miR-140 靶向抑制PD-L1 表达增强宫颈癌HeLa 和Caski 细胞对奥沙利铂的敏感性

摘要] 目的：探究miR-140 能否通过靶向抑制程序性细胞死亡蛋白-1（programmed death-1, PD-L1）表达增强宫颈癌（cervical cancer, CC）细胞对奥沙利铂的敏感性。方法：采用qPCR检测miR-140 在人正常宫颈细胞、CC细胞株及其奥沙利铂耐药细胞株中的表达情况；采用miR-140 模拟物转染细胞，CCK-8 法检测CC正常细胞株及其奥沙利铂耐药细胞株的增殖情况、克隆形成实验检测细胞的克隆形成率。通过Starbase 及TargetScan 在线分析软件预测miR-140 与PD-L1 的靶向结合位点，并通过双荧光素酶报告基因验证miR-140 与PD-L1 的靶向结合关系。采用Annexin V FITC/PI 双染色法和Wb法分别检测过表达miR-140 或同时过表达PD-L1，且在使用奥沙利铂处理后CC细胞的凋亡、迁移及凋亡相关蛋白的表达情况。建立裸鼠CC移植瘤模型，检测miR-140 加强肿瘤对奥沙利铂处理敏感性的效果。结果：miR-140 在奥沙利铂耐药性CC细胞中表达显著下调（P<0.01），过表达miR-140 能够明显上调奥沙利铂耐药CC细胞对奥沙利铂的敏感性（P<0.05），抑制CC细胞的增殖及克隆形成（均P<0.01）。miR-140与PD-L1 3''-UTR靶向结合并抑制其表达。过表达miR-140 显著促进CC细胞的迁移及凋亡（P<0.01），过表达miR-140 的同时过表达PD-L1 明显减弱了miR-140 对CC细胞迁移的抑制及细胞凋亡的促进作用（均P<0.05）。小鼠异体移植瘤模型验证了miR-140 促进肿瘤对奥沙利铂的敏感性。结论：miR-140 通过靶向抑制PD-L1 表达增加CC细胞对奥沙利铂的敏感性，上调miR-140或抑制PD-L1的表达再与奥沙利铂联合处理有可能作为奥沙利铂耐药CC治疗的新策略。

miR-140 inhibits PD-L1 expression to enhance sensitivity of cervical cancer HeLa and Caski cells to oxaliplatin

Abstract] Objective: To investigate whether miR-140 could increase the sensitivity of cervical cancer (CC) to oxaliplatin by downregulating the expression of programmed death-1 (PD-L1). Methods: qPCR was used to analyze miR-140 expression in normal human cervical cells, CC cells and oxaliplatin-resistant CC cells. Cells were transfected with miR-140 mimic, and then, the proliferation of CC cells and oxaliplatin-resistant CC cells was detected by using CCK-8 assay, and the colony formation rate of CC cells was obtained by using colony formation assay. Starbase and TargetScan were used to predict the targeted binding site of miR-140 and PD-L1, and the influence of miR-140 on the expression of PD-L1 was validated by dual luciferase reporter gene assay. Annexin V FITC/PI double staining and Wb assays were used to detect the effect of over-expression of miR-140 or both over-expression of PD-L1 and miR140 on the apoptosis, migration and expression of apoptosis-related proteins in CC cells after treatment with oxaliplatin. Moreover, transplantation tumor of CC cell lines was established in nude mice to assess the effects of miR-140 on enhancing the sensitivity of tumors to oxaliplatin.Results: The expression of miR-140 was significantly decreased in oxaliplatin-resistant CC cells (P<0.01). Over-expression of miR-140 could significantly increase the sensitivity of oxaliplatin-resistant CC cells to oxaliplatin (P<0.05), and inhibit the CC cells proliferation and colony formation (P<0.01). miR-140 showed targeted binding to PD-L1 3''-UTR and inhibited its expression. Over-expression of miR-140 significantly promoted CC cell migration and apoptosis (P<0.01). However, co-transfection of PD-L1 counteracts the ef-fects of miR-140 on cell metastasis and apoptosis (all P<0.05). In addition, xenograft tumor model in mice also verified that miR-140 could promote the sensitivity of tumors to oxaliplatin. Conclusion: miR-140 increases the sensitivity of CC to oxaliplatin through inhibition of PD-L1 expression. Therefore, up-regulation of miR-140 or down-regulation of PD-L1 in combination with oxaliplatin may be a novel strategy for the treatment of Oxaliplatin-resistant CC.