Abstract |
We have used RNA interference (RNAi) screening technology to reveal unknown components of biological signaling pathways including survival mechanisms of estrogen-independent breast cancer cell growth and cancer cell resistance to immune attack. In this chapter, a detailed protocol describing the use of RNAi screening to identify factors important for the proliferation of estrogen-independent MCF7 breast cancer cells will be described. Resistance to therapies that target the estrogen pathway remains a challenge in the treatment of estrogen receptor-positive breast cancer. To address this challenge, small interfering-RNA ( siRNA)-based libraries targeting an estrogen receptor (ER)- and aromatase-centered network, including 631 genes relevant to estrogen signaling, was designed and constructed for RNAi screening. This protocol will include the following parts: (1) selection of RNAi transfection reagent for specific cells; (2) optimization of RNAi screening conditions using Z'-factor; (3) procedure of ER-network gene siRNA library screening using automated machines under optimized experimental conditions; and (4) method of analysis for RNAi screening data to identify specific determinants important for cell proliferation. 46 genes were found to be selectively required for the survival of estrogen-independent MCF7-derived cells.
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Authors | Yong-Wei Zhang, Rochelle E Nasto, Sandra A Jablonski, Ilya G Serebriiskii, Rishi Surana, Joseph Murray, Michael Johnson, Rebecca B Riggins, Robert Clarke, Erica A Golemis, Louis M Weiner |
Journal | Methods in enzymology
(Methods Enzymol)
Vol. 636
Pg. 299-322
( 2020)
ISSN: 1557-7988 [Electronic] United States |
PMID | 32178823
(Publication Type: Journal Article, Research Support, N.I.H., Extramural)
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Copyright | © 2020 Elsevier Inc. All rights reserved. |
Chemical References |
- RNA, Small Interfering
- Receptors, Estrogen
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Topics |
- Breast Neoplasms
(genetics)
- Cell Line, Tumor
- Cell Proliferation
(genetics)
- Female
- Gene Expression Regulation, Neoplastic
- Humans
- MCF-7 Cells
- RNA Interference
- RNA, Small Interfering
(genetics)
- Receptors, Estrogen
(genetics, metabolism)
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