Wang Zhi, Pascal Laura E, Chandran Uma R, Chaparala Srilakshmi, Lv Shidong, Ding Hui, Qi Lin, Wang Zhou
Department of Urology, Xiangya Hospital of Central South University, Changsha, People's Republic of China.
Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Cancer Manag Res. 2020 Jun 10;12:4411-4427. doi: 10.2147/CMAR.S248854. eCollection 2020.
Elongation factor for RNA polymerase II 2 (ELL2) was reported as a putative tumor suppressor in the prostate. ELL2 is frequently down-regulated in prostatic adenocarcinoma specimens, and loss of ELL2 induced murine prostatic intraepithelial neoplasia and enhanced AR-positive prostate cancer cell proliferation. However, the ELL2 gene appears to be amplified in AR-negative neuroendocrine prostate tumors, suggesting a potential oncogenic role for ELL2 in AR-negative prostate cancer cells. In this study, we explored the potential function of ELL2 in PC-3 and DU145, two AR-negative prostate cancer cell lines.
The role of ELL2 in PC-3 and DU145 cells was studied using siRNA-mediated ELL2 knockdown. Genes regulated by ELL2 knockdown in PC-3 cells were identified and analyzed using RNA-Seq and bioinformatics. The expression of representative genes was confirmed by Western blot and/or quantitative PCR. Cell growth was determined by BrdU, MTT and colony formation assays. Cell death was analyzed by 7-AAD/Annexin V staining and trypan blue exclusion staining. Cell cycle was determined by PI staining and flow cytometry.
ELL2 knockdown inhibited the proliferation of PC-3 and DU145 cells. RNA-Seq analysis showed an enrichment in genes associated with cell death and survival following ELL2 knockdown. The interferon-γ pathway was identified as the top canonical pathway comprising of 55.6% of the genes regulated by ELL2. ELL2 knockdown induced an increase in STAT1 and IRF1 mRNA and an induction of total STAT1 and phosphorylated STAT1 protein. Inhibition of cell proliferation by ELL2 knockdown was partly abrogated by STAT1 knockdown. ELL2 knockdown inhibited colony formation and induced apoptosis in both PC-3 and DU145 cells. Furthermore, knockdown of ELL2 caused S-phase cell cycle arrest, inhibition of CDK2 phosphorylation and cyclin D1 expression, and increased expression of cyclin E.
ELL2 knockdown in PC-3 and DU145 cells induced S-phase cell cycle arrest and profound apoptosis, which was accompanied by the induction of genes associated with cell death and survival pathways. These observations suggest that ELL2 is a potential oncogenic protein required for survival and proliferation in AR-negative prostate cancer cells.
RNA聚合酶II 2（ELL2）延伸因子被报道为前列腺中的一种假定肿瘤抑制因子。ELL2在前列腺腺癌标本中经常下调，ELL2缺失可诱导小鼠前列腺上皮内瘤变并增强AR阳性前列腺癌细胞增殖。然而，ELL2基因在AR阴性神经内分泌前列腺肿瘤中似乎发生扩增，提示ELL2在AR阴性前列腺癌细胞中具有潜在致癌作用。在本研究中，我们探讨了ELL2在两种AR阴性前列腺癌细胞系PC-3和DU145中的潜在功能。
使用小干扰RNA（siRNA）介导的ELL2敲低研究ELL2在PC-3和DU145细胞中的作用。使用RNA测序（RNA-Seq）和生物信息学鉴定并分析PC-3细胞中因ELL2敲低而调控的基因。通过蛋白质免疫印迹法（Western blot）和/或定量聚合酶链反应（PCR）确认代表性基因的表达。通过5-溴脱氧尿嘧啶核苷（BrdU）、噻唑蓝（MTT）和集落形成试验测定细胞生长。通过7-氨基放线菌素D（7-AAD）/膜联蛋白V染色和台盼蓝排斥染色分析细胞死亡情况。通过碘化丙啶（PI）染色和流式细胞术测定细胞周期。
ELL2敲低抑制了PC-3和DU145细胞的增殖。RNA-Seq分析显示，ELL2敲低后与细胞死亡和存活相关的基因富集。干扰素-γ途径被确定为最主要的经典途径，由ELL2调控的基因中有55.6%包含在该途径中。ELL2敲低导致信号转导和转录激活因子1（STAT1）和干扰素调节因子1（IRF1）mRNA增加，以及总STAT1和磷酸化STAT1蛋白的诱导。STAT1敲低部分消除了ELL2敲低对细胞增殖的抑制作用。ELL2敲低抑制了PC-3和DU145细胞的集落形成并诱导细胞凋亡。此外，ELL2敲低导致S期细胞周期停滞，抑制细胞周期蛋白依赖性激酶2（CDK2）磷酸化和细胞周期蛋白D1表达，并增加细胞周期蛋白E的表达。
PC-3和DU145细胞中ELL2敲低诱导S期细胞周期停滞和深度凋亡，同时伴随着与细胞死亡和存活途径相关基因的诱导。这些观察结果表明，ELL2是AR阴性前列腺癌细胞存活和增殖所需的潜在致癌蛋白。
