一种基于血清外泌体的新型生物标志物hsa_circ_0002130通过吸附miR-498促进非小细胞肺癌对奥希替尼的耐药性。
A Novel Serum Exosomes-Based Biomarker hsa_circ_0002130 Facilitates Osimertinib-Resistance in Non-Small Cell Lung Cancer by Sponging miR-498.
作者信息
Ma Jing, Qi Guanbin, Li Lei
机构信息
Department of Respiratory and Critical Medicine, Huaihe Hospital of Henan University, Kaifeng, Henan, People's Republic of China.
出版信息
Onco Targets Ther. 2020 Jun 9;13:5293-5307. doi: 10.2147/OTT.S243214. eCollection 2020.
PURPOSE
Exosomes are the effective delivery system for biological compounds, including circular RNAs. In this research, we aimed to explore the role of circular RNA hsa_circRNA_0002130 in osimertinib-resistant non-small cell lung cancer (NSCLC).
MATERIALS AND METHODS
In our study, the relative protein expression of glucose transporter 1 (GLUT1), hexokinase-2 (HK2) and lactate dehydrogenase A (LDHA) was detected by Western blot, while the expression of hsa_circ_0002130 and microRNA-498 (miR-498) was detected by quantitative real-time PCR (qRT-PCR). The biological functions of hsa_circ_0002130 in osimertinib-resistant NSCLC were analyzed by cell viability assay, flow cytometry analysis, luciferase reporter assay, RNA pull-down assay, and tumor xenograft model in vivo. Moreover, glucose uptake, lactate production and extracellular acidification (ECAR) levels were measured by glucose uptake colorimetric assay kit, lactate assay kit II, and Seahorse Extracellular Flux Analyzer XF96 assay, respectively. hsa_circ_0002130 identification and localization were confirmed by RNase R digestion and subcellular localization assay, respectively. Exosomes were isolated from the sera collected from NSCLC patients and identified using a transmission electron microscopy and nanoparticle tracking analysis.
RESULTS
Osimertinib-resistance was closely related to glycolysis. hsa_circ_0002130 was highly expressed in osimertinib-resistant NSCLC cells and hsa_circ_0002130 deletion inhibited osimertinib-resistance both in vitro and in vivo. Moreover, hsa_circ_0002130 targeted miR-498 to regulate GLUT1, HK2 and LDHA. The inhibitory effects of hsa_circ_0002130 deletion on osimertinib-resistant were reversed by downregulating miR-498. Importantly, hsa_circ_0002130 was upregulated in serum exosomes from osimertinib-resistant NSCLC patients.
CONCLUSION
Our findings confirmed that hsa_circ_0002130 served as a promotion role in osimertinib-resistant NSCLC.
目的
外泌体是包括环状RNA在内的生物化合物的有效递送系统。在本研究中，我们旨在探讨环状RNA hsa_circRNA_0002130在奥希替尼耐药的非小细胞肺癌（NSCLC）中的作用。
材料与方法
在我们的研究中，通过蛋白质免疫印迹法检测葡萄糖转运蛋白1（GLUT1）、己糖激酶-2（HK2）和乳酸脱氢酶A（LDHA）的相对蛋白表达，同时通过定量实时聚合酶链反应（qRT-PCR）检测hsa_circ_0002130和微小RNA-498（miR-498）的表达。通过细胞活力测定、流式细胞术分析、荧光素酶报告基因测定、RNA下拉测定和体内肿瘤异种移植模型分析hsa_circ_0002130在奥希替尼耐药NSCLC中的生物学功能。此外，分别通过葡萄糖摄取比色测定试剂盒、乳酸测定试剂盒II和海马细胞外通量分析仪XF96测定来测量葡萄糖摄取、乳酸产生和细胞外酸化（ECAR）水平。分别通过核糖核酸酶R消化和亚细胞定位测定来确认hsa_circ_0002130的鉴定和定位。从NSCLC患者收集的血清中分离出外泌体，并使用透射电子显微镜和纳米颗粒跟踪分析进行鉴定。
结果
奥希替尼耐药与糖酵解密切相关。hsa_circ_0002130在奥希替尼耐药的NSCLC细胞中高表达，hsa_circ_0002130缺失在体外和体内均抑制奥希替尼耐药。此外，hsa_circ_0002130靶向miR-498以调节GLUT1、HK2和LDHA。下调miR-498可逆转hsa_circ_0002130缺失对奥希替尼耐药的抑制作用。重要的是，hsa_circ_0002130在奥希替尼耐药NSCLC患者的血清外泌体中上调。
结论
我们的研究结果证实hsa_circ_0002130在奥希替尼耐药的NSCLC中起促进作用。
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