Sina Rahimi1, Amaneh Mohammadi Roushandeh2, Ammar Ebrahimi3, Ali Akbar Samadani4, Yoshikazu Kuwahara5, Mehryar Habibi Roudkenar6. 1. Department of Medical Biotechnology, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran. 2. Department of Medical Biotechnology, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran; Stem cell and Regenerative Medicine Research Center, Guilan University of Medical Sciences, Rasht, Iran. Electronic address: mohammadi_roushandeh@gums.ac.ir. 3. Department of Medical Biotechnology, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran; Stem cell and Regenerative Medicine Research Center, Guilan University of Medical Sciences, Rasht, Iran. 4. Gastroenterology and Liver Diseases Research Center, Guilan University of Medical Science, Rasht, Iran. 5. Division of Radiation Biology and Medicine, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, Sendai, Japan. 6. Department of Medical Biotechnology, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran; Stem cell and Regenerative Medicine Research Center, Guilan University of Medical Sciences, Rasht, Iran. Electronic address: roudkenar@gums.ac.ir.
Abstract
AIMS: Lipocalin 2 (Lcn2/NGAL) belongs to lipocalin superfamily with diverse functions. The precise function of Lcn2, particularly in cancer development, remains to be elucidated yet. In an attempt to knockout of Lcn2 expression by CRISPR/Cas 9 technology in a highly aggressive and invasive prostate cancer cell line and to evaluate the combination therapy with cisplatin (CDDP), this study was conducted. MAIN METHODS: Control CRISPR/Cas9 plasmid and homology-directed repair plasmid or validated human Lcn2 CRISPR/Cas9 KO plasmids were co-transfected into PC3 cells using fugene HD transfection reagent. The stable cells were selected in the presence of puromycin. Correspondingly, knock out of Lcn2 was evaluated by RT-PCR, ELISA, and immunocytochemistry. PC3-Scr (control) and Lcn2-KO (PC3 cells in which lcn2 has been knocked out) were treated with or without cisplatin (CDDP). Cell proliferative ability was measured by WST-1 and colony-formation assays. Apoptosis was evaluated by DAPI staining, in situ cell death detection (TUNEL) assay, and cell death detection ELISA plus methods. The migration capabilities were studied by wound healing/scratch and transwell assays. KEY FINDINGS: Lcn2 knock out in a highly aggressive and invasive cancer cell like PC3 decreased cell proliferation and increased the sensitivity of CDDP. Conspicuously, loss of Lcn2 expression effectively enhanced CDDP-induced apoptosis in PC3 cells. Lcn2 knock out by CRISPR/Cas9 technology decreased the cell migration capacity of PC3 cells as well. SIGNIFICANCE: Lcn2 not only is a valuable and useful biomarker for diagnosis and prognosis of prostate cancer but also and more importantly is a potential novel emerging therapeutic target.
AIMS: Lipocalin 2 (Lcn2/NGAL) belongs to lipocalin superfamily with diverse functions. The precise function of Lcn2, particularly in cancer development, remains to be elucidated yet. In an attempt to knockout of Lcn2 expression by CRISPR/Cas 9 technology in a highly aggressive and invasive prostate cancer cell line and to evaluate the combination therapy with cisplatin (CDDP), this study was conducted. MAIN METHODS: Control CRISPR/Cas9 plasmid and homology-directed repair plasmid or validated humanLcn2 CRISPR/Cas9 KO plasmids were co-transfected into PC3 cells using fugene HD transfection reagent. The stable cells were selected in the presence of puromycin. Correspondingly, knock out of Lcn2 was evaluated by RT-PCR, ELISA, and immunocytochemistry. PC3-Scr (control) and Lcn2-KO (PC3 cells in which lcn2 has been knocked out) were treated with or without cisplatin (CDDP). Cell proliferative ability was measured by WST-1 and colony-formation assays. Apoptosis was evaluated by DAPI staining, in situ cell death detection (TUNEL) assay, and cell death detection ELISA plus methods. The migration capabilities were studied by wound healing/scratch and transwell assays. KEY FINDINGS:Lcn2 knock out in a highly aggressive and invasive cancer cell like PC3 decreased cell proliferation and increased the sensitivity of CDDP. Conspicuously, loss of Lcn2 expression effectively enhanced CDDP-induced apoptosis in PC3 cells. Lcn2 knock out by CRISPR/Cas9 technology decreased the cell migration capacity of PC3 cells as well. SIGNIFICANCE: Lcn2 not only is a valuable and useful biomarker for diagnosis and prognosis of prostate cancer but also and more importantly is a potential novel emerging therapeutic target.
Authors: Ibrahim AlZaim; Aya Al-Saidi; Safaa H Hammoud; Nadine Darwiche; Yusra Al-Dhaheri; Ali H Eid; Ahmed F El-Yazbi Journal: Cancers (Basel) Date: 2022-03-25 Impact factor: 6.639