Iranian
Biomedical Journal
Abstract:
Background: LncRNAs contribute to the regulation of transcription and translation of genes involved in cellular growth and apoptosis. UCA1 is an important lncRNA known to be associated with most cancers including breast cancer. This work aims to functionally study the role of UCA1 in cellular proliferation and apoptosis using a CRISPR/Cas9 knockout procedure.
Methods: Using the CHOPCHOP software two sgRNAs were designed to target the UCA1gene. After synthesis, these sgRNAs were separately cloned in two CRISPR vectors to construct PX459-sgRNA1 and PX459-sgRNA2 recombinant vectors. MCF-7 breast cancer cells were co-transfected with both recombinant vectors. The expression levels of pro-apoptotic genes P53, BAX, BAK, and FAS as well as the anti-apoptotic genes BCL2 and SURVIVIN, were evaluated and compared between the knockout cells and the control cells. Such comparison study was also performed for the proliferation and apoptosis rates using the MTT assay and fellow cytometry analysis respectively.
Results: The expression of P53, BAX, BAK, and FAS was significantly increased upon UCA1 knockout but the BCL2 and SURVIVIN genes were downregulated [p<0.05]. The MTT assays and fellow cytometry analyses indicated the lower proliferated and higher apoptosis rates respectively. UCA1 knockout in the MCF-7 cell line leads to decreased proliferation and increased apoptosis rates.
Conclusion: The CRISPR/Cas9 mediated knockout of UCA1 seems to be a highly effective procedure to control cellular proliferation in vitro.
Methods: Using the CHOPCHOP software two sgRNAs were designed to target the UCA1gene. After synthesis, these sgRNAs were separately cloned in two CRISPR vectors to construct PX459-sgRNA1 and PX459-sgRNA2 recombinant vectors. MCF-7 breast cancer cells were co-transfected with both recombinant vectors. The expression levels of pro-apoptotic genes P53, BAX, BAK, and FAS as well as the anti-apoptotic genes BCL2 and SURVIVIN, were evaluated and compared between the knockout cells and the control cells. Such comparison study was also performed for the proliferation and apoptosis rates using the MTT assay and fellow cytometry analysis respectively.
Results: The expression of P53, BAX, BAK, and FAS was significantly increased upon UCA1 knockout but the BCL2 and SURVIVIN genes were downregulated [p<0.05]. The MTT assays and fellow cytometry analyses indicated the lower proliferated and higher apoptosis rates respectively. UCA1 knockout in the MCF-7 cell line leads to decreased proliferation and increased apoptosis rates.
Conclusion: The CRISPR/Cas9 mediated knockout of UCA1 seems to be a highly effective procedure to control cellular proliferation in vitro.
Type of Study: Full Length |
Subject:
Molecular Immunology & Vaccines
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