Xie Xiaoque, Huang Nana, Zhang Yiyin, Wei Xiaoli, Gao Mengru, Li Min, Ning Jie, Liu Wei, Zhao Qihong, Wang Hua, Gu Kangsheng
Department of Oncological Radiotherapy, First Affiliated Hospital of Anhui Medical University, Hefei, China.
Department of Oncology, First Affiliated Hospital of Anhui Medical University, Hefei, China.
J Cancer. 2019 Jan 29;10(4):1039-1051. doi: 10.7150/jca.25814. eCollection 2019.
Cisplatin chemoresistance is a clinical obstacle in the treatment of gastric cancer (GC). Enhanced DNA repair capacity may lead to cisplatin resistance. However, the detailed molecular mechanism of GC cisplatin resistance specifically involving nucleotide excision repair (NER) is not clear. However, the mechanism through which the NER pathway contributes to cisplatin resistance in GC is still unclear. In light of the crucial role of microRNAs (miRNAs) in regulating protein expression and biological behavior, we aimed to analyze the expression and function of miR-192-5p in the NER pathway and its role in cisplatin resistance in GC. Comet assays were performed to measure the amount of DNA damage and repair in the SGC7901 and SGC7901/DDP GC cell lines by observing the tail length. MiRNA expression levels in SGC7901/DDP and SGC7901 cells were detected by microarray. Quantitative real-time PCR (qRT-PCR) was carried out to confirm the expression level of miR-192-5p. Lentiviral vector transfection modifies miR-192-5p levels in SGC7901/DDP and SGC7901 cells. The IC values of cisplatin-treated cells were assessed by MTT assays. The protein level was determined by Western blot and immunohistochemistry. With enhanced DNA repair, the expression levels of ERCC3 and ERCC4 in SGC 7901DDP cells increased, while miR-192-5p was significantly downregulated in SGC7901/DDP compared with SGC7901 cells. ERCC3 and ERCC4 were identified as the main targets of miR-192-5p. Forced expression of miR-192-5p in SGC7901/DDP cells significantly inhibited the expression of ERCC3 and ERCC4, making GC cells more sensitive to cisplatin in vitro and in vivo. In contrast, knockdown of miR-192-5p expression in SGC7901 cells increased the expression of ERCC3 and ERCC4, resulting in cisplatin resistance in vitro and in vivo. MiR-192-5p partially reversed GC cisplatin resistance by targeting ERCC3 and ERCC4, which participate in the NER pathway, suggesting that miR-192-5p may be a potential biomarker and therapeutic target for GC cisplatin resistance.
顺铂化疗耐药是胃癌（GC）治疗中的一个临床障碍。DNA修复能力增强可能导致顺铂耐药。然而，GC顺铂耐药具体涉及核苷酸切除修复（NER）的详细分子机制尚不清楚。然而，NER途径导致GC顺铂耐药的机制仍不清楚。鉴于微小RNA（miRNA）在调节蛋白质表达和生物学行为中的关键作用，我们旨在分析miR-192-5p在NER途径中的表达和功能及其在GC顺铂耐药中的作用。通过观察彗星试验中彗尾长度，检测SGC7901和SGC7901/DDP GC细胞系中的DNA损伤和修复量。通过微阵列检测SGC7901/DDP和SGC7901细胞中的miRNA表达水平。进行定量实时PCR（qRT-PCR）以确认miR-192-5p的表达水平。慢病毒载体转染可改变SGC7901/DDP和SGC7901细胞中miR-192-5p的水平。通过MTT试验评估顺铂处理细胞的IC值。通过蛋白质印迹和免疫组织化学测定蛋白质水平。随着DNA修复增强，SGC 7901DDP细胞中ERCC3和ERCC4的表达水平增加，而与SGC7901细胞相比，SGC7901/DDP中miR-192-5p显著下调。ERCC3和ERCC4被确定为miR-192-5p的主要靶标。在SGC7901/DDP细胞中强制表达miR-192-5p可显著抑制ERCC3和ERCC4的表达，使GC细胞在体外和体内对顺铂更敏感。相反，敲低SGC7901细胞中miR-192-5p的表达可增加ERCC3和ERCC4的表达，导致体外和体内顺铂耐药。miR-192-5p通过靶向参与NER途径的ERCC3和ERCC4部分逆转GC顺铂耐药，这表明miR-192-5p可能是GC顺铂耐药的潜在生物标志物和治疗靶点。
