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shRNA screen

Ugur Eskiocak
Soft agar anchorage-independent growth assays have been commonly used as an indicator of cellular transformation in cell culture. Protocols listed here are optimized to allow for all steps, including plasmid purification, virus production, transduction, and soft agar colony formation, to be performed in 96-well plates. These modifications decrease hands-on time, increase fidelity of the assay, and make it possible to screen 500-1000 short-hairpin RNAs (shRNA) in "one-shRNA-one-well" format in parallel...
2019: Methods in Molecular Biology
Yannan Fan, Sehrish K Bazai, Fabrice Daian, Maria Arechederra, Sylvie Richelme, Nuri A Temiz, Annie Yim, Bianca H Habermann, Rosanna Dono, David A Largaespada, Flavio Maina
BACKGROUND & AIMS: The variety of alterations found in hepatocellular carcinoma (HCC) challenges the identification of functionally relevant genes and their combinatorial actions in tumorigenesis. Deregulation of Receptor Tyrosine Kinases (RTKs) is frequent in HCC, yet little is known about the molecular events that cooperate with RTKs and whether these cooperative events play an active role at the root of liver tumorigenesis. METHODS: A forward genetic screen was performed using Sleeping Beauty transposon insertional mutagenesis to accelerate liver tumour formation in a genetic context in which subtly increased MET RTK levels predispose towards tumorigenesis...
December 5, 2018: Journal of Hepatology
Jacob P Turowec, Esther W T Lau, Xiaowei Wang, Kevin R Brown, Frederic A Fellouse, Kamaldeep K Jawanda, James Pan, Jason Moffat, Sachdev Sidhu
Dysregulation of the ErbB family of receptor tyrosine kinases is involved in the progression of many cancers. Antibodies targeting the dimerization domains of family members EGFR and HER2 are approve cancer therapeutics, but efficacy is restricted to a subset of tumors and resistance often develops in response to treatment. A third family member, HER3, heterodimerizes with both EGFR and HER2 and has also been implicated in cancer. Consequently, there is strong interest in developing antibodies that target HER3, but to date, no therapeutics have been approved...
December 6, 2018: Journal of Biological Chemistry
Yunhong Tian, Jianlin Wu, Cristian Chagas, Yichao Du, Huan Lyu, Yunhong He, Shouliang Qi, Yong Peng, Jiani Hu
BACKGROUND: Accurate and early prognosis of disease is essential to clinical decision making, particularly in diseases, such as HCC, that are typically diagnosed at a late stage in the course of disease and therefore carry a poor prognosis. CDCA5 is a cell cycle regulatory protein that has shown prognostic value in several cancers. METHODS: We retrospectively evaluated 178 patients with HCC treated with curative liver resection between September 2009 and September 2012 at Nanchong Central Hospital in Nanchong, Sichuan Province, China...
November 29, 2018: BMC Cancer
Yuqing Tu, Rui Zuo, Nan Ni, Grant Eilers, Duolin Wu, Yuting Pei, Zuoming Nie, Yeqing Wu, Yuehong Wu, Wen-Bin Ou
Oncogenic KIT or PDGFRA receptor tyrosine kinase (TK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GISTs), and the KIT/PDGFRA kinase inhibitor, imatinib, is the standard of care for patients with metastatic GIST. However, approximately 10% of KIT-positive GIST metastases lose KIT expression at the time of clinical progression during imatinib therapy. In the present report, we performed TK-activation screens, using phosphotyrosine-TK double immunoaffinity purification and mass spectrometry, in GIST in vitro models lacking KIT expression...
November 29, 2018: Cell Cycle
Juan Yang, Ji Zhang, Ting Xu, Yanbai Wang, Zhenhai Wang
Objective To construct a short hairpin RNA (shRNA) lentiviral of Mg2+ /Mn2+ -dependent protein phosphatase 1A (PPM1A) gene and establish mouse brain microglia cell line(BV2) which PPM1A was knocked down stably. Methods According to the Coding sequence (CDS) region of PPM1A gene, two pairs of short hairpin RNA (shRNA) sequences (shRNA1, shRNA2) were designed and cloned into GV493 vector, then it was transformed into competent Escherichia coli DH5α strain and cultured. The positive clones were picked for sequencing...
September 2018: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Gejun Zhang, Zi Yu, Shui Fu, Chengcheng Lv, Qingzhuo Dong, Cheng Fu, Chuize Kong, Yu Zeng
Renal cell carcinoma (RCC), which is one of the most diagnosed urological malignancies worldwide, is usually associated with abnormality in both genetic and cellular processes. In the present study, through analyzing The Cancer Genome Atlas (TCGA) dataset, we screened out ERCC6L as a candidate gene that is potentially related to the development of RCC based on its increased expression in ccRCC tissues compared with normal kidney tissues as well as its possible relevance to cancer prognosis. Evidence indicates that ERCC6L is an indispensable component of mammalian cell mitosis, while it fails to disclose the role of ERCC6L in tumorigenesis...
November 21, 2018: Cancer Gene Therapy
Hai-Xia Qu, Lin Cui, Xin-Ying Meng, Zhi-Jie Wang, Yan-Xin Cui, Yun-Peng Yu, Dong Wang, Xiang-Jun Jiang
In the present study, proteins differentially expressed between gastric cancer tissue and para‑tumoral normal gastric tissues were screened, and the function of the highly expressed protein C1QTNF6 in gastric carcinoma was investigated. The differential expression of mRNAs extracted from the tumor and adjacent tissues was analyzed using GeneChip assay. An AGS si‑C1QTNF6 cell line was constructed using shRNA‑C1QTNF6 lentivirus. The cell invasion and migration ability of C1QTNF6‑knockdown cells were determined by Transwell chamber migration and wound healing assays, respectively...
January 2019: International Journal of Molecular Medicine
Xi Ou, Guang-Tao Zhang, Zhe Xu, Jing-Sen Chen, Yong Xie, Ji-Kui Liu, Xiao-Ping Liu
This study aimed to investigate the effects of desumoylating isopeptidase 2 (DESI2) on tumor cell proliferation, apoptosis and invasion of pancreatic cancer, and to assess the signaling pathway involved. Overexpression and silence of DESI2 were designed and the experiments were divided into 5 groups: a normal control group, an interference control group (shRNA-NC); an interference group (sh-DESI2); an overexpression control group (NC), an overexpression group (DESI2). Quantitative real time polymerase chain reaction (qRT-PCR) was used to screen the appropriate interference sequence...
November 8, 2018: Pathology Oncology Research: POR
Jialei Song, Chunmao Yuan, Jue Yang, Tangjingjun Liu, Yao Yao, Xiao Xiao, Babu Gajendran, Dahai Xu, You-Jun Li, Chunlin Wang, Wuling Liu, Min Wen, David Spaner, Jorge Filmus, Eldad Zacksenhaus, Yiguo Zhang, Xiaojiang Hao, Yaacov Ben-David
E26 transformation-specific (ETS) gene family contains a common DNA-binding domain, the ETS domain, responsible for sequence-specific DNA recognition on target promoters. The Fli-1 oncogene, a member of ETS gene family, plays a critical role in hematopoiesis and is overexpressed in diverse hematological malignancies. This ETS transcription factor regulates genes controlling several hallmarks of cancer and thus represents an excellent target for cancer therapy. By screening compounds isolated from the medicinal plant Dysoxylum binectariferum in China, we identified two chemically related flavagline-like compounds including 4'-demethoxy-3',4'-methylenedioxyrocaglaol and rocaglaol that strongly inhibited Fli-1 transactivation ability...
November 2, 2018: FEBS Journal
Yifan Lian, Jiebin Yang, Yikai Lian, Chuangxing Xiao, Xuezhen Hu, Hongzhi Xu
BACKGROUND: Recent studies highlight pseudogene derived long non-coding RNAs (lncRNAs) as key regulators of cancer biology. However, few of them have been well characterized in pancreatic cancer. Here, we aimed to identify the association between pseudogene derived lncRNA DUXAP8 and growth of pancreatic cancer cells. METHODS: We screened for pseudogene derived lncRNAs associated with human pancreatic cancer by comparative analysis of three independent datasets from GEO...
October 26, 2018: Cancer communications
Eisuke Horigome, Michiru Fujieda, Tadashi Handa, Ayaka Katayama, Masashi Ito, Ami Ichihara, Daiki Tanaka, Navchaa Gombodorj, Shinji Yoshiyama, Arito Yamane, Keiichi Yamada, Jun Horiguchi, Kazuo Shinozuka, Tetsunari Oyama, Masahiko Nishiyama, Susumu Rokudai
Purpose: The identification of genes with synthetic lethality in the context of mutant TP53 is a promising strategy for the treatment of basal-like triple negative breast cancer (TNBC). This study investigated regulators of mutant TP53 (R248Q) in basal-like TNBC and their impact on tumorigenesis. Experimental Design: TNBC cells were analyzed by RNA-seq, and synthetic-lethal shRNA knock-down screening, to identify genes related to the expression of mutant TP53 . A tissue microarray of 232 breast cancer samples, that included 66 TNBC cases, was used to assess clinicopathological correlates of tumor protein expression...
October 2, 2018: Oncotarget
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...
November 2018: Journal of the American Society of Nephrology: JASN
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
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...
December 2018: Molecular Medicine Reports
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
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
Franziska Walter, Aisling O'Brien, Caoimhín G Concannon, Heiko Düssmann, Jochen H M Prehn
In response to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen, three ER transmembrane signaling proteins, inositol-requiring enzyme 1 (IRE1), PRKR-like ER kinase (PERK), and activating transcription factor 6α (ATF6α), are activated. These proteins initiate a signaling 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 signaling proteins were silenced through lentivirus-delivered shRNA constructs...
November 23, 2018: Journal of Biological Chemistry
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
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
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