环状 RNA PIP5K1A 通过依赖于 miR-493-5p/ROCK1 轴调控非小细胞肺癌细胞及异种移植鼠模型中的顺铂耐药和恶性进展。
Circ_PIP5K1A regulates cisplatin resistance and malignant progression in non-small cell lung cancer cells and xenograft murine model via depending on miR-493-5p/ROCK1 axis.
机构信息
Department of Clinical Laboratory, The Second Affiliated Hospital of Xi'an Jiaotong University, No.157 West Fifth Road, Xincheng District, Xi'an, 710004, Shaanxi Province, China.
The Public Health, Xi'an Jiaotong University Health Science Center, No.76 Yanta West Road, Xi'an, 710061, Shaanxi Province, China.
出版信息
Respir Res. 2021 Sep 18;22(1):248. doi: 10.1186/s12931-021-01840-7.
BACKGROUND
Chemoresistance limits the therapeutic effect of cisplatin (DDP) on non-small cell lung cancer (NSCLC). Circular RNAs (circRNAs) function as important regulators in chemoresistance. This study aimed to explore the regulation of circRNA Phosphatidylinositol-4-Phosphate 5-Kinase Type 1 Alpha (circ_PIP5K1A) in DDP resistance.
METHODS
The expression analysis of circ_PIP5K1A, micoRNA-493-5p (miR-493-5p) and Rho Associated Coiled-Coil Containing Protein Kinase 1 (ROCK1) was conducted through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell sensitivity was determined using 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell proliferation and cell viability were evaluated by colony formation assay and MTT assay, respectively. Cell cycle and apoptosis detection was performed via flow cytometry. Cell motility was examined by transwell migration or invasion assay. Dual-luciferase reporter assay was applied to confirm the target binding. ROCK1 protein level was assayed via Western blot. In vivo assay was carried out using xenograft model in mice.
RESULTS
Circ_PIP5K1A level was abnormally increased in DDP-resistant NSCLC tissues and cells. Silencing circ_PIP5K1A reduced DDP resistance, proliferation, cell cycle progression and cell motility in DDP-resistant NSCLC cells. Circ_PIP5K1A directly interacted with miR-493-5p in NSCLC cells. The function of circ_PIP5K1A was dependent on the negative regulation of miR-493-5p. MiR-493-5p directly targeted ROCK1 and circ_PIP5K1A regulated the ROCK1 level via acting as a sponge of miR-493-5p. Overexpression of miR-493-5p inhibited chemoresistance and cancer progression by downregulating ROCK1 expression in DDP-resistant NSCLC cells. Circ_PIP5K1A regulated DDP sensitivity in vivo via the miR-493-5p/ROCK1 axis.
CONCLUSION
These findings suggested that circ_PIP5K1A upregulated the ROCK1 expression to promote DDP resistance and cancer progression in NSCLC by sponging miR-493-5p.
背景
化疗耐药限制了顺铂（DDP）在非小细胞肺癌（NSCLC）中的治疗效果。环状 RNA（circRNA）作为重要的调节剂在化疗耐药中发挥作用。本研究旨在探讨环状 RNA 磷酸肌醇-4-磷酸 5-激酶 1A（circ_PIP5K1A）在 DDP 耐药中的调控作用。
方法
通过逆转录定量聚合酶链反应（RT-qPCR）分析 circ_PIP5K1A、微小 RNA-493-5p（miR-493-5p）和 Rho 相关卷曲螺旋蛋白激酶 1（ROCK1）的表达。使用 3-(4,5-二甲基噻唑-2-y1)-2,5-二苯基四氮唑溴盐（MTT）测定细胞敏感性。通过集落形成试验和 MTT 试验分别评估细胞增殖和细胞活力。通过流式细胞术检测细胞周期和细胞凋亡。通过 Transwell 迁移或侵袭试验检测细胞迁移能力。双荧光素酶报告试验用于确认靶标结合。通过 Western blot 检测 ROCK1 蛋白水平。在体内实验中，使用异种移植模型在小鼠中进行实验。
结果
circ_PIP5K1A 水平在 DDP 耐药 NSCLC 组织和细胞中异常升高。沉默 circ_PIP5K1A 降低了 DDP 耐药性、增殖、细胞周期进展和 DDP 耐药 NSCLC 细胞的迁移能力。circ_PIP5K1A 直接与 NSCLC 细胞中的 miR-493-5p 相互作用。circ_PIP5K1A 的功能依赖于 miR-493-5p 的负调控。miR-493-5p 直接靶向 ROCK1，circ_PIP5K1A 通过作为 miR-493-5p 的海绵来调节 ROCK1 水平。在 DDP 耐药 NSCLC 细胞中过表达 miR-493-5p 可通过下调 ROCK1 表达来抑制化疗耐药和癌症进展。circ_PIP5K1A 通过 miR-493-5p/ROCK1 轴在体内调节 DDP 敏感性。
结论
这些发现表明，circ_PIP5K1A 通过海绵吸附 miR-493-5p 上调 ROCK1 表达，促进 NSCLC 中的 DDP 耐药和癌症进展。
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