Wang Xianfang, Shi Jun, Chen Ying, Wang Caihong, Shi Huifang, Xie Xuefang
Department of Laboratory, People's Hospital of Rizhao No. 126 Tai'an Road Rizhao 276800 Shandong China
Department of Pharmacy, Rizhao Maternal and Child Health Care Hospital China.
RSC Adv. 2019 Nov 26;9(66):38735-38744. doi: 10.1039/c9ra06993c. eCollection 2019 Nov 25.
Long noncoding RNAs (lncRNAs) are implicated in the development of chemoresistance in many cancers. However, the effect and mechanism of lncRNA antisense noncoding RNA in the INK4 locus (ANRIL) on cisplatin (CDDP) resistance in non-small cell lung cancer (NSCLC) remain unclear. The levels of ANRIL, microRNA (miR)-656-3p and sex-determining region Y-related high-mobility group box 4 (SOX4) in NSCLC tissues and cells were detected by quantitative real-time polymerase chain reaction or western blotting. Cell viability, apoptosis, migration and epithelial-to-mesenchymal transition (EMT) were assessed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT assay), flow cytometry, trans-well assays and western blotting, respectively. The xenograft model was established using CDDP-resistant NSCLC cells. The target association between miR-656-3p and ANRIL or SOX4 was validated by luciferase reporter assay and RNA immunoprecipitation. ANRIL expression was increased in CDDP-resistant NSCLC tissues and cells. Knockdown of ANRIL decreased cell viability, migration and EMT but induced apoptosis in CDDP-resistant NSCLC cells. Moreover, silencing of ANRIL reduced xenograft tumor growth . miR-656-3p was targeted by ANRIL and its exhaustion attenuated the suppressive role of ANRIL knockdown in CDDP resistance in NSCLC cells. SOX4 acted as a target of miR-656-3p and was positively regulated by ANRIL. Collectively, interference of ANRIL repressed CDDP resistance through promoting apoptosis and inhibiting cell viability, migration and EMT by up-regulating miR-656-3p and down-regulating SOX4, indicating a new target to improve the chemotherapeutic efficacy in NSCLC.
长链非编码RNA（lncRNA）与多种癌症的化疗耐药性发展有关。然而，INK4基因座反义非编码RNA（ANRIL）在非小细胞肺癌（NSCLC）顺铂（CDDP）耐药中的作用及机制仍不清楚。通过定量实时聚合酶链反应或蛋白质免疫印迹法检测NSCLC组织和细胞中ANRIL、微小RNA（miR）-656-3p和性别决定区Y相关高迁移率族蛋白4（SOX4）的水平。分别使用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT法）、流式细胞术、Transwell法和蛋白质免疫印迹法评估细胞活力、凋亡、迁移和上皮-间质转化（EMT）。使用对CDDP耐药的NSCLC细胞建立异种移植模型。通过荧光素酶报告基因检测和RNA免疫沉淀验证miR-656-3p与ANRIL或SOX4之间的靶向关系。ANRIL在对CDDP耐药的NSCLC组织和细胞中表达增加。敲低ANRIL可降低对CDDP耐药的NSCLC细胞的活力、迁移和EMT，但诱导其凋亡。此外，沉默ANRIL可减少异种移植肿瘤的生长。miR-656-3p是ANRIL的靶标，其耗尽减弱了ANRIL敲低对NSCLC细胞CDDP耐药的抑制作用。SOX4是miR-656-3p的靶标，并受ANRIL正向调控。总体而言，干扰ANRIL通过上调miR-656-3p和下调SOX4促进凋亡并抑制细胞活力、迁移和EMT，从而抑制CDDP耐药，这表明其可能是提高NSCLC化疗疗效的新靶点。
