【摘要】 目的：探究mi R-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mi R-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mi R-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mi R-216a-5p mimic+pcDNA DUSP10组和mi R-216a-5p mimic+pcDNA NC组。通过qRT-PCR检测mi R-216a-5p和DUSP10 m RNA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mi R-216a-5p和DUSP10的靶向关系。通过GFP-m RFP-LC3检测自噬体。结果：与8Gy NC-mimic组相比，8Gy mi R-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 mi R-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mi R-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，mi R-216a-5p-mimic组的DUSP10 m RNA和蛋白表达水平均降低（P<0.001）。与mi R-216a-5p mimic+pcDNA NC组相比，mi R-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：mi R-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。更多还原

【Abstract】 Objective: To investigate the regulatory mechanism of mi R-216a-5p on autophagy and radiosensitivity of gastric cancer cell and its regulatory effect on dual-specificity phosphatase 10(DUSP10). Method: 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. mi R-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 mi R-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 mi R-216a-5p mimic+pcDNA DUSP10 group and mi R-216a-5p mimic+pcDNA NC group. The m RNA levels of mi R-216a-5p and DUSP10 were detected by qRT-PCR. Cell proliferation was detected by 5-ethynyl-2’-deoxyuridine(Ed U) 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 mi R-216a-5p and DUSP10 was detected by luciferase reporter gene. Autophagosomes were detected by GFP-m RFP-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 mi R-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 the8Gy NC-mimic group, the number of autophagosomes and the expression level of LC3II protein in the 8Gy mi R-216a-5p-mimic group decreased, while the expression level of p62 protein increased(P<0.001). After co-culture with mi R-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 m RNA and protein expression levels in the mi R-216a-5p-mimic group were lower(P<0.001). Compared with the mi R-216a-5p mimic+pcDNA NC group, the number of colonies and autophagosomes in the mi R-216a-5p mimic+pcDNA DUSP10 group increased, while the apoptosis rate was decreased(P<0.001). Conclusion: mi R-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.更多还原