Long non-coding RNA HOTAIR knockdown enhances radiosensitivity through regulating microRNA-93/ATG12 axis in colorectal cancer
Long non-coding RNA HOTAIR knockdown enhances radiosensitivity through regulating microRNA-93/ATG12 axis in colorectal cancer
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DOI：
10.1038/s41419-020-2268-8
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发表时间：
2020-03-06
影响因子：
9
通讯作者：
Ge, Hong
中科院分区：
文献类型：
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作者：
Liu, Yingqiang;Chen, Xijuan;Ge, Hong
结直肠癌（CRC）是一个全球性的医疗保健问题。放射抵抗是CRC放射治疗的一个巨大挫折。本文就长链非编码RNA HOTAIR在结直肠癌发生和放射抵抗中的作用及其分子机制进行了深入研究。通过定量逆转录聚合酶链反应（RT-qPCR）测定ATG 12 mRNA、HOTAIR和microRNA-93（miR-93）水平。Western blotting法检测细胞中LC 3 I、LC 3 II、p62、ATG 12、cleaved caspase 3、Bax和Bcl-2的蛋白水平，免疫组化法检测组织中LC 3 I、LC 3 II、p62、ATG 12、cleaved caspase 3、Bax和Bcl-2的蛋白水平。细胞存活分数，活力和凋亡率分别通过克隆形成存活测定，CCK-8测定和流式细胞术分析。通过生物信息学分析和荧光素酶报告基因检测HOTAIR、miR-93和ATG 12之间的关系。建立小鼠异种移植瘤模型以研究HOTAIR敲低对体内CRC辐射抗性的影响。我们发现，HOTAIR的表达显着上调，血浆中的CRC患者放疗后和CRC细胞照射后。HOTAIR敲低、miR-93过表达或ATG 12沉默削弱了CRC中的细胞活力，诱导细胞凋亡，抑制细胞自噬，并增强细胞放射敏感性。HOTAIR通过下调miR-93发挥其功能。此外，HOTAIR作为miR-93的分子海绵调节ATG 12的表达。在结直肠癌组织中，ATG 12蛋白表达显著上调，并与miR-93和HOTAIR表达相关。此外，HOTAIR敲低通过调节miR-93/ATG 12轴增强CRC异种移植肿瘤的放射敏感性。综上所述，HOTAIR基因敲低可通过调控miR-93/ATG 12轴增强结直肠癌的放射敏感性，进一步阐明了HOTAIR在结直肠癌放射抵抗中的作用及其分子基础。
Colorectal cancer (CRC) is a global healthcare problem. Radioresistance is a huge setback for CRC radiotherapy. In this text, the roles and molecular mechanisms of long non-coding RNA HOTAIR in CRC tumorigenesis and radioresistance were further investigated. ATG12 mRNA, HOTAIR, and microRNA-93 (miR-93) levels were measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. Protein levels of LC3 I, LC3 II, p62, ATG12, cleaved caspase 3, Bax, and Bcl-2 were detected by western blotting assay in cells and were examined by immunohistochemistry (IHC) assay in tissues. Cell survival fractions, viability, and apoptotic rates were determined by clonogenic survival assay, CCK-8 assay, and flow cytometry analysis, respectively. The relationships of HOTAIR, miR-93, and ATG12 were tested by bioinformatics analysis and luciferase reporter assay. Mouse xenograft tumor models were established to investigate the influence of HOTAIR knockdown on CRC radioresistance in vivo. We found that HOTAIR expression was markedly upregulated in plasma from CRC patients after radiotherapy and CRC cells after irradiation. HOTAIR knockdown, miR-93 overexpression, or ATG12 silencing weakened cell viability, induced cell apoptosis, inhibited cell autophagy, and enhanced cell radiosensitivity in CRC. HOTAIR exerted its functions by downregulating miR-93. Moreover, HOTAIR functioned as a molecular sponge of miR-93 to regulate ATG12 expression. ATG12 protein expression was markedly upregulated and associated with miR-93 and HOTAIR expression in CRC tissues. Furthermore, HOTAIR knockdown enhanced radiosensitivity of CRC xenograft tumors by regulating miR-93/ATG12 axis. In conclusion, HOTAIR knockdown potentiated radiosensitivity through regulating miR-93/ATG12 axis in CRC, further elucidating the roles and molecular basis of HOTAIR in CRC radioresistance.