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RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA
Published on: April 10, 2018
Long non-coding RNA NKILA regulates expression of HSP90α, NF-κB and β-catenin proteins in the MCF-7 breast cancer
Mehmet Ali Tibatan1, Evren Önay Uçar2
1Department of Biotechnology, Institute of Graduate Studies in Sciences, Istanbul University, Istanbul, Turkey. matibatan@gmail.com.
Abstract:
Non-coding RNAs are increasingly being investigated and have shown great potential for diagnosis, prognosis and treatment of cancer. Thus, we have investigated a possible regulatory mechanism between NF-κB suppressor-NKILA, and HSP90, NF-κB, and β-catenin molecules in MCF-7 breast cancer cells. HSP90 is an important stress protein and together with β-catenin and NF-κB molecules can be responsible for cancer cell development. However, there is no comprehensive data available on the novel molecule NKILA unlike for HSP90, β-catenin and NF-κB alone. Therefore, we suggest there might be a correlation between NKILA and these proteins. To investigate the NKILA role on HSP90, NF-κB and β-catenin proteins we inhibited the NKILA by using transfection in MCF-7 breast cancer cells. NKILA-siRNA transfected cells were incubated for 5 h. Then, cells were collected and proteins were extracted to be separated by SDS-PAGE. The aforementioned proteins of siRNA transfected group were evaluated by quantification and comparison of their relative expression levels with the control group by immunoblotting. Results showed, HSP90 and NF-κB/p105, NF-κB/p65 and NF-κB/p50 subunits significantly increased while the level of β-catenin decreased after NKILA inhibition. For the first time we have demonstrated that HSP90 and expression levels of beta-catenin are associated with NKILA levels which may be closely related to the canonical NF-κB pathway in MCF-7 cells. These novel findings may have significant implications in cancer cells development and possibly present important hints for the future studies of the cancer cell targeted therapy.
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