Cisplatin is the first-line chemotherapeutic drug for non-small cell lung cancer (NSCLC), and emerging evidences suggests that targeting circular RNAs (circRNAs) is an effective strategy to increase cisplatin-sensitivity in NSCLC, but the detailed mechanisms are still not fully delineated. Cell proliferation, viability and apoptosis were examined by using the cell counting kit-8 (CCK-8) assay, trypan blue staining assay and Annexin V-FITC/PI double staining assay, respectively. The expression levels of cancer associated genes were measured by using the Real-Time qPCR and Western Blot analysis at transcriptional and translated levels. Dual-luciferase reporter gene system assay was conducted to validated the targeting sites among hsa_circRNA_103809, miR-377-3p and 3′ untranslated region (3’UTR) of GOT1 mRNA. The expression status, including expression levels and localization, were determined by immunohistochemistry (IHC) assay in mice tumor tissues. Here we identified a novel hsa_circRNA_103809/miR-377-3p/GOT1 signaling cascade which contributes to cisplatin-resistance in NSCLC in vitro and in vivo. Mechanistically, parental cisplatin-sensitive NSCLC (CS-NSCLC) cells were subjected to continuous low-dose cisplatin treatment to generate cisplatin-resistant NSCLC (CR-NSCLC) cells, and we found that hsa_circRNA_103809 and GOT1 were upregulated, while miR-377-3p was downregulated in CR-NSCLC cells but not in CS-NSCLC cells. In addition, hsa_circRNA_103809 sponged miR-337-3p to upregulate GOT1 in CS-NSCLC cells, and knock-down of hsa_circRNA_103809 enhanced the inhibiting effects of cisplatin on cell proliferation and viability, and induced cell apoptosis in CR-NSCLC cells, which were reversed by downregulating miR-377-3p and overexpressing GOT1. Consistently, overexpression of hsa_circRNA_103809 increased cisplatin-resistance in CS-NSCLC cells by regulating the miR-377-3p/GOT1 axis. Finally, silencing of hsa_circRNA_103809 aggravated the inhibiting effects of cisplatin treatment on NSCLC cell growth in vivo. Analysis of data suggested that targeting the hsa_circRNA_103809/miR-377-3p/GOT1 pathway increased susceptibility of CR-NSCLC cells to cisplatin, and this study provided novel targets to improve the therapeutic efficacy of cisplatin for NSCLC treatment in clinic.

中文翻译：

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新型环状RNA hsa_circRNA_103809 / miR-377-3p / GOT1通路调节非小细胞肺癌（NSCLC）的顺铂耐药性

顺铂是用于非小细胞肺癌（NSCLC）的一线化疗药物，新兴证据表明，靶向环状RNA（circRNA）是提高NSCLC顺铂敏感性的有效策略，但详细的机制仍不完全划定的。分别通过细胞计数试剂盒8（CCK-8）测定，锥虫蓝染色测定和膜联蛋白V-FITC / PI双重染色测定来检测细胞增殖，活力和凋亡。通过使用实时qPCR和蛋白质印迹分析在转录和翻译水平上测量癌症相关基因的表达水平。进行了双重荧光素酶报告基因系统分析，以验证GOT1 mRNA的hsa_circRNA_103809，miR-377-3p和3'非翻译区（3'UTR）之间的靶向位点。表情状态 通过免疫组织化学（IHC）测定在小鼠肿瘤组织中的表达水平和定位。在这里，我们确定了一个新颖的hsa_circRNA_103809 / miR-377-3p / GOT1信号级联，该级联有助于在体外和体内对NSCLC的顺铂耐药。从机理上讲，对顺式敏感的NSCLC（CS-NSCLC）亲本细胞进行连续低剂量顺铂处理以产生耐顺铂的NSCLC（CR-NSCLC）细胞，我们发现hsa_circRNA_103809和GOT1上调，而miR-377- 3p在CR-NSCLC细胞中下调，但在CS-NSCLC细胞中则未下调。此外，hsa_circRNA_103809使miR-337-3p在CS-NSCLC细胞中上调，而敲除hsa_circRNA_103809则增强了顺铂对细胞增殖和生存能力的抑制作用，并诱导了CR-NSCLC细胞的细胞凋亡，通过下调miR-377-3p和过表达GOT1来逆转它们。一致地，通过调节miR-377-3p / GOT1轴，hsa_circRNA_103809的过表达增加了CS-NSCLC细胞中顺铂耐药性。最后，hsa_circRNA_103809的沉默加剧了顺铂治疗对体内NSCLC细胞生长的抑制作用。数据分析表明，靶向hsa_circRNA_103809 / miR-377-3p / GOT1途径可提高CR-NSCLC细胞对顺铂的敏感性，这项研究为提高顺铂在NSCLC治疗中的临床疗效提供了新的靶点。hsa_circRNA_103809的沉默加剧了顺铂治疗对体内NSCLC细胞生长的抑制作用。数据分析表明，靶向hsa_circRNA_103809 / miR-377-3p / GOT1途径可提高CR-NSCLC细胞对顺铂的敏感性，这项研究为提高顺铂在NSCLC治疗中的临床疗效提供了新的靶点。hsa_circRNA_103809的沉默加剧了顺铂治疗对体内NSCLC细胞生长的抑制作用。数据分析表明，靶向hsa_circRNA_103809 / miR-377-3p / GOT1途径可提高CR-NSCLC细胞对顺铂的敏感性，这项研究为提高顺铂在NSCLC治疗中的临床疗效提供了新的靶点。