miR-373-3p通过靶向ATG7参与调控胶质母细胞瘤细胞自噬和舒尼替尼敏感性

Micro RNA-373-3p is involved in regulation of autophagy and sunitinib sensitivity of glioblastoma cells via targeting autophagy-related gene 7

目的探究微小RNA(miR)-373-3p对胶质母细胞瘤细胞自噬和舒尼替尼敏感性的影响及其机制。方法体外常规培养U251细胞,采用50μmol/L舒尼替尼作用72 h构建耐舒尼替尼U251细胞株。(1)采用实时荧光定量PCR(RT-qPCR)检测U251、耐舒尼替尼U251细胞miR-373-3p的表达。将耐舒尼替尼U251细胞分为空白对照组、无义序列组、miR-373-3p模拟物组,后2组细胞分别转染miR-373-3p无义序列、miR-373-3p模拟物,采用RT-qPCR检测细胞miR-373-3p的表达。将细胞分为U251组、耐舒尼替尼U251组、无义序列+耐舒尼替尼U251组、miR-373-3p模拟物+耐舒尼替尼U251组,转染后采用CCK-8法检测细胞活性,TUNEL染色检测细胞凋亡率,免疫荧光染色检测细胞微管相关蛋白轻链3(LC3)的表达,Western blotting检测细胞凋亡、自噬相关蛋白的表达。(2)构建pGL3-自噬相关基因7(ATG7)野生型(WT)和pGL3-ATG7突变型(MUT)质粒,双荧光素酶报告分析系统检测miR-373-3p模拟物组、无义序列组细胞荧光素酶活性。将细胞分为U251组、耐舒尼替尼U251组、无义序列+耐舒尼替尼U251组、miR-373-3p模拟物+耐舒尼替尼U251组,转染后采用RT-qPCR、Western blotting分别检测细胞ATG7 mRNA和蛋白的表达。(3)将耐舒尼替尼U251细胞分为空白对照组、ATG7阴性对照组、ATG7过表达组,转染后采用RT-qPCR、Western blotting分别检测细胞ATG7 mRNA和蛋白的表达。将U251细胞、耐舒尼替尼U251细胞分为U251组、耐舒尼替尼U251组、无义序列+耐舒尼替尼U251组、miR-373-3p模拟物+耐舒尼替尼U251组、miR-373-3p模拟物+ATG7阴性对照+耐舒尼替尼U251组、miR-373-3p模拟物+ATG7过表达+耐舒尼替尼U251组,转染后采用CCK-8法检测细胞活性,TUNEL染色检测细胞凋亡率,Western blotting检测细胞凋亡和自噬相关蛋白的表达。结果(1)与U251细胞比较,耐舒尼替尼U251细胞miR-373-3p的表达下调,差异有统计学意义(P<0.05)。与空白对照组、无义序列组比较,miR-373-3p模拟物组细胞miR-373-3p的表达升高,差异有统计学意义(P<0.05)。与U251组比较,耐舒尼替尼U251组细胞活性增加,凋亡率下降,B淋巴细胞瘤-2(Bcl-2)蛋白的表达升高,Bcl-2相关X蛋白(Bax)蛋白的表达和活化半胱氨酸天冬氨酸蛋白酶-3(Caspase 3)/Caspase 3值降低,Beclin 1蛋白的表达、LC3Ⅱ/LC3Ⅰ值增加,p62蛋白的表达减少。与无义序列+耐舒尼替尼U251组比较,miR-373-3p模拟物+耐舒尼替尼U251组细胞活性降低、凋亡率提高,Bcl-2蛋白的表达降低,Bax蛋白的表达和活化Caspase 3/Caspase 3值升高,Beclin 1蛋白的表达、LC3Ⅱ/LC3Ⅰ值降低,p62蛋白的表达增加,差异均有统计学意义(P<0.05);与U251组比较,耐舒尼替尼U251组LC3荧光颗粒增多且荧光亮度升高;与无义序列+耐舒尼替尼U251组比较,miR-373-3p模拟物+耐舒尼替尼U251组细胞荧光颗粒减少且亮度减弱。(2)对于pGL3-ATG7 WT质粒,miR-373-3p模拟物组荧光素酶活性低于无义序列组,差异有统计学意义(P<0.05)。与U251组比较,耐舒尼替尼U251组ATG7 mRNA和蛋白的表达增加;与无义序列+耐舒尼替尼U251组比较,miR-373-3p模拟物+耐舒尼替尼U251组ATG7 mRNA和蛋白的表达降低,差异均有统计学意义(P<0.05)。(3)与空白对照组、ATG7阴性对照组比较,ATG7过表达组细胞ATG7 mRNA和蛋白的表达均增加,差异均有统计学意义(P<0.05)。与无义序列+耐舒尼替尼U251组比较,miR-373-3p模拟物+耐舒尼替尼U251组细胞活性降低、凋亡率较高,Bcl-2蛋白的表达降低,活化Caspase 3/Caspase 3值、Bax蛋白的表达升高,Beclin 1蛋白的表达、LC3Ⅱ/LC3Ⅰ值降低,p62蛋白的表达升高;与miR-373-3p模拟物+ATG7阴性对照+耐舒尼替尼U251组比较,miR-373-3p模拟物+ATG7过表达+耐舒尼替尼U251组细胞活性增加,凋亡率降低,Bcl-2蛋白的表达升高,活化Caspase 3/Caspase 3值、Bax蛋白的表达降低,Beclin 1蛋白的表达、LC3Ⅱ/LC3Ⅰ值升高,p62蛋白的表达减少,差异均有统计学意义(P<0.05)。结论miR-373-3p通过调控自噬而增强胶质母细胞瘤细胞舒尼替尼敏感性,其机制可能与靶向ATG7有关。

Objective To investigate the influence and mechanism of micro RNA(miR)-373-3p in autophagy and sunitinib sensitivity of glioblastoma cells.Methods U251 cells were routinely cultured in vitro;and some U251 cells were subjected to 50μmol/L sunitinib treatment for 72 h to construct sunitinib-resistant U251 cell line.(1)Real-time reverse transcription quantitative PCR(RT-qPCR)was used to detect the miR-373-3p expression in U251 and sunitinib-resistant U251 cells.Sunitinib-resistant U251 cells were divided into blank control group,nonsense sequence group and miR-373-3p mimic group;cells in the latter 2 groups were transfected with nonsense sequence and miRNA-337-3p mimic,respectively;miR-373-3p expression was detected by RT-qPCR.Cells were divided into U251 group,sunitinib-resistant U251 group,sunitinib-resistant U251+nonsense sequence group,and sunitinib-resistant U251+miR-373-3p mimic group;after each transfection,CCK-8 assay was used to evaluate the cell viability;TUNEL was used to detect the apoptotic rate;immunofluorescent assay was used to detect the expression of microtubule-associated protein light chain 3(LC3);Western blotting was used to detect the expressions of apoptosis-and autophagy-associated proteins.(2)The pGL3-autophagy-related gene 7(ATG7)wild-type(WT)and pGL3-ATG7 mutant type(MUT)plasmids were established;dual-luciferase reporter system was used to detect the cell luciferase activity in the miR-373-3p mimic group and nonsense sequence group.Cells were divided into U251 group,sunitinib-resistant U251 group,sunitinib-resistant U251+nonsense sequence group,and sunitinib-resistant U251+miR-373-3p mimic group;after each transfection,RT-qPCR and Western blotting were used to detect the mRNA and protein expressions of ATG7 in the cells.(3)The sunitinib-resistant U251 cells were divided into blank control group,ATG7 negative control group,and ATG7 overexpression group;after each transfection,RT-qPCR and Western blotting were used to detect the ATG7 mRNA and protein expressions.U251 and sunitinib-resistant U251 cells were divided into U251 group,sunitinib-resistant U251 group,sunitinib-resistant U251+nonsense sequence group,sunitinib-resistant U251+miR-373-3p mimic group,sunitinib-resistant U251+miR-373-3p mimic+ATG7 negative control group,and sunitinib-resistant U251+miR-373-3p mimic+ATG7 overexpression group;after each transfection,CCK-8 assay was used to evaluate the cell apoptosis,TUNEL was used to examine the apoptotic rate,and Western blotting was employed to detect the expressions of apoptosis-and autophagy-associated proteins.Results(1)As compared with that in the U251 cells,miR-373-3p was lowly expressed in sunitinib-resistant U251 cells,with statistic difference(P<0.05).As compared with that in the blank control group and nonsense sequence group,miR-373-3p expression was significantly elevated in the miR-373-3p mimic group(P<0.05).As compared with the U251 group,the sunitinib-resistant U251 group had significantly increased cell viability,significantly decreased cell apoptotic rate,statistically increased B lymphocytoma-2(Bcl-2)and Beclin 1 protein expressions,and significantly increased LC3II/LC3I values,significantly decreased Bcl-2 associated X protein(Bax)and p62 protein expressions and cleaved Caspase3/Caspase 3 values(P<0.05).As compared with the sunitinib-resistant U251+nonsense sequence group,the sunitinib-resistant U251+miR-373-3p mimic group had significantly decreased cell viability,significantly increased cell apoptotic rate,statistically decreased Bcl-2 and Beclin 1 protein expressions,and significantly decreased LC3II/LC3I values,significantly increased Bax and p62 protein expressions and cleaved Caspase3/Caspase 3 values(P<0.05).As compared with the U251 group,the sunitinib-resistant U251 group had increased number of fluorescent particles labeled with LC3 and enhanced fluorescent intensity;as compared with the sunitinib-resistant U251+nonsense sequence group,the sunitinib-resistant U251+miR-373-3p mimic group had decreased number of fluorescent particles labeled with LC3 and reduced fluorescent intensity.(2)The luciferase activity of pGL3-ATG7 WT plasmids in the miR-373-3p mimic group was signficantly lower than that in nonsense sequence group(P<0.05).As compared with those in the U251 group,ATG7 mRNA and protein expressions were both significantly increased in the sunitinib-resistant U251 group(P<0.05);as compared with those in the sunitinib-resistant U251+nonsense sequence group,ATG7 mRNA and protein expressions were both significantly decreased in the sunitinib-resistant U251+miR-373-3p mimic group(P<0.05).(3)As compared with the blank control group and ATG7 negative control group,the ATG7 overexpression group had significantly increased ATG7 mRNA and protein expressions(P<0.05).As compared with the sunitinib-resistant U251+nonsense sequence group,the sunitinib-resistant U251+miR-373-3p mimic group had significantly decreased cell viability,significantly increased cell apoptotic rate,statistically decreased Bcl-2 and Beclin 1 protein expressions,significantly decreased LC3II/LC3I values,significantly increased Bax and p62 protein expressions,and significantly increased cleaved Caspase3/Caspase 3 values(P<0.05).As compared with the sunitinib-resistant U251+miR-373-3p mimic+ATG7 negative control group,the sunitinib-resistant U251+miR-373-3p mimic+ATG7 overexpression group had significantly increased cell viability,significantly decreased apoptotic rate,statistically increased Bcl-2 and Beclin 1 protein expressions,significantly increased LC3II/LC3I values,significantly decreased Bax and p62 protein expressions,and significantly decreased cleaved Caspase3/Caspase 3 values(P<0.05)Conclusion MiR-373-3p can enhance sunitinib sensitivity by regulating autophagy in glioblastoma cells,whose mechanism might be related to targeting ATG7.