DGCR8/miR-106 Axis Enhances Radiosensitivity of Head and Neck Squamous Cell Carcinomas by Downregulating RUNX3.
DGCR8/miR-106 Axis Enhances Radiosensitivity of Head and Neck Squamous Cell Carcinomas by Downregulating RUNX3.
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Dgcr8/mir-106轴通过下调runx3增强头颈鳞状细胞癌的放射敏感性
DOI：
10.3389/fmed.2020.582097
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发表时间：
2020
影响因子：
3.9
通讯作者：
Liu Z
中科院分区：
文献类型：
--
作者：
Zhang C;Chen H;Deng Z;Long D;Xu L;Liu Z
目的：头颈部鳞状细胞癌（Head and Neck Squamous Cell Carcinoma，HNSCC）是世界上第六大常见的恶性肿瘤，其放疗效果与人乳头瘤病毒（Human Papillomavirus，HPV）感染密切相关。因此，我们的研究目的是分析HPV E7的机制及其对HNSCC细胞放射敏感性的影响。研究方法：通过实时定量PCR（RT-qPCR）检测DiGeorge综合征关键区基因8（DGCR 8）、has-miR-106 a和Runt相关转录因子3（RUNX 3）的mRNA表达。Western blot检测DGCR 8、E7、RUNX 3、caspase-3/cleaved caspase-3、PARP/cleaved PARP和γ H2 AX蛋白表达。免疫荧光法检测DGCR 8的表达水平。使用Starbase数据库（http：//starbase.sysu.edu.cn/）分析has-miR-106 a-5 p与DGCR 8之间的相关性。采用TargetScan数据库（http：//www.targetscan.org/vert_72/）计算结合位点的预测。通过克隆形成试验和细胞计数试剂盒-8（CCK-8）试验评价放射敏感性。结果如下：我们的研究发现HPV阳性的HNSCC细胞中HPV E7和DGCR 8的mRNA和蛋白表达水平高于HPV阴性的细胞。DGCR 8在E7过表达的FaDu和UM-SCC-4中表达增加，而DGCR 8在E7沉默的UM-SCC-47和UPCI-SCC-090中表达降低。FaDu和UM-SCC-4中DGCR 8过表达后，miR-106 a表达增加。然而，miR-106 a在E7沉默的UM-SCC-47和UPCI-SCC-090中的表达降低。在辐射条件下，克隆形成实验发现，DGCR 8或miR-106 a沉默后的FaDu和UM-SCC-4细胞中形成的克隆数少于对照组，而DGCR 8或miR-106 a过表达的UM-SCC-47和UPCI-SCC-090细胞中形成的克隆数多于对照组。荧光素酶报告基因分析证实miR-106 a靶向RUNX 3 mRNA的3′非翻译区（UTR）。miR-106 a过表达导致RUNX 3表达降低，miR-106 a沉默增加RUNX 3表达。用miR-106 a抑制剂进行的挽救实验恢复了辐射抗性并减少了辐射条件下的DNA损伤。结论：我们的研究表明，HPV E7激活DGCR 8/miR-106 a/RUNX 3轴可增强放射敏感性，并为靶向治疗干预提供指导。
Purpose: Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent malignant tumor worldwide, and the radiotherapy effect is strongly associated with human papillomavirus (HPV) infection. Therefore, the aim of our study was to analyze the mechanism of HPV E7 and its effects on radiosensitivity in HNSCC cells. Methods: The mRNA expression of DiGeorge syndrome critical region gene 8 (DGCR8), has-miR-106a, and Runt-related transcription factor 3 (RUNX3) was examined by quantitative real-time PCR (RT-qPCR). The protein expression of DGCR8, E7, RUNX3, caspase-3/cleaved caspase-3, poly(ADP-ribose) polymerase (PARP)/cleaved PARP, and γH2AX was measured by Western blot. The expression level of DGCR8 was measured by immunofluorescence assay. Starbase database (http://starbase.sysu.edu.cn/) was used to analyze the correlation between has-miR-106a-5p and DGCR8. TargetScan database (http://www.targetscan.org/vert_72/) was adopted to calculate the prediction of binding sites. Radiosensitivity was evaluated through clone formation assays and Cell Counting Kit-8 (CCK-8) assays. Results: In our study, we found that the mRNA and protein expression levels of HPV E7 and DGCR8 in HPV-positive HNSCC cells were higher than those in HPV-negative cells. The expression of DGCR8 was increased in FaDu and UM-SCC-4 with E7 overexpression, while the expression of DGCR8 was decreased in UM-SCC-47 and UPCI-SCC-090 with E7 silence. The miR-106a expression was increased after DGCR8 overexpression in FaDu and UM-SCC-4. However, the miR-106a expression was decreased in UM-SCC-47 and UPCI-SCC-090 with E7 silence. In radiation conditions, clone formation assays found that less clones formed in FaDu and UM-SCC-4 cells subsequent to silencing DGCR8 or miR-106a than that in the control group, and more clones were formed in UM-SCC-47 and UPCI-SCC-090 cells overexpressing DGCR8 or miR-106a than that in the control group. Luciferase reporter gene assays verified that miR-106a targeted the 3′ untranslated region (UTR) of RUNX3 mRNA. MiR-106a overexpression resulted in a decrease in RUNX3 expression, and miR-106a silence increased RUNX3 expression. Rescue experiments conducted with miR-106a inhibitor restored radiation resistance and reduced DNA damage in radiation condition. Conclusions: Our study indicated that HPV E7 activated DGCR8/miR-106a/RUNX3 axis to enhance radiation sensitivity and provided directions for targeted therapeutic interventions.
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作者：
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通讯作者：
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影响因子：
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通讯作者：
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