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Amber A Bokhari, Laura R Lee, Dewayne Raboteau, Jane Turbov, Isabel V Rodriguez, J Wesley Pike, Chad A Hamilton, G Larry Maxwell, Gustavo C Rodriguez, Viqar Syed
Here, we evaluated the expression of CYP24A1, a protein that inactivates vitamin D in tissues. CYP24A1 expression was increased in advanced-stage endometrial tumors compared to normal tissues. Similarly, endometrial cancer cells expressed higher levels of CYP24A1 than immortalized endometrial epithelial cells. RT-PCR and Western blotting were used to examine CYP24A1 mRNA and protein levels in endometrial cancer cells after 8, 24, 72, and 120 h of exposure to progesterone, progestin derivatives and calcitriol, either alone or in combination...
October 18, 2016: Oncotarget
Binyi Lin, Tianchi Chen, Qijun Zhang, Xiaoxiao Lu, Zhiyun Zheng, Jun Ding, Jinfeng Liu, Zhe Yang, Lei Geng, Liming Wu, Lin Zhou, Shusen Zheng
To investigate the potential oncogene promoting recurrence of hepatocellular carcinoma (HCC) following liver transplantation (LT), throughput RNA sequencing was performed in a subgroup of HCC patients. The up-regulated FAM83D in HCC tissues was found and further verified in 150 patients by real-time PCR and immunohistochemistry. FAM83D overexpression significantly correlated with high HCC recurrence rate following LT and poor HCC characteristics such as high AFP, poor differentiation. Of cancer stem cells (CSCs) markers, CD44 expression was effectively suppressed when FAM83D was knocked down by siRNA...
October 18, 2016: Oncotarget
Yuanyuan Ji, Zhidong Wang, Zongfang Li, Na Huang, Haiyan Chen, Baohua Li, Bo Hui
Emerging evidence confirms that insulin-like growth factor -II (IGF-II), oncogenes C-myc and N-ras are an essential regulator for development and growth in hepatocellular carcinoma (HCC). Although our previous study also indicated that IGF-II might upregulate levels of oncogenes C-myc and N-ras in hepatoma carcinoma cells, the molecular mechanism had not been fully elucidated. Herein, we successfully silenced IGF-II expression in SMCC-7721 cells by small RNA interference. Functional analysis showed that knockdown of IGF-II significantly suppressed growth and proliferation of SMMC-7721 cells and decreased C-myc and N-ras mRNA and protein levels...
October 18, 2016: Cytokine
Haibo Wang, Xiaokun Han, Eric Kunz, M Elizabeth Hartnett
Purpose: This study addresses the hypothesis that age-related stresses upregulate Thy-1 in choroidal endothelial cells (CECs) and contribute to CEC activation and migration, processes important in choroidal neovascularization (CNV). Methods: Measurements were made of Thy-1 protein (Western blot) in CECs and Thy-1 mRNA (real time quantitative PCR) in CECs treated with VEGF, CCL11, or PBS or in RPE/choroids from young or old donors or lasered or nonlasered mice. Immunolabeled Thy-1 in ocular sections was compared from young versus old human donor eyes or those with or without neovascular AMD or from lasered versus nonlasered mice...
October 1, 2016: Investigative Ophthalmology & Visual Science
Fei Wang, Dinglan Wu, Fengrong He, Housheng Fu, Jianbing Xu, Jianxiang Chen, Shufang Zhang, Weifu Wang
PURPOSE: Cofilin 1 is a type of cytoskeletal protein. The overexpression of this gene has been regarded to hold a special relationship with the development and progress of some cancers. However, the detailed position of Cofilin 1 in human bladder cancer has not been investigated intensively. METHODS: In this study, we mainly explored the relationship between human bladder cancer and the expression of Cofilin 1. The expression of Cofilin 1 in bladder cancer tissues and paracancerous tissues of patients was evaluated with quantitative polymerase chain reaction, Western blot, and immunohistochemical staining...
October 21, 2016: Tumori
Yanina S Bogliotti, Marcela Vilarino, Pablo J Ross
Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo...
October 5, 2016: Journal of Visualized Experiments: JoVE
Aeri Yoo, Hyun-Min Park, Soon-Suk Kang, Eun-Soon Kim, Hungwon Tchah, Jae Yong Kim
Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group...
September 27, 2016: Journal of Visualized Experiments: JoVE
I Nakachi, B A Helfrich, M A Spillman, E A Mickler, C J Olson, J L Rice, C D Coldren, L E Heasley, M W Geraci, R S Stearman
Src kinase is recognized as a key target for molecular cancer therapy. However, methods to efficiently select patients responsive to Src inhibitors are lacking. We explored the sensitivity of ovarian cancer cell lines to the Src kinase inhibitor saracatinib to identify predictive markers of drug sensitivity using gene microarrays. Pituitary tumor transforming gene 1 (PTTG1) was selected as a potential biomarker as mRNA levels were correlated with saracatinib resistance, as well as higher PTTG1 protein expression...
October 20, 2016: Clinical and Translational Science
Somayeh Shatizadeh Malekshahi, Vahid Salimi, Ehsan Arefian, Ghazal Fatemi-Nasab, Sarvin Adjaminejad-Fard, Jila Yavarian, Talat Mokhtari-Azad
We attempted to generate siRNAs with two active strands, which can simultaneously knock down the expression of mRNA and viral genomic RNA. In this study, short hairpin RNAs (shRNAs) against N and F genes were used. Expression of F and N mRNA transcripts as well as genomic RNA was determined with relative real-time RT-PCR. The RSV load in infected cell culture supernatant was determined by absolute quantitative real-time PCR. We found that (i) in the presence of shRNA-N, a greater reduction in viral genomic RNA was found; (ii) the level of expression at MOI 0...
October 20, 2016: Molecular Biotechnology
Gang Ren, Nicholas Cairl, Ji Young Kim, Cynthia M Smas
This article describes qPCR analysis for the Adig/Smaf1 gene in multiple in vitro adipocyte differentiation models including white and brown adipogenesis, cell lines and primary cultures. The article also contains qPCR data for transcript levels of Adig/Smaf1 in a wide panel of murine tissues. Expression of Adig/Smaf1 transcript in white and brown adipose tissue in fasted and refed mice is reported and also data for Adig/Smaf1 transcript expression in genetically obese ob/ob mice. Data on the effects of siRNA-mediated knockdown of Srebp1c on Adig/Smaf1 transcript levels in 3T3-L1 adipocytes are shown...
December 2016: Data in Brief
Davod Pashoutan Sarvar, Karim Shamsasenjan, Parvin Akbarzadehlaleh
Mesenchymal stromal/stem cells (MSCs) are involved in tissue homeostasis through direct cell-to-cell interaction, as well as secretion of soluble factors. Exosomes are the sort of soluble biological mediators that obtained from MSCs cultured media in vitro. MSC-derived exosomes (MSC-DEs) which produced under physiological or pathological conditions are central mediators of intercellular communications by conveying proteins, lipids, mRNAs, siRNA, ribosomal RNAs and miRNAs to the neighbor or distant cells. MSC-DEs have been tested in various disease models, and the results have revealed that their functions are similar to those of MSCs...
September 2016: Advanced Pharmaceutical Bulletin
Qian-Qian Luo, Zhong-Ming Qian, Yu-Fu Zhou, Meng-Wan Zhang, Dang Wang, Li Zhu, Ya Ke
The inconsistent of responses of IRP1 and HIF-1 alpha to hypoxia and the similar tendencies in the changes of IRP1 and pCREB contents led us to hypothesize that pCREB might be involved in the regulation of IRP1 under hypoxia. Here, we investigated the role of pCREB in IRP1 expression in HepG2 cells under hypoxia using quantitative PCR, western blot, immunofluorescence, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). We demonstrated that 1) Hypoxia increased pCREB levels inside of the nucleus; 2) Putative CREs were found in the IRP1 gene; 3) Nuclear extracts of HepG2 cells treated with hypoxia could bind to CRE1 and CRE3, and 100-fold competitor of putative CREs could abolish the binding activity to varying degrees; 4) pCREB was found in the CRE1 and CRE3 DNA-protein complexes of EMSA; 5) CRE1 and CRE3 binding activity of IRP1 depended on CREB activation but not on HIF-1; 6) Increased IRP1 expression under hypoxia could be prevented by LY294002; 7) ChIP assays demonstrated that pCREB binds to IRP1 promoter; and 8) HIF-1 and/or HIF-2 siRNA had no effect on the expression of pCREB and IRP1 proteins in cells treated with hypoxia for 8 hours...
2016: International Journal of Biological Sciences
Chihua Wu, Jing Luo
BACKGROUND Long non-coding RNA (lncRNA) UCA1 is an oncogene in breast cancer. The purpose of this study was to investigate the role of UCA1 in tamoxifen resistance of estrogen receptor positive breast cancer cells. MATERIAL AND METHODS Tamoxifen sensitive MCF-7 cells were transfected for UCA1 overexpression, while tamoxifen resistant LCC2 and LCC9 cells were transfected with UCA siRNA for UCA1 knockdown. qRT-PCR was performed to analyze UCA1 expression. CCK-8 assay, immunofluorescence staining of cleaved caspase-9, and flow cytometric analysis of Annexin V/PI staining were used to assess tamoxifen sensitivity...
October 21, 2016: Medical Science Monitor: International Medical Journal of Experimental and Clinical Research
Gary K Scott, David Chu, Ravneet Kaur, Julia Malato, Daniel E Rothschild, Katya Frazier, Serenella Eppenberger-Castori, Byron Hann, Ben Ho Park, Christopher C Benz
ERα phosphorylation at hinge site S294 (pS294) was recently shown to be essential for ER-dependent gene transcription and mediated by an unknown cyclin-dependent kinase (CDK). This study was undertaken to identify the exact CDK pathway mediating pS294 formation, and to determine if this phosphorylation event occurs with, and can be targeted to treat, the ligand-independent growth of breast cancers expressing endocrine-refractory ESR1 mutations. Using a newly developed anti-pS294 monoclonal antibody, a combination of CDK specific siRNA knockdown studies and a broad panel of CDK selective inhibitors against ligand (E2)-stimulated MCF7 cells, we first identified CDK2 as the primary mediator of pS294 formation and showed that CDK2-selective inhibitors like Dinaciclib, but not CDK4/6 inhibitors like Palbociclib, can selectively prevent pS294 formation and repress ER-dependent gene expression...
October 18, 2016: Oncotarget
Hisato Kawakami, Shengbing Huang, Krishnendu Pal, Shamit K Dutta, Debabrata Mukhopadhyay, Frank A Sinicrope
Oncogenic BRAFV600E mutations activate MAP kinase signaling and are associated with treatment resistance and poor prognosis in patients with colorectal cancer (CRC). In BRAFV600E mutant CRCs, treatment failure may be related to BRAFV600E -mediated apoptosis resistance that occurs by an as yet undefined mechanism. We found that BRAFV600E can upregulate anti-apoptotic MCL-1 in a gene dose-dependent manner using CRC cell lines isogenic for BRAF. BRAFV600E -induced MCL-1 upregulation was confirmed by ectopic BRAFV600E expression that activated MEK/ERK signaling to phosphorylate (MCL-1Thr163) and stabilize MCL-1...
October 7, 2016: Molecular Cancer Therapeutics
Takanori Kanazawa, Takahiro Endo, Naoki Arima, Hisako Ibaraki, Yuuki Takashima, Yasuo Seta
This study aimed to build an innovative system to deliver a systemic small interfering RNA (siRNA) treatment for rheumatoid arthritis. We combined arginine-histidine-cysteine based oligopeptide-modified polymer micelles with siRNA targeting the nuclear factor κB subunit, RelA (siRelA). This is a key molecule in the control of inflammation. We tested the cellular uptake of siRNA and its effects on inflammatory cytokine levels in vitro using synoviocytes, and siRNA distribution and therapeutic effects in vivo in mice with collagen-induced arthritis (CIA)...
October 17, 2016: International Journal of Pharmaceutics
M R Williams, T Nakatsuji, J A Sanford, A F Vrbanac, R L Gallo
Bacteria that reside on the skin can influence the behavior of the cutaneous immune system, but the mechanisms responsible for these effects are incompletely understood. Colonization of the skin by Staphylococcus aureus (S. aureus) is increased in atopic dermatitis (AD) and can result in increased severity of the disease. In this study we show that S. aureus stimulates human keratinocytes to increase their endogenous protease activity, including specific increases in trypsin activity. This increased protease activity coincided with increased expression of mRNA for kallikreins (KLKs), with KLK6, 13, and 14 showing the greatest induction after exposure to S...
October 17, 2016: Journal of Investigative Dermatology
Zhao-Hui Wang, Xia-Qing Guo, Qi-Shun Zhang, Jian-Lei Zhang, Yan-Li Duan, Guo-Fei Li, Dong-Lin Zheng
BACKGROUND AND AIM: Long non-coding RNAs have been confirmed to play a critical role in various cancers. In the present study, the effect of long non-coding RNA (lncRNA) CCAT1 on glioma cell proliferation and its potential mechanism were investigated. METHODS AND RESULTS: Real-time PCR results showed that lncRNA-CCAT1 expression was significantly upregulated in glioma cancer tissues and cell lines compared with controls. After inhibiting CCAT1 expression in glioma cell line U251 with siRNA-CCAT1 (si-CCAT1), the cell viability and cell colony formation were decreased, the cell cycle was arrested in G1 phase, and the cell apoptosis was increased...
October 17, 2016: Biochemical and Biophysical Research Communications
Dongkyu Kim, Sook Hee Ku, Hyosuk Kim, Ji Hoon Jeong, Minhyung Lee, Ick Chan Kwon, Donghoon Choi, Sun Hwa Kim
Gene therapy is aimed at selectively knocking up or knocking down the target genes involved in the development of diseases. In many human diseases, dysregulation of disease-associated genes is occurred concurrently: some genes are abnormally turned up and some are turned down. In the field of non-viral gene therapy, plasmid DNA (pDNA) and small interfering RNA (siRNA) are suggested as representative regulation tools for activating and silencing the expression of genes of interest, representatively. Herein, we simultaneously loaded both siRNA (Src homology region 2 domain-containing tyrosine phosphatase-1 siRNA, siSHP-1) for anti-apoptosis and pDNA (hypoxia-inducible vascular endothelial growth factor expression vector, pHI-VEGF) for angiogenesis in a single polymeric nanocarrier and used to synergistically attenuate ischemia-reperfusion (IR)-induced myocardial infarction, which is mainly caused by dysregulating of cardiac apoptosis and angiogenesis...
October 17, 2016: Journal of Controlled Release: Official Journal of the Controlled Release Society
Yoo Seob Shin, Sung Un Kang, Ju Kyeong Park, Yang Eun Kim, Yeon Soo Kim, Seung Joon Baek, Seong-Ho Lee, Chul-Ho Kim
BACKGROUND AND PURPOSE: Aberrant expression of β-catenin is highly associated with progression of various cancers including head and neck cancer (HNC). Green tea is most commonly used beverage in the world and one of the more bioactive compounds is the antioxidant epigallocatechin gallate (EGCG). This study was performed to investigate the mechanism by which EGCG inhibits the growth of HNC, focusing on the modulation of the expression and activity of β-catenin. METHODS: In vitro effects of EGCG on the transcription, translation, or degradation of β-catenin were investigated...
November 15, 2016: Phytomedicine: International Journal of Phytotherapy and Phytopharmacology
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