| 尹建浩,陈 威,王 琼,袁达伟,朱 琨,李 康,线胤生,张 勇,许 刚.上调miR-216a-5p通过靶向双特异性磷酸酶10抑制胃癌细胞自噬并增强放射敏感性[J].,2022,(22):4206-4214 |
| 上调miR-216a-5p通过靶向双特异性磷酸酶10抑制胃癌细胞自噬并增强放射敏感性 |
| Up-regulation of miR-216a-5p Inhibits Autophagy and Enhances Radiosensitivity in Gastric Cancer Cell by Targeting Dual-Specificity Phosphatase 10 |
| 投稿时间：2022-04-26 修订日期：2022-05-22 |
| DOI：10.13241/j.cnki.pmb.2022.22.002 |
| 中文关键词: 胃癌 miR-216a-5p 双特异性磷酸酶10 自噬 放射敏感性 |
| 英文关键词: Gastric cancer miR-216a-5p Dual-specificity phosphatase 10 Autophagy Radiosensitivity |
| 基金项目:国家自然科学基金项目（81372280）；陕西省自然科学基础研究计划项目（2019JQ-960） |
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| 中文摘要: |
| 摘要 目的：探究miR-216a-5p对胃癌细胞自噬和放射敏感性的调控机制及其对双特异性磷酸酶10（DUSP10）的调控作用。方法：采用直线加速器6-MV X射线照射SGC-7901细胞，剂量率为0.8Gy/min，总剂量为8Gy。用Lipofectamine 2000试剂将miR-216a-5p mimic、NC mimic、pcDNA DUSP10或pcDNA NC转染到SGC-7901细胞中。转染后，将细胞分为miR-216a-5p mimic组和NC mimic组，每组又分为0Gy和8Gy两个亚组。在拯救实验中，将细胞分为miR-216a-5p mimic+pcDNA DUSP10组和miR-216a-5p mimic+pcDNA NC组。通过qRT-PCR检测miR-216a-5p和DUSP10 mRNA水平。通过5-乙炔基-2'-脱氧尿苷（EdU）掺入实验和集落形成测定检测细胞增殖。通过流式细胞仪评估细胞凋亡。通过Western blot检测DUSP10、Bax、Bad、Bcl-2、LC3和p62的蛋白表达。通过免疫荧光法检测γH2AX的表达，用于评估细胞中的DNA双链断裂（DSB）。通过荧光素酶报告基因检测miR-216a-5p和DUSP10的靶向关系。通过GFP-mRFP-LC3检测自噬体。结果：与8Gy NC-mimic组相比，8Gy miR-216a-5p-mimic组的集落数量、EdU阳性率和Bcl-2蛋白表达水平降低，而γH2AX阳性率、细胞凋亡率和Bax和Bad蛋白表达水平升高（P<0.01）。与8Gy NC-mimic组相比，8Gy miR-216a-5p-mimic组的自噬体数量和LC3II蛋白表达水平降低，而p62蛋白表达水平升高（P<0.001）。与miR-216a-5p-mimic共培养后，与DUSP10-3'-UTR-MUT组相比，DUSP10-3'-UTR-WT的相对荧光素酶活性显著降低（P<0.001）。与NC-mimic组相比，miR-216a-5p-mimic组的DUSP10 mRNA和蛋白表达水平均降低（P<0.001）。与miR-216a-5p mimic+pcDNA NC组相比，miR-216a-5p mimic+pcDNA DUSP10组的集落数量和自噬体数量升高，而细胞凋亡率降低（P<0.001）。结论：miR-216a-5p通过抑制DUSP10来抑制细胞增殖、增加放射诱导的细胞凋亡并抑制放射诱导的自噬，从而增强胃癌细胞的放射敏感性。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the regulatory mechanism of miR-216a-5p on autophagy and radiosensitivity of gastric cancer cell and its regulatory effect on dual-specificity phosphatase 10 (DUSP10). Methods: SGC-7901 cells were irradiated with linear accelerator 6-MV X-ray at a dose rate of 0.8 Gy/min. The total dose is 8Gy. miR-216a-5p-mimic, NC-mimic, pcDNA-DUSP10 or pcDNA-NC were transfected into SGC-7901 cells with Lipofectamine 2000 reagent. After transfection, the cells were divided into miR-216a-5p-mimic group and NC-mimic group, and each group was divided into two subgroups, 0Gy and 8Gy. In the rescue experiment, the cells were divided into miR-216a-5p mimic+pcDNA DUSP10 group and miR-216a-5p mimic+pcDNA NC group. The mRNA levels of miR-216a-5p and DUSP10 were detected by qRT-PCR. Cell proliferation was detected by 5-ethynyl-2'-deoxyuridine (EdU) incorporation test and colony formation assay. The apoptosis was assessed by flow cytometry. The protein expressions of DUSP10, Bax, Bad, Bcl-2, LC3 and p62 were detected by Western blot. The expression of γH2AX was detected by immunofluorescence method to evaluate DNA double-strand break (DSB) in cells. The targeting relationship between miR-216a-5p and DUSP10 was detected by luciferase reporter gene. Autophagosomes were detected by GFP-mRFP-LC3. Results: Compared with the 8Gy NC-mimic group, the number of colonies, EdU positive rate and Bcl-2 protein expression level in the 8Gy miR-216a-5p-mimic group decreased, while the positive rate of γH2AX, the rate of apoptosis and the expression levels of Bax and Bad proteins increased (P<0.01). Compared with the 8Gy NC-mimic group, the number of autophagosomes and the expression level of LC3II protein in the 8Gy miR-216a-5p-mimic group decreased, while the expression level of p62 protein increased (P<0.001). After co-culture with miR-216a-5p-mimic, the relative luciferase activity of DUSP10-3'-UTR-WT was significantly reduced compared with the DUSP10-3'-UTR-MUT group (P<0.001). Compared with the NC-mimic group, the DUSP10 mRNA and protein expression levels in the miR-216a-5p-mimic group were lower (P<0.001). Compared with the miR-216a-5p mimic+pcDNA NC group, the number of colonies and autophagosomes in the miR-216a-5p mimic+pcDNA DUSP10 group increased, while the apoptosis rate was decreased (P<0.001). Conclusion: miR-216a-5p inhibits cell proliferation, increases radiation-induced apoptosis and inhibits radiation-induced autophagy by inhibiting DUSP10, thereby enhancing the radiosensitivity of gastric cancer cells. |
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