Abstract
This research provides a glimmer of hope that the knockout of HCP5 leads to a therapy response to considerably prolong the life of patients with OC. RT-PCR evaluated the expression of lncRNA HCP5 in the ovarian cancer OVCAR-3 cell line. CRISPR knockout cell lines validated by western blot. Small genomic deletions at the targeted locus were induced. CCK-8 colony formation assays were used to analyze the effect of HCP5 knockout on the proliferation capacity of OVCAR-3 cells. Transwell migration and invasion assayed. Furthermore, the Sphere-formation assay isolated the most aggressive population of cancer stem cells. Bioinformatic analysis showed a significant correlation between lncRNA HCP5 up-regulation and OVCAR-3 cell proliferation. The ChIP technique assesses specific sites of interaction between transcription factors and DNA. Real-time PCR assays explored the relationship between HCP5, Hsa-miR-9-5p, CXCR4, CDH1, caspase-3, p53, bcl2 and survivin. PCR carried out amplification of the 448-bp band for sgRNA1 and sgRNA2 after the use of particular primers for HCP5. the number of breast cancer cells that moved to the bottom chamber reduced considerably after transfection with PX461-sgRNA1/2 vectors compared to the Blank control groups (P < 0.05). MTT assay designated growth curves that showed the rate of OVCAR-3 growth was significantly repressed (***P < 0.001) when compared with control OVCAR-3 cells after HCP5 knockdown. Also, the survival results of W.T cells in 24, 48 and 72 h showed 92%, 87% and 85%, respectively. This is while the cells of the CRISPR/Cas9 group in which LncRNA HCP5 was knocked out had 42% (*P < 0.05), 23%(**P < 0.01) and 14% (**P < 0.01) survival, respectively. The expression levels of caspase-3, Hsa-miR-9-5p, P53 genes in the HCP5 deletion of CRISPR/Cas9 group significantly increased than the W.T. control group; the deletion group showed a considerable reduction in HCP5 expression compared to the blank control group (3.6-fold, p < 0.01). Whereas BCL2, SURVIVIN, CXCR4, CDH1 genes expression markedly increased than in HCP5 knockout cells (5.8-fold, p < 0.05). These results indicate that CRISPR/Cas9‐mediated HCP5 disruption on OVCAR-3 cell lines promotes anti‐tumor biomarkers, suppressing ovarian cancer progression. Consistent with these results, HCP5 is one of the most critical lnc for the efficient proliferation and migration of OVCAR-3 cell lines.
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Acknowledgements
The authors would like to thank the staff members of Biotechnology Research Center of Islamic Azad University, Shahrekord Branch, Iran for their help and support. The raw data of the manuscript was deposited at https://doi.org/10.6084/m9.figshare.2474946.
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TPG, RJ, and FE conceived and designed the study. ZM, MK, VB performed the experiments and analyzed the data. RR, and BM wrote the manuscript. All authors read and approved the final manuscript.
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The study was conducted in accordance with the Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran principles. It was approved by the Medical Research Ethics Committee of Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.
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Moradi, Z., Kazemi, M., Jamshidi-Khalifelou, R. et al. CRISPR du-HITI an attractive approach to targeting Long Noncoding RNA HCP5 as inhibitory factor for proliferation of ovarian cancer cell. Funct Integr Genomics 24, 61 (2024). https://doi.org/10.1007/s10142-024-01324-z
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DOI: https://doi.org/10.1007/s10142-024-01324-z