Objective: To establish enzalutamide-resistant human prostate cancer cell lines and screen out the lncRNA and mRNA expression profiles associated with enzalutamide resistance.
Methods: Human prostate cancer cell lines LNCAP and C4-2B were cultured with 10 μmol/L enzalutamide for 6 months in vitro for the establishment of enzalutamide-resistant subclones LNCAP-ENZA and C4-2B-ENZA. The IC50 value and enzalutamide resistance index of each cell line were examined by MTT assay, the expressions of enzalutamide-related genes FL-AR, AR-V7 and HnRNPA1 were determined by Western blot, and the lncRNA and mRNA differential expressions of C4-2B and C4-2B-ENZA were detected by high-throughout lncRNA microarray.
Results: Compared with LNCAP and C4-2B, the IC50 values of enzalutamide-resistant subclones LNCAP-ENZA (60.83 μmol/L) and C4-2B-ENZA (88.32 μmol/L) were increased significantly (P < 0.05) and the enzalutamide-resistance indexes of the LNCAP-ENZA and C4-2B-ENZA cells were 4.94 and 4.67, respectively. The expressions of AR-V7 and HnRNPA1 were markedly up-regulated in the LNCAP-ENZA and C4-2B-ENZA cells as compared with those in the LNCAP and C4-2B cells, but that of FL-AR showed no significant change. A total of 1 440 lncRNAs and 1 236 mRNAs were identified as differentially expressed in the C4-2B-ENZA cells.
Conclusions: Enzalutamide -resistant human prostate cancer cell subclones LNCAP-ENZA and C4-2B-ENZA were successfully established and enzalutamide resistance-associated lncRNA and mRNA were identified, which may provide some molecular evidence for the management of enzalutamide-resistant human prostate cancer.
目的： 构建人前列腺癌恩杂鲁胺耐药细胞株并筛选恩杂鲁胺耐药对lncRNA和mRNA表达谱的影响。方法： 体外以恩杂鲁胺10 μmol/L浓度诱导培养人前列腺癌LNCaP及C4-2B细胞株6个月，建立耐药细胞株LNCaP-ENZA 及C4-2B-ENZA。MTT法检测各细胞株IC50值和耐药指数，Western印迹检测前列腺癌恩杂鲁胺耐药相关基因FL-AR、AR-V7、HnRNPA1在各细胞株中的表达。应用高通量芯片技术筛选C4-2B和C4-2B-ENZA细胞中差异表达的lncRNA与mRNA。 结果： 恩杂鲁胺耐药细胞株LNCaP-ENZA和C4-2B-ENZA的IC50值分别为60.83 、88.32 μmol/L，与亲本细胞(分别为12.31、18.96 μmol/L)相比显著增高(P<0.05)，其耐药指数分别为4.94和4.67。Western印迹结果表明LNCaP-ENZA和C4-2B-ENZA中恩杂鲁胺耐药相关基因AR-V7、HnRNPA1的蛋白表达量相较与LNCaP和C4-2B细胞显著升高，而FL-AR的表达量未见显著改变。通过lncRNA芯片技术筛选后发现与C4-2B相比，在C4-2B-ENZA细胞中显著差异表达的lncRNA有1 440个，其中上调倍数最大者为DPP10-AS1(fold change=33.6), 下调最大倍数为G090385(fold change=186.41);mRNA有1 236个 ，其中上调倍数最大者为BCHE（fold change=193.70），下调倍数最大者为ANKRD1（fold change=182.90）。.
Keywords: C4-2B cell line; LNCaP cell line; enzalutamide resistance; lncRNA; mRNA; prostate cancer.