of transgenic animal technology. Suppressor of variegation 3 ∼ 9 homolog 1(SUV39H1) gene plays a prominent role on repressive heterochromatin and transcription. To understand if exogenous transgenic gene expression was affected by SUV39H1 epigenetic modification, in this paper, the effective shRNA fragments targeting SUV39H1 gene were first screened, their roles on expression of exogenous transgenic genes were determined by using Bcap-37 cell line with stable expressing IFNα-2b gene as a model, the preliminary regulation mechanism of SUV39H1 gene was investigated...
September 4, 2018: Animal Biotechnology
https://www.readbyqxmd.com/read/30135191/dose-and-schedule-determine-distinct-molecular-mechanisms-underlying-the-efficacy-of-the-p53-mdm2-inhibitor-hdm201
#14
Sébastien Jeay, Stéphane Ferretti, Philipp Holzer, Jeanette Fuchs, Emilie A Chapeau, Markus Wartmann, Dario Sterker, Vincent Romanet, Masato Murakami, Grainne Kerr, Eric Y Durand, Swann Gaulis, Marta Cortes-Cros, Stephan Ruetz, Therese-Marie Stachyra, Joerg Kallen, Pascal Furet, Jens Würthner, Nelson Guerreiro, Ensar Halilovic, Astrid Jullion, Audrey Kauffmann, Emil Kuriakose, Marion Wiesmann, Michael R Jensen, Francesco Hofmann, William R Sellers
Activation of p53 by inhibitors of the p53-MDM2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. Here, we report distinct mechanisms by which the novel, potent, and selective inhibitor of the p53-MDM2 interaction HDM201 elicits therapeutic efficacy when applied at various doses and schedules. Continuous exposure of HDM201 led to induction of p21 and delayed accumulation of apoptotic cells. By comparison, high-dose pulses of HDM201 were associated with marked induction of PUMA and a rapid onset of apoptosis...
August 22, 2018: Cancer Research
https://www.readbyqxmd.com/read/30120307/mechanistic-role-of-transglutaminase-2-in-focal-adhesions
#15
Evelyn Png, Aihua Hou, Louis Tong
Transglutaminase (TG)-2 interacts with matrix proteins and integrins, forming focal adhesions (FA) to initiate cell migration, thus playing a vital role in wound healing. Previously we showed that TG-2 influenced phosphorylation of paxillin and other FA proteins. Here, we aimed to investigate the molecular mechanism of TG-2 regulation of paxillin. Human corneal epithelial cells expressing shRNA against TG-2 (shTG) and scrambled sequence control (shRNA) were cultured. TG-2 was pulled down by anti-paxillin antibody, but not MAP3K12...
August 17, 2018: Scientific Reports
https://www.readbyqxmd.com/read/30118697/brms1l-suppresses-ovarian-cancer-metastasis-via-inhibition-of-the-%C3%AE-catenin-wnt-pathway
#16
Penglong Cao, Shuai Zhao, Zhigang Sun, Nan Jiang, Yuhong Shang, Yingxin Wang, Juebin Gu, Shijun Li
A low level of breast cancer metastasis suppressor 1-like (BRMS1L) has been implicated in tumour metastasis involving breast cancer and other cancers. It remains unclear whether BRMS1L is involved in epithelial ovarian cancer (EOC) metastasis and what the molecular mechanism of BRMS1L is in suppressing EOC metastasis. In this study, we examined the mRNA expression and protein level of BRMS1L by screening EOC patients. Our results show that BRMS1L expression is downregulated in EOC patients compared to that in normal people and negatively correlated to pathological stages of EOC...
October 1, 2018: Experimental Cell Research
https://www.readbyqxmd.com/read/30108332/unbiased-shrna-screening-using-a-combination-of-facs-and-high-throughput-sequencing-enables-identification-of-novel-modifiers-of-polycomb-silencing
#17
Kenichi Nishioka, Hitomi Miyazaki, Hidenobu Soejima
Polycomb silencing is an important and rapidly growing field that is relevant to a broad range of aspects of human health, including cancer and stem cell biology. To date, the regulatory mechanisms for the fine-tuning of Polycomb silencing remain unclear, but it is likely that there is a series of unidentified factors that functionally modify or balance the silencing. However, a practical gene screening strategy for identifying such factors has not yet been developed. The failure of screening strategies used thus far is probably due to the effect of the loss-of-function phenotypes of these factors on cell cycle progression...
August 14, 2018: Scientific Reports
https://www.readbyqxmd.com/read/30102978/pharmacological-targeting-of-p38-map-kinase-6-map2k6-inhibits-the-growth-of-esophageal-adenocarcinoma
#18
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...
November 2018: Cellular Signalling
https://www.readbyqxmd.com/read/30092848/histone-demethylase-jmjd6-regulates-cellular-migration-and-proliferation-in-adipose-derived-mesenchymal-stem-cells
#19
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
https://www.readbyqxmd.com/read/30061382/estrogen-receptor-1-is-a-key-regulator-of-hiv-1-latency-that-imparts-gender-specific-restrictions-on-the-latent-reservoir
#20
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...
August 14, 2018: Proceedings of the National Academy of Sciences of the United States of America
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