NF2/YAP信号通路通过FSP1诱导CD24高表达的三阴性乳腺癌细胞铁死亡

doi:10.3877/cma.j.issn.1674-0807.2024.04.003

中华乳腺病杂志(电子版) ›› 2024, Vol. 18 ›› Issue (04) : 206 -211. doi: 10.3877/cma.j.issn.1674-0807.2024.04.003

论著

NF2/YAP信号通路通过FSP1诱导CD24高表达的三阴性乳腺癌细胞铁死亡

蒲卢兰¹, 李静佳², 陈宇¹, 周瑜清¹, 荣欣欣¹, 侯令密², 周方方¹^,^†(

)

^1. 637000　四川南充，川北医学院基础医学与法医学院
^2. 637000　四川南充，川北医学院附属医院甲状腺乳腺外科/乳腺癌生物靶向实验室

收稿日期:2023-11-14 出版日期:2024-08-01
通信作者: 周方方
基金资助:
四川省科技厅自然科学基金面上项目(2022NSFSC0775); 四川省基层卫生事业发展研究中心资助项目(SWFZ22-C-82); 南充市科技局市校科技战略合作项目(20SXQT0052); 川北医学院校级科研发展计划项目(CBY22-QNA11)

NF2/YAP signaling pathway induces ferroptosis in triple negative breast cancer cells with high expression of CD24 by FSP1

Lulan Pu¹, Jingjia Li², Yu Chen¹, Yuqing Zhou¹, Xinxin Rong¹, Lingmi Hou², Fangfang Zhou¹^,^†()

^1. School of Basic Medicine and Forensic Medicine, North Sichuan Medical College, Nanchong 637000, China
^2. Department of Breast and Thyroid Surgery, Biological Targeting Laboratory of Breast Cancer, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, China

Received:2023-11-14 Published:2024-08-01
Corresponding author: Fangfang Zhou

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引用本文：

蒲卢兰, 李静佳, 陈宇, 周瑜清, 荣欣欣, 侯令密, 周方方. NF2/YAP信号通路通过FSP1诱导CD24高表达的三阴性乳腺癌细胞铁死亡[J]. 中华乳腺病杂志(电子版), 2024, 18(04): 206-211.

Lulan Pu, Jingjia Li, Yu Chen, Yuqing Zhou, Xinxin Rong, Lingmi Hou, Fangfang Zhou. NF2/YAP signaling pathway induces ferroptosis in triple negative breast cancer cells with high expression of CD24 by FSP1[J]. Chinese Journal of Breast Disease(Electronic Edition), 2024, 18(04): 206-211.

目的

研究NF2和YAP表达对CD24高表达的三阴性乳腺癌细胞铁死亡的影响，探讨铁死亡的相关分子机制。

方法

培养CD24^high和CD24^lowMDA-MB-231细胞，通过台盼蓝染色(TBA)实验检测经半胱氨酸饥饿、铁死亡诱导剂Erastin和RSL3处理后细胞的死亡情况，TBA法检测丙二醛(MDA)含量，分析细胞铁死亡水平。使用Western blot检测NF2和YAP在CD24^high和CD24^lowMDA-MB-231细胞中的蛋白表达情况，并用慢病毒分别进行敲除和过表达，观察细胞铁死亡水平，并采用实时定量RT-PCR和Western blot检测成纤维细胞特异蛋白1(FSP1)的表达变化。

结果

经半胱氨酸饥饿、铁死亡诱导剂Erastin和RSL3分别处理后MDA-MB-231细胞死亡率增加，CD24^low细胞死亡率高于CD24^high细胞(t＝14.548，P<0.001；t＝8.310，P＝0.001；t＝8.600，P＝0.001)。同时，TBA法结果显示，在含或不含半胱氨酸条件下，CD24^high和CD24^lowMDA-MB-231细胞的MDA含量差异有统计学意义(t＝－3.920，P＝0.017；t＝11.566，P<0.001)；与对照组(DMSO)相比，CD24^high和CD24^low细胞经铁死亡诱导剂Erastin和RSL3处理后MDA水平增加，其中CD24^low细胞的MDA含量比CD24^high更高(t＝10.763，P＝0.006；t＝24.067，P<0.001)。Western blot实验结果表明，NF2与YAP在CD24^low和CD24^high细胞的蛋白含量差异有统计学意义(t＝－4.331，P＝0.012；t＝4.219，P＝0.013)，CD24^low细胞中YAP的蛋白表达更高(t＝4.219，P＝0.013)，而CD24^high细胞中NF2的蛋白表达更高(t＝－4.331，P＝0.012)。在CD24^highMDA-MB-231细胞中构建NF2敲除或(和)YAP过表达细胞株，并用实时定量RT-PCR和Western blot检测，结果显示铁死亡关键蛋白FSP1在4组间的表达差异有统计学意义(F＝30.297，P<0.001)。同时，检测NF2敲除或(和)YAP过表达后CD24^highMDA-MB-231细胞的铁死亡情况，结果表明敲除NF2或(和)过表达YAP后加入铁死亡诱导剂Erastin均可在一定程度上促进细胞死亡(F＝38.911，P<0.050)，其中在NF2敲除联合YAP过表达条件下，CD24^highMDA-MB-231细胞死亡率最高，而在Erastin的基础上添加铁死亡抑制剂Fer-1后细胞的死亡率下降，4组间比较差异无统计学意义(F＝0.256，P＝0.855)。

结论

在CD24高表达的三阴性乳腺癌细胞中敲除NF2联合过表达YAP会抑制FSP1表达，促进细胞铁死亡。

关键词: 乳腺肿瘤, 铁死亡, 成纤维细胞特异蛋白1, NF2, YAP

Objective

To observe the impact of NF2 and YAP expression on ferroptosis of triple negative breast cancer cells with high expression of CD24 and explore the related molecular mechanism.

Methods

MDA-MB-231 breast cancer cells with high expression of CD24 (CD24^high) and low expression of CD24 (CD24^low) were cultured. The cells were treated with ferroptosis agonists Erastin, RSL3 and cysteine starvation. The cell death was detected by Trypan blue staining (TBA). Malondialdehyde(MDA)content was measured using the TBA method to analyze the level of cellular ferroptosis. Western blot analysis was used to detect the protein expression of NF2 and YAP in CD24^high and CD24^low MDA-MB-231 cells. Lentiviral vectors were used to knock out and over-express NF2 and YAP, and changes in cellular ferroptosis were observed. The real-time quantitative RT-PCR and Western blot analysis were used to detect the expression of fibroblast-specific protein 1(FSP1)after NF2 knock out and YAP over-expression.

Results

After treatment with cysteine starvation, the ferroptosis inducers Erastin and RSL3, the death rate of MDA-MB-231 cells increased, with the death rate of CD24^low cells significantly higher than that of CD24^high cells (t=14.548, P<0.001; t=8.310, P=0.001; t=8.600, P=0.001). Meanwhile, the TBA method results showed that there was a significant difference in MDA content between CD24^high and CD24^low MDA-MB-231 cells under conditions with or without cysteine (t=-3.920, P=0.017; t=11.566, P<0.001); compared with the control group with DMSO, the MDA levels of CD24^high and CD24^low cells significantly increased after treatment with the ferroptosis inducers Erastin and RSL3, with the MDA content of CD24^low cells significantly higher than that of CD24^high cells (t=10.763, P=0.006; t=24.067, P<0.001). Western blot experiments showed that there was a significant difference in the protein content of NF2 and YAP between CD24^low and CD24^high MDA-MB-231 cells (t=-4.331, P=0.012; t=4.219, P=0.013), with higher expression of YAP protein in CD24^low cells (t=4.219, P=0.013) and higher expression of NF2 protein in CD24^high cells (t=-4.331, P=0.012). NF2 knockout or (and) YAP overexpression cell lines were constructed in CD24^high cells, and real-time quantitative RT-PCR and Western blot analysis were used to detect the expression of the ferroptosis key protein FSP1 among the four groups (F=30.297, P<0.001). Meanwhile, the ferroptosis situation of CD24^high MDA-MB-231 cells after NF2 knockout or (and) YAP overexpression was detected, and the results showed that knocking out NF2 or (and) overexpressing YAP could promote cell death to a certain extent after adding the ferroptosis inducer Erastin (F=38.911, P<0.050), with the highest death rate of CD24^high MDA-MB-231 cells under the condition of NF2 knockout combined with YAP overexpression. The addition of the ferroptosis inhibitor Fer-1 after Erastin led to a decrease in the death rate of cells, with no statistically significant difference among the four groups (F=0.256, P=0.855).

Conclusions

Knocking out NF2 and overexpressing YAP in CD24^high triple negative breast cancer cells can inhibit the expression of FSP1 and promote cell ferroptosis.

Key words: Breast neoplasms, Ferroptosis, Fibroblast-specific protein 1, NF2, YAP

表1 实时荧光定量反转录PCR引物序列

引物名称	引物序列(5’→3’)
NF2	正向：GTGCCATACTAAGCAGCTTGC
NF2	反向：ACACTACAGCTCGGAAATCCA
YAP	正向：GTCGGAACCCTTCCCTCATTA
YAP	反向：GGCCTTCACTGACAGGTAGTA
FSP1	正向：ATCGTCCGAGATGATGTTGATGGG
FSP1	反向：ATTACTGCTACTCCGTACACCACTG
GAPDH	正向：TGACTTCAACAGCGACACCCA
GAPDH	反向：CACCCTGTTGCTGTAGCCAAA

表1 实时荧光定量反转录PCR引物序列

引物名称	引物序列(5’→3’)
NF2	正向：GTGCCATACTAAGCAGCTTGC
NF2	反向：ACACTACAGCTCGGAAATCCA
YAP	正向：GTCGGAACCCTTCCCTCATTA
YAP	反向：GGCCTTCACTGACAGGTAGTA
FSP1	正向：ATCGTCCGAGATGATGTTGATGGG
FSP1	反向：ATTACTGCTACTCCGTACACCACTG
GAPDH	正向：TGACTTCAACAGCGACACCCA
GAPDH	反向：CACCCTGTTGCTGTAGCCAAA

表2 半胱氨酸饥饿处理后CD24^low与CD24^high细胞死亡率比较

组别	实验次数	含半胱氨酸	半胱氨酸饥饿
CD24^low	3	4.60±0.63	28.93±0.40
CD24^high	3	3.83±0.81	12.60±1.82
t值		1.301	14.548
P值		0.263	<0.001

表2 半胱氨酸饥饿处理后CD24^low与CD24^high细胞死亡率比较

组别	实验次数	含半胱氨酸	半胱氨酸饥饿
CD24^low	3	4.60±0.63	28.93±0.40
CD24^high	3	3.83±0.81	12.60±1.82
t值		1.301	14.548
P值		0.263	<0.001

表3 铁死亡诱导剂处理CD24^low与CD24^high细胞死亡率比较

组别	实验次数	DMSO	Erastin	RSL3
CD24^low	3	4.93±1.36	38.15±2.92	39.51±2.16
CD24^high	3	3.61±1.12	14.73±3.91	18.90±1.79
t值		1.292	8.310	8.600
P值		0.266	0.001	0.001

表3 铁死亡诱导剂处理CD24^low与CD24^high细胞死亡率比较

组别	实验次数	DMSO	Erastin	RSL3
CD24^low	3	4.93±1.36	38.15±2.92	39.51±2.16
CD24^high	3	3.61±1.12	14.73±3.91	18.90±1.79
t值		1.292	8.310	8.600
P值		0.266	0.001	0.001

表4 半胱氨酸饥饿处理后CD24^low与CD24^high细胞MDA水平比较

组别	实验次数	含半胱氨酸	半胱氨酸饥饿
CD24^low	3	0.32±0.02	1.63±0.06
CD24^high	3	0.37±0.01	0.96±0.08
t值		-3.920	11.566
P值		0.017	<0.001

表4 半胱氨酸饥饿处理后CD24^low与CD24^high细胞MDA水平比较

组别	实验次数	含半胱氨酸	半胱氨酸饥饿
CD24^low	3	0.32±0.02	1.63±0.06
CD24^high	3	0.37±0.01	0.96±0.08
t值		-3.920	11.566
P值		0.017	<0.001

表5 铁死亡诱导剂处理后CD24^low与CD24^high细胞MDA水平比较

组别	实验次数	DMSO	Erastin	RSL3
CD24^low	3	0.42±0.04	1.76±0.02	1.67±0.02
CD24^high	3	0.40±0.03	1.28±0.08	1.22±0.03
t值		0.970	10.763	24.067
P值		0.387	0.006	<0.001

表5 铁死亡诱导剂处理后CD24^low与CD24^high细胞MDA水平比较

组别	实验次数	DMSO	Erastin	RSL3
CD24^low	3	0.42±0.04	1.76±0.02	1.67±0.02
CD24^high	3	0.40±0.03	1.28±0.08	1.22±0.03
t值		0.970	10.763	24.067
P值		0.387	0.006	<0.001

图1 Western blot检测CD24^low和CD24^highMDA-MB-231细胞中NF2和YAP蛋白表达情况

图2 NF2敲除联合YAP过表达降低CD24^highMDA-MB-231细胞中FSP1表达注：a代表对照组；b代表YAP过表达组(YAP-OV)；c代表NF2敲除组(NF2-KO)；d代表NF2敲除联合YAP过表达组(YAP-OV＋NF2-KO)

表6 NF敲除联合YAP过表达CD24^high细胞加药处理后死亡率比较

组别	实验次数	DMSO	Erastin	Erastin＋Fer-1
对照	3	5.74±0.50	19.89±0.68	6.37±2.50
YAP-OV	3	6.64±1.33	29.43±0.53	6.68±0.78
NF2-KO	3	5.86±1.34	36.31±6.10	7.42±1.48
YAP-OV＋NF2-KO	3	6.19±0.60	46.28±0.57	7.01±0.71
F值		0.469	38.911	0.256
P值		0.712	<0.001	0.855

表6 NF敲除联合YAP过表达CD24^high细胞加药处理后死亡率比较

组别	实验次数	DMSO	Erastin	Erastin＋Fer-1
对照	3	5.74±0.50	19.89±0.68	6.37±2.50
YAP-OV	3	6.64±1.33	29.43±0.53	6.68±0.78
NF2-KO	3	5.86±1.34	36.31±6.10	7.42±1.48
YAP-OV＋NF2-KO	3	6.19±0.60	46.28±0.57	7.01±0.71
F值		0.469	38.911	0.256
P值		0.712	<0.001	0.855

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NF2/YAP signaling pathway induces ferroptosis in triple negative breast cancer cells with high expression of CD24 by FSP1

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NF2/YAP signaling pathway induces ferroptosis in triple negative breast cancer cells with high expression of CD24 by FSP1