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Src羧基端激酶结合蛋白影响线粒体分裂及NK细胞杀伤肺癌细胞活性的分子机制探讨

《现代肿瘤医学》[ISSN:1672-4992/CN:61-1415/R]

期数:
2021年23期
页码:
4073-4080
栏目:
论著（基础研究）
出版日期:
2021-10-29

文章信息/Info

Title:
The molecular mechanism of csk-binding protein affecting mitochondrial division and NK cell killing lung cancer cell activity
作者:
董 良1; 李洪霖1; 杨 清2; 段 铮1; 孙明月1; 许彦超3; 马纯政1
1.河南省中医院肿瘤科；2.医务科，河南 郑州 450000; 3.河南中医药大学，河南 郑州 450000
Author(s):
DONG Liang1; LI Honglin1; YANG Qing2; DUAN Zheng1; SUN Mingyue1; XU Yanchao3; MA Chunzheng1
1.Department of Oncology;2.Department of Medical Affairs,Henan Provincial Hospital of Traditional Chinese Medicine,Henan Zhengzhou 450000，China;3.Henan University of Traditional Chinese Medicine,Henan Zhengzhou 450000，China.
关键词:
Src羧基端激酶结合蛋白; 肺癌; 线粒体分裂; NK细胞; 杀伤
Keywords:
csk-binding protein; lung cancer; mitochondrial division; NK cells; killing
分类号:
R734.2
DOI:
10.3969/j.issn.1672-4992.2021.23.001
文献标识码:
A
摘要:
目的：探究Src羧基端激酶结合蛋白（csk-binding protein，CBP）在人肺癌组织中的表达水平，及其对线粒体分裂和自然杀伤（natural killer,NK)细胞杀伤肺癌细胞活性的影响。方法：免疫组织化学染色和实时荧光定量PCR（qRT-PCR）检测肺癌组织及癌旁组织中CBP的表达；含CBP慢病毒感染人肺癌细胞株A549，通过荧光倒置显微镜、qRT-PCR 和Western blot检测细胞感染效果；CCK-8法检测各组A549细胞活性，Mito-Tracker染色观察各组A549细胞内线粒体形态和长度变化，Western blot检测各组A549细胞线粒体动态相关蛋白Mfn1、Mfn2、Drp1的表达，免疫荧光染色检测各组A549细胞内细胞色素C（Cyt C）的表达，流式细胞术检测各组A549细胞凋亡情况；从人外周血单个核细胞（peripheral blood mononuclear cell,PBMC）中分离NK细胞并进行鉴定，乳酸脱氢酶释放实验检测NK细胞对各组A549细胞的杀伤率。结果：肺癌组织中CBP的表达水平较癌旁组织中明显下降（P＜0.01）；感染CBP过表达慢病毒的A549细胞中CBP mRNA和蛋白的相对表达量均升高，培养48 h、72 h、96 h后细胞增殖活性下降，线粒体呈椭圆状或短杆状，长度明显缩短，线粒体分裂相关蛋白Mfn1、Mfn2蛋白表达减少，Drp1蛋白表达增加，有大量Cyt C从线粒体释放，细胞凋亡率升高，同时NK细胞对A549细胞的杀伤率明显提高，差异均具有统计学意义（P＜0.01）；而在使用线粒体分裂蛋白抑制剂Mdivi-1预先处理A549细胞后再感染CBP过表达慢病毒，线粒体分裂受到抑制，Mfn1、Mfn2蛋白表达增加，Drp1蛋白表达减少，Cyt C释放减少，细胞凋亡率降低，NK细胞对A549细胞的杀伤率也受到抑制，差异均具有统计学意义（P＜0.01）。结论：CBP在人肺癌组织中低表达，在A549细胞中过表达CBP能够促进线粒体分裂与细胞凋亡，并提高NK细胞对A549细胞的杀伤率。
Abstract:
Objective:To explore the expression level of csk-binding protein(CBP) in human lung cancer tissues and its effect on mitochondrial division and NK cell killing lung cancer cell activity.Methods:Immunohistochemical staining and real-time fluorescent quantitative PCR(qRT-PCR) were used to detect the expression of CBP in lung cancer tissues and adjacent tissues.CBP-containing lentivirus was used to infect human lung cancer cell line A549,and the effect of cell infection was detected by fluorescence inverted microscope,qRT-PCR and Western blot.CCK-8 method was used to detect the activity of A549 cells in each group.Mito-Tracker staining was used to observe the changes of mitochondrial morphology and length in A549 cells of each group.Western blot was used to detect the expression of mitochondrial dynamic related proteins Mfn1,Mfn2 and Drp1 in each group of A549 cells.Immunofluorescence staining was used to detect the expression of cytochrome C(Cyt C) in A549 cells in each group.Flow cytometry was used to detect the apoptosis of A549 cells in each group.NK cells were isolated and identified from human peripheral blood mononuclear cells(PBMC).Lactate dehydrogenase release experiment was used to detect the killing rate of NK cells to A549 cells in each group.Results:The expression level of CBP in lung cancer tissues was significantly lower than that in adjacent tissues(P＜0.01).The relative expression levels of CBP mRNA and protein in A549 cells infected with CBP overexpressing lentivirus increased.After culturing 48 h,72 h,96 h,the cell proliferation activity decreased.The mitochondria were oval or short rod-shaped.The length of mitochondria was significantly shortened.The protein expression of Mfn1 and Mfn2 decreased,and the protein expression of Drp1 increased.A large amount of Cyt C was released from mitochondria,and the rate of apoptosis increased,at the same time,the killing rate of NK cells to A549 cells was significantly increased,and the difference was statistically significant(P＜0.01).However,after pre-treatment of A549 cells with the inhibitor of mitochondrial division protein Mdivi-1 and infection with CBP overexpression lentivirus,mitochondrial division was inhibited,the protein expression of Mfn1 and Mfn2 increased,the protein expression of Drp1 decreased,the release of Cyt C decreased,the cell apoptosis rate was reduced,and the killing rate of NK cells on A549 cells was also inhibited.The difference was statistically significant(P＜0.01).Conclusion:CBP is low expressed in human lung cancer tissues.Overexpression of CBP in A549 cells can promote mitochondrial division and apoptosis,and increase the killing rate of NK cells to A549 cells.

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备注/Memo

备注/Memo:
National Natural Science Foundation of China(No.81804057）；国家自然科学基金青年项目（编号：81804057）；河南省科技厅科技攻关课题（编号：212102311118）；河南省中医管理局课题（编号：2019JDZX031）；河南省中医药管理局中医临床基地项目（编号：2017JDZX016)
更新日期/Last Update: 2021-10-29