吉林大学学报(医学版)  2020, Vol. 46 Issue (04): 693-698     DOI: 10.13481/j.1671-587x.20200405

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于雷, 王策, 韩冰, 李鑫, 韩雨辰, 孙宇莹, 郭湘舒, 刘威武, 王志成
YU Lei, WANG Ce, HAN Bing, LI Xin, HAN Yuchen, SUN Yuying, GUO Xiangshu, LIU Weiwu, WANG Zhicheng
线粒体靶向KillerRed增强辐射诱导HeLa细胞自噬作用及其机制
Enhancement of mitochondria-targeted KillerRed in autophagy caused by radiation in HeLa cells and its mechanism
吉林大学学报(医学版), 2020, 46(04): 693-698
Journal of Jilin University (Medicine Edition), 2020, 46(04): 693-698
10.13481/j.1671-587x.20200405

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收稿日期: 2019-10-22
线粒体靶向KillerRed增强辐射诱导HeLa细胞自噬作用及其机制
于雷1,2 , 王策 , 韩冰3 , 李鑫1,4 , 韩雨辰1 , 孙宇莹1 , 郭湘舒1 , 刘威武3 , 王志成1     
1. 吉林大学公共卫生学院 国家卫健委放射生物学重点实验室, 吉林 长春 130021;
2. 吉林大学第二医院放疗科, 吉林 长春 130041;
3. 吉林大学第二医院放射线科, 吉林 长春 130041;
4. 吉林省肿瘤医院放疗医技科, 吉林 长春 130012
[摘要]: 目的 探讨线粒体靶向KillerRed(mtKR)增强辐射诱导HeLa细胞线粒体失能及细胞自噬作用,并阐明其相关分子机制。方法 线粒体靶向质粒PTEN诱导激酶1(Pink1)-mtKR转染HeLa细胞后,进行可见光和4 Gy X射线照射,实验分为对照组、空载体组、Pink1-mtKR组、对照+4 Gy X射线照射组、空载体+4 GyX射线照射组和Pink1-mtKR+4 GyX射线照射组。X线照射后24 h,采用流式细胞术检测线粒体膜电位(MMP)和细胞自噬率,采用生化试剂盒检测线粒体呼吸链复合物Ⅰ和Ⅲ水平,采用Western blotting法检测自噬分子P62、Pink1、帕金蛋白(parkin)和线粒体外膜转换酶20(Tom20)的表达量。结果 Pink1-mtKR瞬时转染HeLa细胞后,给予可见光联合4 Gy X射线照射,与对照组比较,Pink1-mtKR组细胞MMP、线粒体呼吸链复合物Ⅰ和Ⅲ水平均明显降低(P < 0.05),细胞自噬率明显升高(P < 0.05),Pink1-mtKR+4 GyX射线照射组细胞MMP、线粒体呼吸链复合物Ⅰ和Ⅲ水平进一步降低(P < 0.05),自噬率进一步升高(P < 0.05)。与对照组比较,Pink1-mtKR组和Pink1-mtKR+4 Gy X射线照射组细胞总蛋白中Pink1和parkin蛋白表达量无明显变化,而P62蛋白表达量增加,Pink1-mtKR组和Pink1-mtKR+4 Gy X射线照射组细胞线粒体蛋白中Pink1、parkin和Tom 20蛋白表达量均明显增加。结论 mtKR可增强辐射诱导的线粒体失能和自噬,其机制可能涉及到Pink1/parkin自噬通路的调控。
关键词: 线粒体    活性氧    辐射    细胞自噬    线粒体靶向KillerRed    
Enhancement of mitochondria-targeted KillerRed in autophagy caused by radiation in HeLa cells and its mechanism
YU Lei1,2 , WANG Ce , HAN Bing3 , LI Xin1,4 , HAN Yuchen1 , SUN Yuying1 , GUO Xiangshu1 , LIU Weiwu3 , WANG Zhicheng1     
1. NHC Key laboratory of Radiobiology, School of Public Health, Jilin University, Changchun 130021, China;
2. Department of Radiotherapy, Second Hospital, Jilin University, Chanchun 130041, China;
3. Department of Radiology, Second Hospital, Jilin University, Chanchun 130041, China;
4. Department of Radiotherapy and Medical Technology, Jilin Cancer Hospital, Changchun 130012, China
[ABSTRACT]: Objective To explore the enhancement of mitochondira-targeted KillerRed(mtKR) on the mitochondrial dysfunciton and autophagy caused by radiation in the HeLa cells, and to clarify the relative molecular mechanisms. Methods After mitochondria-targeted expression vectors Pink-mtKR were transfected into the HeLa cells, the cells were irradiated by visible light and 4 Gy X-ray. The cells were divided into control, empty vector, Pink1-mtKR, control + 4 Gy X-ray irradiation, empty vector + 4 Gy X-ray irradiation and Pink1-mtKR+4 Gy X-ray irradiation groups. After the cells were irradiated with X-ray for 24 h, the mitochondrial membrane potentials (MMP) and the autophgic rates were detected by flow cytometry, the levels of mitochondrial respiratory chain complex Ⅰ and Ⅲ were measured by biochemical assay, and the expression amounts of P62, Pink1, parkin and Tom20 proteins were measured by Western blotting method. Results After Pink1-mtKR plasmids were transfected into the HeLa cells, the cells were irradiated by visible light and 4 Gy X-ray; compared with control group, the MMP and the levels of mitochondrial respiratory chain complex Ⅰand Ⅲ in Pink1-mtKR group were significantly decreased (P < 0.05), and the autohpagic rate was significantly increased (P < 0.05); the MMP and the levels of mitochondrial respiratory chain complex Ⅰ and Ⅲ in Pink1-mtKR + 4 Gy X-ray irradiation group were decreased obviously (P < 0.05), and the autohpagic rate was significantly increased(P < 0.05). Compared with control group, the expression amounts of Pink1 and parkin proteins in total protein of the cells in Pink1-mtKR group and Pink1-mtKR+4 Gy X-ray irradiation group had no obvious changes, but the expression amounts of the P62 protein was increased; the expression amounts of Tom20, Pink1 and parkin in mitochondrial protein in Pink1-mtKR group and Pink1-mtKR+4 Gy X-ray irradiation group were increased obviously. Conclusion MtKR may enhance the mitochondrial dysfunction and autohpagy caused by radiation, and its mechamism may be related to the regulation of Pink1/parkin autophgy pathway.
KEYWORDS: mitochondrion    reactive oxygen species    radiation    autophagy    mitochondrion-targeted KillerRed    

活性氧(reactive oxygen species, ROS)是一类由线粒体产生的活性分子,作用于线粒体后可以导致线粒体膜电位(mitochondrial membrance potential, MMP)的降低和线粒体呼吸链的损伤,进而引起线粒体失能,并诱导细胞自噬[1-3]。本课题组前期研究[4-5]显示:线粒体靶向KillerRed(mitochondrion-targeted KillerRed, mtKR)可以增强辐射所致的细胞增殖抑制和凋亡,但其具体机制尚未明确。在本研究中,以构建的mtKR质粒Pink1-mtKR转染宫颈癌HeLa细胞,采用可见光和4 Gy X射线联合照射后,检测细胞MMP和氧化呼吸链复合物Ⅰ和Ⅲ的活性等功能指标,并检测自噬和PTEN诱导假定激酶1/帕金蛋白(PTEN-inducedputativekinase1/parkin,Pink1/parkin)调控通路相关蛋白表达,阐明mtKR对辐射诱导的线粒体失能和细胞自噬的作用,探讨其相关的分子调控机制,为增强宫颈癌放疗效果提供新思路。

1 材料与方法 1.1 细胞、主要试剂和仪器

人宫颈癌HeLa细胞由本实验室保存。MEM完全培养基(美国Gibico公司),Hieff TransTM脂质体核酸转染试剂(上海翊圣生物科技有限公司),青、链霉素(美国Thermo Fisher Scientific公司),罗丹明123、线粒体呼吸链复合体Ⅰ和Ⅲ水平检测试剂盒(北京索莱宝科技有限公司),单丹磺酰尸胺(monodansylcadaverin, MDC,美国Sigma公司),线粒体分离试剂盒(碧云天生物技术有限公司),Pink1、parkin和线粒体外膜转位酶20(transcocase of outer mitochondrial membrance 20, Tom20)多克隆一抗(美国ImmunoWay公司),P62多克隆一抗(美国CST公司),热休克蛋白60(heat shock protein 60,Hsp60)和GAPDH兔多克隆一抗(美国Bioworld公司),辣根过氧化物酶标记的二抗(美国Santa Cruz公司),其他试剂均为国产分析纯。多功能微孔板检测仪(美国Biotek公司),X射线辐照仪(型号X-RAD 320iX,美国Precision X-ray股份有限公司)。

1.2 实验分组和照射

HeLa细胞分为对照组、空载体组、Pink1-mtKR组、对照+4 Gy X射线照射组、空载体+4 GyX射线照射组和Pink1-mtKR+4 Gy X射线照射组。无菌条件下避光可见光源照射细胞[5],时间为30 min,12 h后X射线照射,条件为电压180 kV,电流12.0 mA,靶皮距70 cm,剂量率1.0 Gy·min-1,单次照射剂量为4 Gy。

1.3 流式细胞术检测细胞MMP和自噬率

取对数生长期的HeLa细胞接种于24孔板中,细胞密度为0.7×105个/孔,瞬时转染空载体和Pink-mtKR质粒,30 h后给予30 min可见光照射,12 h后进行4 Gy X射线照射,于24 h时收集各组细胞,PBS洗涤3次。检测MMP时,加入300 μL PBS,重悬细胞,加入罗丹明123(终浓度5 μmol·L-1)于37℃避光反应30min,上机检测,以平均荧光强度(mean fluorescence intensity, MFI)表示MMP。加入10 mL MDC (5 mmol·L-1),37℃静置30 min后,1 000 r·min-1离心5 min,弃废液,加入4%多聚甲醛1 mL避光条件下固定15 min,离心后加入PBS洗涤1次,上机检测细胞自噬率。

1.4 细胞中线粒体呼吸链复合物Ⅰ和Ⅲ水平检测

HeLa细胞瞬时转染后,进行可见光照射和4 Gy X射线照射,24 h后按5 ×107个细胞加入1.0 mL提取液的比例加入提取液,用匀浆器在冰上匀浆,按照试剂盒说明书提取线粒体蛋白,并分别按照试剂盒说明书检测线粒体呼吸链复合物Ⅰ和Ⅲ水平。

1.5 Western blotting法检测细胞中Pink1、parkin、P62和Tom20蛋白表达量

HeLa细胞进行瞬时转染后,进行可见光和4 Gy X射线照射,24 h后分别提取总蛋白和线粒体蛋白,并测定蛋白浓度。40 μg蛋白变性后上样,浓缩胶80 V,分离胶120 V,SDS-PAGE电泳后转膜缓冲液4℃中过夜湿转;5%脱脂奶粉封闭1 h后,加入Pink1、parkin、P62和Tom20一抗(采用TBST配置,1:1 000)、GAPDH和HSP60一抗(1:500),37℃孵育2 h,TBST洗涤3次,每次10 min,加入辣根过氧化物酶标记的二抗(1:3 000)后37℃孵育1 h,TBST洗涤3次,ECL试剂盒进行反应,暗室中曝光,并拍照观察目的蛋白表达量。

1.6 统计学分析

采用SPSS24.0统计软件进行统计学分析。各组细胞MMP、自噬率和线粒体呼吸链复合物Ⅰ和Ⅲ水平以x±s表示,经正态性检验均符合正态分布,多组间样本均数比较采用方差分析,2组间比较采用Student’ s t检验。以P<0.05表示差异有统计学意义。

2 结果 2.1 各组细胞MMP

可见光与对照组比较,照射后24 h,Pink1-mtKR组HeLa细胞MMP明显降低(P<0.05),对照+ 4 GyX射线照射组、空载体+4 GyX射线照射组和Pink1-mtKR+4 GyX射线照射组HeLa细胞MMP降低更明显(P<0.05);与对照+4 GyX射线照射组比较,Pink1-mtKR + 4GyX射线照射组细胞MMP明显降低(P<0.05),且低于Pink1-mtKR组(P<0.05)。见表 1图 1

表 1 可见光和X射线照射后24 h各组细胞MMP及线粒体呼吸链复合物Ⅰ和Ⅲ水平 Tab. 1 MMP and levels of mitochondrial respiratory chain complex Ⅰ and Ⅲ of cells in various groups at 24 h after irradiation with visible light and 4 Gy X-ray 
(n=3, x±s)
Group MMP(MFI) Mitochondrial respiratory complex Ⅰ [cB/(nmol·L-1)] Mitochondrial respiratory complexe Ⅲ [cB/(nmol·L-1)]
Control 47.01±0.66 1.91±0.14 0.25±0.02
Empty vector 39.34±2.62 1.92±0.18 0.25±0.01
Pink1-mtKR 29.03±3.91* 1.18±0.09* 0.14±0.07*
Control+4 Gy X-ray irradiation 22.18±0.76* 0.93±0.13* 0.12±0.02*
Empty vector+4 Gy X-ray irradiation 21.41±0.95* 1.09±0.12* 0.14±0.04*
Pink1-mtKR +4 Gy X-ray irradiation 19.85±1.09*△# 0.26±0.11**△# 0.03±0.01**△#
*P<0.05,**P<0.01 vs control group; P<0.05 vs control+4 Gy X-ray irradiation group; #P<0.05 vs Pink1-mtKR group.
A: Control group; B: Empty vector group; C: Pink1-mtKR group; D: Control+4 Gy X-ray irradiation group; E: Empty vector+4 Gy X-ray irradiation group; F: Pink1-mtKR+4 Gy X-ray irradiation group. 图 1 可见光和4 Gy X射线照射后流式细胞术检测各组HeLa细胞MMP Fig. 1 MMP of HeLa cells in various groups detected by flow cytometry after irradiation with visible light and 4 Gy X-ray
2.2 各组细胞中线粒体呼吸链复合物Ⅰ和Ⅲ水平

与对照组比较,可见光照射后24 h,Pink1-mtKR组线粒体呼吸链复合物Ⅰ和Ⅲ水平明显降低(P<0.05),对照+4 GyX射线照射组、空载体+ 4 GyX射线照射组和Pink1-mtKR+4 GyX射线照射组细胞中线粒体呼吸链复合物Ⅰ和Ⅲ水平降低更明显(P<0.05或P<0.01);与对照+4 GyX射线照射组比较,Pink1-mtKR + 4 GyX射线照射组细胞中线粒体呼吸链复合物Ⅰ和Ⅲ水平明显降低(P<0.05),且低于Pink1-mtKR组(P<0.05)。见表 1

2.3 各组细胞自噬率

与对照组比较,可见光照射后12 h,空载体组和Pink1-mtKR组细胞自噬率明显变化(P>0.05);照射后24 h,Pink1-mtKR组细胞自噬率明显升高(P<0.05)。可见光和4 GyX射线联合照射后12和24 h,与对照组比较,对照+4GyX射线照射组、空载体+4GyX射线照射组和Pink1-mtKR+4GyX射线照射组细胞自噬率均明显升高(P<0.05)。与对照+4 Gy X射线照射组比较,照射后24 h,Pink1-mtKR+4 GyX射线照射组细胞自噬率明显升高(P<0.05);照射后12和24 h,Pink1-mtKR +4 GyX射线照射组细胞自噬率明显高于Pink1-mtKR组(P<0.05)。见表 2图 2

表 2 可见光和X射线照射后12和24 h各组HeLa细胞自噬率 Tab. 2 Autophagic rates of HeLa cells in HeLa various groups at 12 and 24 h after irradiation wtihvisible light and 4 Gy X-ray 
(n=3, x±s η/%)
Group Autophagic rate
(t/h) 12 24
Control 5.25±0.80 6.17±0.38
Empty vector 4.94±0.37 6.63±0.93
Pink1-mtKR 6.07±0.20 9.01±0.25*
Control+4 Gy X-ray irradiation 9.37±0.40* 20.51±0.51*
Empty vector+4 Gy X-ray irradiation 8.80±0.94* 20.37±0.61*
Pink1-mtKR+4 Gy X-ray irradiation 10.91±0.65*# 27.93±2.54*△#
*P<0.05 vs control group; P<0.05 vs control+4 Gy X-ray irradiation group;P<0.05 vs Pink1-mtKR group.
A-F: 12 h; G-L: 24 h; A, G: Control group; B, H: Empty vector group; C, I: Pink1-mtKR group; D, J: Control+4 Gy X-ray irradiation group; E, K: Empty vector+4 Gy X-ray irradiation group; F, L: Pink1-mtKR+4 Gy X-ray irradiation group. 图 2 可见光和X射线照射后12和24 h流式细胞术检测各组HeLa细胞自噬率 Fig. 2 Autophgic rates of HeLa cells in various groups detected by flow cytometry after irradiation with visible light and 4 Gy X-ray
2.4 各组细胞中自噬相关蛋白表达量

在总蛋白中,各组细胞中Pink1、parkin和Tom20蛋白表达量无明显变化,但Pink1-mtKR组P62蛋白表达量增加,Pink1-mtKR+4 GyX射线照射组增加更明显;在线粒体蛋白中,Pink1-mtKR组和Pink1-mtKR + 4 GyX射线照射组细胞中Pink1、parkin和Tom20蛋白表达量均增加。见图 3

A: Total protein; B: Mitochondrial protein; Lane 1:Control group; Lane 2: Empty vector group; Lane 3: Pink1-mtKR group; Lane 4: Control+4 Gy X-ray irradiationgroup; Lane 5: Empty vector+4 Gy X-ray irradiationgroup; Lane 6: Pink1-mtKR+4 Gy X-ray irradiationgroup. 图 3 Western blotting法检测各组HeLa细胞中自噬相关蛋白表达电泳图 Fig. 3 Electrophoregram of expressions of autophagic proteins in HeLa cells in various groups detected by Western blotting method
3 讨论

宫颈癌是妇科常见的恶性肿瘤,对于中晚期宫颈癌患者,放射治疗是首选治疗方式[6-7]。放射治疗主要通过射线的直接作用而损伤细胞;射线间接作用时,主要通过自由基对细胞产生作用。ROS主要由氧化磷酸化途径产生,此过程依赖于线粒体氧化呼吸链复合物Ⅰ和Ⅲ水平。低水平ROS能够发挥细胞正常生理功能,而高水平ROS则诱导氧化应激,损伤线粒体,甚至损伤线粒体DNA[8]。正常生理条件下,ROS可以被抗氧化系统清除,一旦ROS产生过量,则会损伤线粒体和细胞[9-10]。而线粒体是作为细胞主要的能量和代谢源,也是ROS的重要靶点。如何通过靶向线粒体改变其功能而提高治疗肿瘤效果,已引起研究者的广泛关注。基于过量ROS可导致氧化损伤的理论,放疗可以通过诱导ROS而致细胞凋亡和自噬,达到杀伤肿瘤细胞的目的[11-13],大量的实验证据也证明了这一点。如何诱导足量的ROS并且靶向线粒体,最终杀伤肿瘤细胞,是一个重要的研究方向。

研究[4-5]显示:基于线粒体靶向的序列Pink1介导mtKR蛋白,可以实现线粒体靶向诱导ROS产生,且增加辐射诱导的HeLa细胞增殖抑制,可能与促进凋亡有关,但其他机制仍需进一步研究。线粒体主要功能包括产生ATP,90%的ATP由线粒体产生,并在线粒体膜上产生MMP,MMP降低时主要发生线粒体失能表型,而线粒体失能会导致后续的细胞凋亡、自噬和侵袭过程[14-15]。在本研究中,转染了Pink1-mtKR质粒的HeLa细胞中,线粒体呼吸链复合物Ⅰ和Ⅲ水平较对照组和空载体组明显降低,且MMP也明显降低,证明Pink1-mtKR在可见光诱导下可以引起HeLa细胞的线粒体失能。线粒体失能在氧化应激中发挥重要作用,ROS的产生进一步损伤线粒体的电子传递链[16]

已有文献[17-19]显示:线粒体ROS产生和线粒体脂质的氧化在细胞自噬中起重要作用,是自噬的重要参与者。Pink1存在于大多数线粒体膜中,但通常会降解。当发生线粒体损伤时,Pink1不会降解,会使parkin募集并磷酸化,从而引发线粒体自噬,Pink1敲除小鼠表现出线粒体呼吸活动受损和对氧化应激的敏感性增加[20]。上述结果表明:Pink1/parkin在线粒体自噬和ROS调节的线粒体生成中起作用。许多线粒体自噬的调节因子可以受到ROS的调节,通过ROS诱导MMP降低,可看作是线粒体自噬的一种信号。本研究通过mtKR诱导HeLa细胞线粒体失能,阐明了mtKR在光诱导下导致HeLa细胞自噬的相关机制,即KR靶向线粒体,通过ROS导致MMP去极化,Pink1在线粒体外膜大量累积,招募parkin并使其磷酸化,从而泛素化各种外膜蛋白,进而招募P62等蛋白,以启动线粒体自噬。

本研究结果表明:Pink1-mtKR转染的HeLa细胞经可见光和X射线照射后可以诱导MMP和线粒体呼吸链复合物Ⅰ、Ⅲ水平均降低,mtKR可增加由辐射诱导的细胞自噬,且与Pink1/parkin线粒体自噬途径有关。mtKR引起的线粒体失能和最终的细胞死亡为肿瘤的辐射增敏提供了一个新的思路。

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