Armstrong Cameron M, Liu Chengfei, Lou Wei, Lombard Alan P, Evans Christopher P, Gao Allen C
Department of Urology, University of California Davis, Sacramento, California.
University of California Davis, UC Davis Comprehensive Cancer Center, Sacramento, California.
Prostate. 2017 Jun;77(9):1020-1028. doi: 10.1002/pros.23358.
Docetaxel is one of the primary drugs used for treating castration resistant prostate cancer (CRPC). Unfortunately, over time patients invariably develop resistance to docetaxel therapy and their disease will continue to progress. The mechanisms by which resistance develops are still incompletely understood. This study seeks to determine the involvement of miRNAs, specifically miR-181a, in docetaxel resistance in CRPC.
Real-time PCR was used to measure miR-181a expression in parental and docetaxel resistant C4-2B and DU145 cells (TaxR and DU145-DTXR). miR-181a expression was modulated in parental or docetaxel resistant cells by transfecting them with miR-181a mimics or antisense, respectively. Following transfection, cell number was determined after 48 h with or without docetaxel. Cross resistance to cabazitaxel induced by miR-181a was also determined. Western blots were used to determine ABCB1 protein expression and rhodamine assays used to assess activity. Phospho-p53 expression was assessed by Western blot and apoptosis was measured by ELISA in C4-2B TaxR and PC3 cells with inhibited or overexpressed miR-181a expression with or without docetaxel.
miR-181a is significantly overexpressed in TaxR and DU145-DTXR cells compared to parental cells. Overexpression of miR-181a in parental cells confers docetaxel and cabazitaxel resistance and knockdown of miR-181a in TaxR cells re-sensitizes them to treatment with both docetaxel and cabazitaxel. miR-181a was not observed to impact ABCB1 expression or activity, a protein which was previously demonstrated to be highly involved in docetaxel resistance. Knockdown of miR-181a in TaxR cells induced phospho-p53 expression. Furthermore, miR-181a knockdown alone induced apoptosis in TaxR cells which could be further enhanced by the addition of DTX.
Overexpression of mir-181a in prostate cancer cells contributes to their resistance to docetaxel and cabazitaxel and inhibition of mir-181a expression can restore treatment response. This is due, in part, to modulation of p53 phosphorylation and apoptosis.
多西他赛是用于治疗去势抵抗性前列腺癌（CRPC）的主要药物之一。不幸的是，随着时间的推移，患者不可避免地会对多西他赛治疗产生耐药性，其疾病将继续进展。耐药性产生的机制仍未完全了解。本研究旨在确定微小RNA（miRNA），特别是miR-181a，在CRPC多西他赛耐药中的作用。
采用实时定量聚合酶链反应（Real-time PCR）检测亲本细胞以及多西他赛耐药的C4-2B和DU145细胞（TaxR和DU145-DTXR）中miR-181a的表达。分别用miR-181a模拟物或反义寡核苷酸转染亲本细胞或多西他赛耐药细胞，调节miR-181a的表达。转染后，在有或没有多西他赛的情况下，48小时后测定细胞数量。还测定了miR-181a诱导的对卡巴他赛的交叉耐药性。使用蛋白质免疫印迹法测定ABCB1蛋白表达，使用罗丹明测定法评估活性。通过蛋白质免疫印迹法评估磷酸化p53的表达，并在有或没有多西他赛的情况下，用酶联免疫吸附测定法（ELISA）在miR-181a表达被抑制或过表达的C4-2B TaxR和PC3细胞中测量细胞凋亡。
与亲本细胞相比，TaxR和DU145-DTXR细胞中miR-181a显著过表达。亲本细胞中miR-181a的过表达赋予了对多西他赛和卡巴他赛的耐药性，而TaxR细胞中miR-181a的敲低使它们对多西他赛和卡巴他赛治疗重新敏感。未观察到miR-181a影响ABCB1的表达或活性，ABCB1蛋白先前已被证明与多西他赛耐药高度相关。TaxR细胞中miR-181a的敲低诱导了磷酸化p53的表达。此外，单独敲低miR-181a可诱导TaxR细胞凋亡，加入多西他赛后可进一步增强凋亡。
前列腺癌细胞中miR-181a的过表达导致其对多西他赛和卡巴他赛耐药，抑制miR-181a表达可恢复治疗反应。这部分是由于p53磷酸化和细胞凋亡的调节。
