Effects and mechanism of PLOD2 on oxaliplatin chemoresistance in ovarian cancer
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摘要:
目的：探讨赖氨酸羟基化酶2(procollagenlysine 2⁃xoglutarate 5⁃dioxygenase 2，PLOD2)对卵巢癌奥沙利铂化疗耐药的影响及机制。方法：应用慢病毒转染将PLOD2干扰和过表达质粒转染至卵巢癌A2780和OV1063细胞株，嘌呤霉素筛选出稳定转染质粒的细胞株；qRT⁃PCR和Western blot检测PLOD2表达的变化；CCK⁃8实验、克隆形成实验、流式细胞术检测干扰或过表达PLOD2对卵巢癌细胞奥沙利铂作用下增殖和细胞凋亡的影响；建立异种移植瘤裸鼠模型，观察 PLOD2 表达下调后奥沙利铂对肿瘤生长抑制作用的变化；通过 qRT⁃PCR 和 Western blot 检测 PLOD2 干扰和过表达对乳腺癌耐药蛋白(breast cancer resistance protein，BCRP)、多药耐药蛋白1(multidrug resistance protein 1，MRP1)mRNA水平和蛋白水平的影响。结果：在稳定转染PLOD2 干扰或过表达质粒后，PLOD2 mRNA和蛋白表达水平分别较转染空载体质粒组明显下调或升高。在相同药物浓度条件下，奥沙利铂对卵巢癌PLOD2干扰组细胞的增殖抑制率显著高于对照组，半数抑制浓度IC50下降，克隆形成能力下降， 细胞凋亡率增加；同样，在相同药物浓度条件下，奥沙利铂对卵巢癌PLOD2过表达组细胞的增殖抑制率显著低于对照组，IC50 升高，克隆形成能力增强，细胞凋亡率下降。PLOD2 干扰组奥沙利铂作用下的瘤体体积和瘤重均低于对照组。稳定转染 PLOD2干扰质粒或过表达质粒后，BCRP、MRP1 mRNA和蛋白表达水平明显下调或上调。结论：PLOD2可增强卵巢癌对奥沙利铂的化疗耐药性，这一作用可能与药转蛋白BCRP和MRP1表达上调有关。
Abstract:
Objective：To investigate the effects and mechanism of PLOD2 on oxaliplatin chemoresistance in ovarian cancer. Methods：A2780 cells and OV1063 cells were infected with the lentivirus encoding shRNA against PLOD2 or lentivirus encoding OERNA against PLOD2 respectively. The stably transfected cell lines were selected by puromycin. The expression levels of PLOD2 were detected by qRT⁃PCR and Western blot. CCK⁃8 proliferation test，clone formation assay and flow cytometry were used to detect effects of oxaliplatin on cell proliferation and apoptosis. The xenograft nude mouse tumor model was used to observe inhibition effect of oxaliplatin on tumor growth after PLOD2 downregulation；The mRNA and protein expressions of BCRP and MRP1 were detected by qRT ⁃ PCR and Western blot. Results：The mRNA and protein expressions of shPLOD2(transfected with PLOD2 shRNA)and OEPLOD2(transfected with PLOD2 OERNA)were significantly lower or higher compared with the control groups(transfected with the scrambled shRNA or OERNA). At the same concentration of oxaliplatin，the shPLOD2 group showed higher proliferation inhibition rate，the IC50 value of the shPLOD2 was lower，the ability of clone formation of shPLOD2 group decreased significantly，and the apoptosis rate increased significantly than those in the control group. In contrast，at the same concentration of oxaliplatin，the OEPLOD2 group showed lower proliferation inhibition rate，the IC50 value of the OEPLOD2 was higher，the ability of clone formation of OEPLOD2 group increased significantly，and the apoptosis rate decreased significantly than those in the control group. Compared withthe shControl group，the volume and weight of transplanted tumors in the shPLOD2 group under oxaliplatin treatment were significantly decreased. The mRNA and protein expressions of chemoresistance ⁃ related gene BCRP and MRP1 decreased after interference with PLOD2 expression. However，the mRNA and protein expressions of chemoresistance ⁃ related gene BCRP and MRP1 increased after PLOD2 overexpression. Conclusion：The study suggests that PLOD2 can intensify the oxaliplatin chemoresistance of ovarian cancer， which may be related to the upregulation of BCRP and MRP1 expression.
