|Survivin Gene Disruption via CRISPR/Cas9 Induces Apoptosis Through Down-regulation of FBXO5 and RRM2 in Prostate Cancer Cell Line PC3|
|Leila Farhadi1,2, Shohreh Fakhari1, Farzad Soleimani2, Ali Jalili1,2|
|1Cancer and Immunology Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran
2Cellular and Molecular Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran
CJMB 2021; 8: 191-197
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Keywords : Prostate cancer, Survivin, Apoptosis, Crispr/cas9
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Objectives: Prostate cancer (PC) is a complex and heterogeneous disease that arises from both genetic and epigenetic alterations. Survivin acts as a bifunctional controller of apoptosis restraint and is up-regulated in numerous human cancers involving PC. CRISPR/Cas9 was illustrated as a profoundly specific and effective method for altering genes involving oncogenes.
Materials and Methods: Colony polymerase chain reaction (PCR) was performed to identify the transformed colonies. Plasmid purification was performed from desired colonies due to the manufacture plasmid extraction kit protocol. A plasmid involving Cas9 and sgRNAs was co-transfected into PC3 cells using lipofectamine 3000. A vector with no cloned gRNA was utilized for scrambling. The efficacy of transfection and the expression levels of ribonucleotide reductase (RNR) small subunit M2 (RRM2) and FBXO5 were identified by quantitative reverse transcription-PCR. Cell proliferation and apoptosis were assayed by XTT assay and Annexin V-PE/7-AAD, respectively. Data were assayed by utilizing one-way ANOVA and Tukey"s test using SPSS (version 20, USA), and P < 0.05 was considered statistically significant.
Results: The results revealed that lipofectamine 3000 is an efficient approach for delivering on the CRISPR/Cas9 system in PC3 cells. The knocked out of survivin by the CRISPR/Cas9 significantly decreased the proliferation and induced apoptosis of transfected PC3 cells compared to the scrambling vector. Finally, CRISPR/Cas9 systems significantly down-regulated the expression levels of RRM2 and FBXO5 at mRNA levels (fold change 0.406, P = 0.0002).
Conclusions: In general, targeting survivin by CRISPR/Cas9 led to the down-regulation of RRM2 and FBXO5, as well as the induction of apoptosis in PC3 cells. Thus, more research using further PC cell lines and primary cells is necessary.
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