Mesothelin对乳腺癌细胞MDA-MB-231生物学行为的影响

doi:10.11958/20230452

天津医药 ›› 2023, Vol. 51 ›› Issue (10): 1032-1039.doi: 10.11958/20230452

Mesothelin对乳腺癌细胞MDA-MB-231生物学行为的影响

李宇翔¹(), 李葛¹, 高振芳¹, 侯春梅¹, 韩根成¹, 孙慧燕², 王立生³

1.军事科学院军事医学研究院军事认知与脑科学研究所（邮编100850）
2.河北燕达医学研究院
3.青岛大学附属医院分子诊断与再生医学实验室

收稿日期:2023-03-28 修回日期:2023-06-28 出版日期:2023-10-15 发布日期:2023-10-18
作者简介:李宇翔（1989），男，助理研究员，主要从事肿瘤免疫逃逸与肿瘤治疗机制方面研究。E-mail：liyuxiang_sd@163.com
基金资助:
国家自然科学基金资助项目(82301992);科技创新2030-“脑科学与类脑研究”重大项目(2021ZD0201600);河北省重点研发计划资助项目(19272405D)

Effects of Mesothelin on proliferation, apoptosis, migration and invasion of breast cancer MDA-MB-231 cells

LI Yuxiang¹(), LI Ge¹, GAO Zhenfang¹, HOU Chunmei¹, HAN Gencheng¹, SUN Huiyan², WANG Lisheng³

1. Institute of Military Cognition and Brain Sciences, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100850, China
2. Hebei Yanda Medical Research Institute
3. Laboratory of Molecular Diagnosis and Regenerative Medicine, Medical Research Center, Affiliated Hospital of Qingdao University

Received:2023-03-28 Revised:2023-06-28 Published:2023-10-15 Online:2023-10-18

李宇翔, 李葛, 高振芳, 侯春梅, 韩根成, 孙慧燕, 王立生. Mesothelin对乳腺癌细胞MDA-MB-231生物学行为的影响[J]. 天津医药, 2023, 51(10): 1032-1039.

LI Yuxiang, LI Ge, GAO Zhenfang, HOU Chunmei, HAN Gencheng, SUN Huiyan, WANG Lisheng. Effects of Mesothelin on proliferation, apoptosis, migration and invasion of breast cancer MDA-MB-231 cells[J]. Tianjin Medical Journal, 2023, 51(10): 1032-1039.

摘要/Abstract

摘要：

目的探讨间皮素（Mesothelin）对三阴性乳腺癌（TNBC）细胞增殖、凋亡、迁移及侵袭能力的影响。方法免疫化学染色检测TNBC组织、癌旁组织及乳腺癌细胞系中Mesothelin的表达。将过表达Mesothelin慢病毒质粒（Mesothelin组）及对照Scramble质粒（Scramble组）转染TNBC细胞系MDA-MB-231细胞，未感染细胞作为Control组。流式细胞术检测病毒感染效率；实时荧光定量PCR、流式细胞术及Western blot检测Mesothelin表达；CCK-8检测细胞增殖情况；利用Annexin V-APC/PI检测细胞凋亡；Western blot检测B淋巴细胞瘤-2（Bcl-2）、Bcl-2关联X蛋白（Bax）、E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）、波形蛋白（Vimentin）表达；划痕实验检测细胞迁移能力；Transwell实验检测细胞的侵袭能力。结果 Mesothelin在TNBC肿瘤组织中表达高于癌旁组织（P<0.05），在乳腺癌细胞系中有表达。流式细胞术验证发现慢病毒感染效率达到90%以上。与Scramble组相比，Mesothelin组Mesothelin mRNA和蛋白表达水平升高，细胞增殖率升高，细胞凋亡率降低，Bax、E-cadherin蛋白表达减少，Bcl-2、N-cadherin和Vimentin蛋白表达增加，细胞的迁移及侵袭能力增强（P<0.05）。结论 Mesothelin能够促进乳腺癌细胞增殖，抑制细胞凋亡，增加细胞的迁移及侵袭能力。

关键词: 三阴性乳腺癌, 细胞增殖, 细胞凋亡, 细胞运动, 间皮素

Abstract:

Objective To investigate the effect of Mesothelin on proliferation, apoptosis, migration and invasion of human triple-negative breast cancer cells (TNBC). Methods Immunohistochemical staining was used to detect the expression of Mesothelin in TNBC tissue, paracancer tissue and breast cancer cell lines. TNBC cell line MD-MB-231 cells were transfected with the overexpressed Mesothelin lentivirus plasmid (the mesothelin group) and the control Scramble plasmid (the Scramble group). MD-MB-231 cells with no infected lentivirus were used as the control group. Flow cytometry was used to detect the efficiency of virus infection. The expression of Mesothelin was detected by qPCR, flow cytometry and Western blot assay. Cell proliferation was detected by CCK-8. Annexin V-APC/PI was used to detect apoptosis. The expression of anti-B lymphoblastoma-2 (Bcl-2), anti-Bcl-2 associated X protein (Bax), E-cadherin, N-cadherin and Vimentin were detected by Western blot assay. Cell migration ability was detected by scratch test. The invasive ability of cells was detected by Transwell assay. Results The expression of Mesothelin was significantly higher in TNBC tumor tissue than that in paracancer tissue (P<0.05), and Mesothelin was expressed in breast cancer cell line. Flow cytometry showed that the efficiency of lentivirus infection was more than 90%. Compared with the Scramble group, the expression level of Mesothelin mRNA was increased in the Mesothelin group, and the cell proliferation rate was increased, while the cell apoptosis rate was decreased. The expression of Bax and E-cadherin protein decreased, the expression of Bcl-2, N-cadherin and Vimentin protein increased, and the migration and invasion ability of cells were significantly enhanced (P<0.05). Conclusion Mesothelin can promote the proliferation, inhibit the apoptosis and increase the ability of migration and invasion of human triple-negative breast cancer MDA-MB-231 cells.

Key words: triple negative breastneoplasms, cell proliferation, apoptosis, cell movement, Mesothelin

中图分类号:

R737.9

图/表 13

图1 Mesothelin在乳腺癌组织和癌旁组织中的表达（DAB染色）

Fig.1 Mesothelin expression in carcinoma tissue and corresponding para-carcinoma tissue of breast cancer patients (DAB staining)

图2 Mesothelin在乳腺癌细胞系中的表达（DAB染色，×200）

Fig.2 Mesothelin expression in breast cancer cell lines (DAB staining, ×200)

图3 慢病毒转染效率的检测

Fig.3 Detection of transfection efficiency of lentivirus

表1 各组细胞中Mesothelin阳性细胞比例、Mesothelin mRNA和蛋白表达水平比较 (n=3,$\bar{x}±s$)

Tab.1 Comparison of the proportion of mesothelin positive cells, relative mRNA and protein expression of mesothelin between three groups of MDA-MB-231 cells

组别	Mesothelin阳性细胞比例/%	Mesothelin mRNA	Mesothelin 蛋白
Control组	8.67±0.97	1.03±0.10	0.17±0.02
Scramble组	9.74±1.69	2.09±0.33	0.17±0.03
Mesothelin组	91.60±1.99^ab	35.02±3.98^ab	0.88±0.04^ab
F	2 627.000^＊＊	210.600^＊＊	517.000^＊＊

图4 慢病毒感染MDA-MB-231细胞检测Mesothelin的表达

Fig.4 Lentivirus mediated overexpression of Mesothelin in MDA-MB-231 cells

表2 各组细胞不同时间点增殖率比较 (n=3,%,$\bar{x}±s$)

Tab.2 Comparison of proliferation in different time points between three groups of cells

组别		0 h		24 h
Control组		103.96±5.09		205.41±7.25
Scramble组		101.30±2.03		200.78±11.19
Mesothelin组		102.41±5.93		217.59±7.27
F		0.247		2.942
组别	48 h		72 h		96 h
Control组	255.92±8.16		331.49±20.42		522.72±15.75
Scramble组	235.03±18.07		290.10±17.78		475.78±52.99
Mesothelin组	263.15±17.13		529.60±32.30^ab		756.71±49.05^ab
F	2.797		83.020^＊＊		37.320^＊＊

表3 各组细胞凋亡、迁移和侵袭情况比较 (n=3,$\bar{x}±s$)

Tab.3 Comparison of apoptosis rate, migration and invasion between three groups of cells

组别	24 h凋亡率/%		48 h凋亡率/%		12 h愈合率/%
Control组	7.18±0.96		12.72±0.79		12.68±1.39
Scramble组	8.51±0.55		13.77±1.04		12.73±3.50
Mesothelin组	3.44±0.36^ab		8.43±0.84^ab		27.40±0.42^ab
F	46.380^＊＊		29.870^＊＊		45.050^＊＊
组别		24 h愈合率/%		24 h侵袭细胞数/（个/视野）
Control组		24.86±4.59		13.67±0.58
Scramble组		25.94±8.15		17.33±2.31
Mesothelin组		60.03±6.55^ab		69.67±8.50^ab
F		27.590^＊＊		113.200^＊＊

图5 过表达Mesothelin对MDA-MB-231细胞凋亡的影响

Fig.5 Effect of mesothelin overexpression on the apoptosis of MDA-MB-231 cells

图6 过表达Mesothelin对MDA-MB-231细胞凋亡相关蛋白表达的影响

Fig.6 Effect of Mesothelin overexpression on the expression of apoptosis-related protein in MDA-MB-231 cells

表4 各组细胞中Bax、Bcl-2、E-cadherin、N-cadherin和Vimentin表达水平比较 (n=3,$\bar{x}±s$)

Tab.4 Comparison of relative expression levels of Mesothelin, Bax, Bcl-2, E-cadherin, N-cadherin and Vimentin between three groups of cells

组别	Bax	Bcl-2	E-cadherin	N-cadherin	Vimentin
Control组	1.13±0.03	0.67±0.03	0.70±0.02	0.54±0.03	0.50±0.04
Scramble组	1.07±0.02	0.60±0.05	0.71±0.01	0.47±0.03	0.52±0.06
Mesothelin组	0.49±0.04^ab	1.48±0.03^ab	0.57±0.00^ab	0.98±0.04^ab	1.62±0.15^ab
F	411.100^＊＊	431.900^＊＊	88.370^＊＊	224.400^＊＊	137.500^＊＊

图7 过表达Mesothelin对MDA-MB-231细胞迁移能力的影响（×50）

Fig.7 Effect of Mesothelin overexpression on the migration of MDA-MB-231 cells (×50)

图8 过表达Mesothelin对MDA-MB-231细胞侵袭能力的影响（结晶紫染色，×200）

Fig.8 Effect of Mesothelin overexpression on the invasion of MDA-MB-231 cells (Crystal violet staining, ×200)

图9 过表达Mesothelin对MDA-MB-231细胞EMT相关蛋白表达的影响

Fig.9 Effect of Mesothelin overexpression on the expression of EMT-related proteins in MDA-MB-231 cells

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Mesothelin对乳腺癌细胞MDA-MB-231生物学行为的影响

Effects of Mesothelin on proliferation, apoptosis, migration and invasion of breast cancer MDA-MB-231 cells

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