FMRP通过激活RAS/MAPK信号通路抑制结直肠肿瘤细胞的铁死亡

doi:10.12122/j.issn.1673-4254.2024.05.10

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (5): 885-893.doi: 10.12122/j.issn.1673-4254.2024.05.10

FMRP通过激活RAS/MAPK信号通路抑制结直肠肿瘤细胞的铁死亡

王南¹(), 石斌², 马小兰¹, 吴伟超³, 曹佳⁴()

^1.宁夏医科大学总医院，硕士培养站，宁夏银川 750004
^2.宁夏医科大学总医院，急诊科，宁夏银川 750004
^4.宁夏医科大学总医院，医学科学研究院外科学研究室，宁夏银川 750004
^3.银川市第一人民医院耳鼻喉科，宁夏银川 750004

收稿日期:2023-12-27 出版日期:2024-05-20 发布日期:2024-06-06
通讯作者: 曹佳 E-mail:979377808@qq.com;caojiamed@126.com
作者简介:王南，硕士，住院医师，E-mail: 979377808@qq.com
基金资助:
宁夏回族自治区重点研发计划项目(2022CMG03124);宁夏回族自治区重点研发（引才专项）项目(2021BEB04046);宁夏回族自治区第六批自治区青年科技人才托举工程项目(NXKJTJ2021119)

High expression of fragile X mental retardation protein inhibits ferroptosis of colorectal tumor cells by activating the RAS/MAPK signaling pathway

Nan WANG¹(), Bin SHI², Xiaolan MAN¹, Weichao WU³, Jia CAO⁴()

^1.Master's Cultivation Station, Ningxia Medical University General Hospital, Yinchuan 750004, China
^2.Department of Emergency Medicine, Ningxia Medical University General Hospital, Yinchuan 750004, China
^4.Department of Surgical Sciences of Medical Science Research Institute, Ningxia Medical University General Hospital, Yinchuan 750004, China
^3.Department of Otolaryngology, Yinchuan First People's Hospital, Yinchuan 750004, China

Received:2023-12-27 Online:2024-05-20 Published:2024-06-06
Contact: Jia CAO E-mail:979377808@qq.com;caojiamed@126.com

摘要/Abstract

摘要：

目的探讨脆性X智力障碍蛋白（FMRP）调控结直肠肿瘤（CRC）细胞逃避铁死亡的作用机制。方法使用RT-qPCR和Western blotting方法验证FMRP在CRC细胞中的表达；利用TCGA数据库分析FMRP参与调控CRC进展的生物功能及信号通路；采用慢病毒表达系统和siRNA干扰技术，分别构建FMRP过表达载体（Lv-FMRP）和敲低载体（siFMRP-1、siFMRP-2、siFMRP-3），细胞实验设Control组、NC组、siFMRP-1组、siFMRP-2组、siFMRP-3组、Lv-NC组、Lv-FMRP组；采用CCK8和平板克隆实验检测细胞增殖；采用MDA/ROS/GSH/Fe²⁺试剂盒检测细胞铁死亡水平；采用JC-1荧光染色法检测线粒体膜电位变化；采用Western blot检测铁死亡相关蛋白及RAS/MAPK信号通路相关蛋白表达；利用裸鼠皮下成瘤实验观察FMRP对肿瘤生长的影响。结果与正常肠粘膜上皮细胞NCM460相比，FMRP在CRC细胞中明显高表达（P<0.05）；TCGA数据库联合生信分析显示FMRP与活性氧调控、氧化应激诱导细胞死亡、线粒体呼吸等生物过程相关，与谷胱甘肽代谢通路相关；体外实验结果显示，与Control组相比，FMRP敲低组细胞增殖能力降低，GSH含量降低，MDA和ROS含量升高，Fe²⁺荧光强度增强（P<0.05），SLC7A11/GPX4蛋白表达降低，线粒体膜电位JC-1荧光强度升高，而FMRP过表达组与上述结果相反；体内实验结果显示，敲低FMRP抑制瘤体生长，抑制SLC7A11表达（P<0.01）；进一步检测RAS/MAPK信号通路，发现敲低FMRP导致ERK、MEK、MAPK、RAS蛋白磷酸化水平降低，过表达FMRP上述蛋白磷酸化水平升高（P<0.05）。结论 FMRP通过激活RAS/MAPK信号通路抑制细胞铁死亡促进CRC恶性进展。

关键词: FMRP, 铁死亡, RAS/MAPK信号通路, 结直肠肿瘤

Abstract:

Objective To investigate the mechanism by which fragile X mental retardation protein (FMRP) regulates ferroptosis evasion in colorectal cancer (CRC) cells. Methods We examined FMRP expression levels in CRC cell lines using RT-qPCR and Western blotting and analyzed the biological functions and signaling pathways involved in FMRP-mediated regulation of CRC progression using the TCGA database. A lentiviral FMRP overexpression vector (Lv-FMRP) and 3 knockdown vectors (siFMRP-1, siFMRP-2, and siFMRP-3) were constructed, and their effects on proliferation of HCT116 cells were examined using CCK8 assay and plate clone formation assay; the changes in cell ferroptosis level was determined using MDA/ROS/GSH/Fe²⁺ kits, mitochondrial membrane potential changes were detected using JC-1 fluorescence staining, and the expressions of proteins associated with ferroptosis and the RAS/MAPK signaling pathway were detected using Western blotting. The subcutaneous tumorigenic potential of the transfected cells was evaluated in nude mice. Results Compared with normal colonic mucosal epithelial NCM460 cells, the CRC cell lines had significantly higher FMRP expression level. Bioinformatics analysis suggested the involvement of FMRP in regulation of reactive oxygen, oxidative stress-induced cell death, mitochondrial respiration, and glutathione metabolism pathways. In the cell experiments, FMRP knockdown significantly inhibited proliferation of HCT116 cells, lowered cellular GSH content, increased MDA and ROS levels, Fe²⁺fluorescence intensity, and mitochondrial membrane potential, and decreased SLC7A11/GPX4 protein expressions and the phosphorylation levels of ERK, MEK, MAPK, and RAS proteins; FMRP overexpression resulted in the opposite changes in the cells. In the tumor-bearing nude mice, HCT116 cells with FMRP knockdown showed attenuated tumorigenic potential with lowered xenograft growth rate and reduced SLC7A11 expression in the xenograft. Conclusion The high expression of FMRP inhibits ferroptosis in CRC cells and promotes progression of CRC by activating the RAS/MAPK signaling pathway.

Key words: fragile X mental retardation protein, ferroptosis, RAS/MAPK signaling pathway, colorectal cancer

王南, 石斌, 马小兰, 吴伟超, 曹佳. FMRP通过激活RAS/MAPK信号通路抑制结直肠肿瘤细胞的铁死亡[J]. 南方医科大学学报, 2024, 44(5): 885-893.

Nan WANG, Bin SHI, Xiaolan MAN, Weichao WU, Jia CAO. High expression of fragile X mental retardation protein inhibits ferroptosis of colorectal tumor cells by activating the RAS/MAPK signaling pathway[J]. Journal of Southern Medical University, 2024, 44(5): 885-893.

图/表 8

表1 siRNA序列

Tab.1 Sequences of the small interfering RNAs

Gene	Sense (5'-3')	Antisense (5'-3')
hFMR1-1	GAGGAUGAUAAAGGGUGAGUUTT	AACUCACCCUUUAUCAUCCUCTT
hFMR1-2	CGAGAUUUCAUGAACAGUUUATT	UAAACUGUUCAUGAAAUCUCGTT
hFMR1-3	GCGUUUGGAGAGAUUACAAAUTT	AUUUGUAAUCUCUCCAAACGCTT

表2 抗体信息

Tab.2 Antibody information

Antibody name	Company	Dilution rate
Anti-GAPDH	Cell Signaling technology	1∶1000
Anti-FMRP	Abcam	1∶1000
Anti-HO-1	Abmart	1∶1000
Anti-GPX4	Abmart	1∶1000
Anti-SLC7A11	Abmart	1∶1000
Anti-ERK	Cell signaling technology	1∶1000
Anti-p-ERK	Cell signaling technology	1∶1000
Anti-MAPK	Cell signaling technology	1∶500
Anti-p-MAPK	Cell signaling technology	1∶1000
Anti-RAS	Cell signaling technology	1∶500
Anti-p-RAS	Cell signaling technology	1∶500
Anti-MEK	Cell signaling technology	1∶1000
Anti-p-MRK	Cell signaling technology	1∶1000

图1 FMRP在CRC中的表达

Fig.1 Expression levels of FMRP in colorectal cancer (CRC) cell lines and tissues. A: RT-PCR for detecting the expression of FMRP in different CRC cell lines. B: Western blotting for detecting FMRP protein expression in different CRC cell lines. C: Expression of FMRP in colorectal cancer tissues and normal tissues. D: Relationship between FMRP expression and disease-free survival of CRC patients. *P<0.05 vs NCM460 group.

图2 生信分析筛选FMRP富集的生物功能及信号通路

Fig.2 Bioinformatic analysis of biological functions and signaling pathways enriched in FMRP. A: Two groups of differential genes. B: Up-regulated and down-regulated differential genes. C: GO functional enrichment bands. D: GSEA-GO enrichment analysis. E: Circular enrichment map of differential genes in iron ion homeostasis. F: GSEA-KEGG analysis map.

图3 FMRP对细胞增殖能力的影响

Fig.3 Effect of FMRP knockdown or overexpression on HCT116 cell proliferation. A: Western blotting for verifying the efficiency of FMRP knockdown or overexpression. B: RT-PCR of FMRP mRNA expression in HCT116 cells. C: CCK8 assay of the proliferation of the transfected HCT116 cells. D: Plate clone formation assay of cell proliferation. *P<0.05, **P<0.01 vs Control or Lv-NC group.

图4 FMRP对细胞铁死亡的影响

Fig.4 Effect of FMRP knockdown or overexpression on ferroptosis of HCT116 cells. A: GSH contents in transfected HCT116 cells. B: MDA contents in the transfected cells. C: ROS level in the transfected cells. D: Fe2+ level in the transfected cells (scale bar=50 μm). E: Western blots of the ferroptosis markers in the transfected cells. F: Immunofluorescence detection of SLC7A11 in HCT116 cells with FMRP knockdown (scale bar=20 μm). G: Immunofluorescence detection of mitochondrial membrane potential in HCT116 cells with FMRP knockdown (scale bar=20 μm). *P<0.05, **P<0.01 vs Control or Lv-NC group.

图5 FMRP对裸鼠移植瘤的生长的影响

Fig.5 Effect of FMRP knockdown on growth of transplanted tumor in nude mice. A: The size of subcutaneously transplanted tumor in nude mice. B: Changes in tumor weight in nude mice. C: Changes in tumor volume in nude mice. D: Immunohistochemistry for FMRP and SLC7A11 in the tumor tissue (scale bar=250 μm). *P<0.05, **P<0.01 vs Control.

图6 FMRP对RAS/MAPK信号通路的影响

Fig.6 Effect of FMRP knockdown or overexpression on RAS/MAPK signaling pathway in HCT116 cells detected by Western blotting. *P<0.05, **P<0.01 vs Control or Lv-NC group.

参考文献 28

1	Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-49. DOI: 10.3322/caac.21660
2	Chudy-Onwugaje K, Huang WY, Su LJ, et al. Aspirin, ibuprofen, and reduced risk of advanced colorectal adenoma incidence and recurrence and colorectal cancer in the PLCO Cancer Screening Trial[J]. Cancer, 2021, 127(17): 3145-55. DOI: 10.1002/cncr.33623
3	Shamay-Ramot A, Khermesh K, Porath HT, et al. Fmrp interacts with Adar and regulates RNA editing, synaptic density and locomotor activity in zebrafish[J]. PLoS Genet, 2015, 11(12): e1005702. DOI: 10.1371/journal.pgen.1005702
4	Shah S, Molinaro G, Liu BT, et al. FMRP control of ribosome translocation promotes chromatin modifications and alternative splicing of neuronal genes linked to autism[J]. Cell Rep, 2020, 30(13): 4459-72.e6. DOI: 10.1016/j.celrep.2020.02.076
5	Pedini G, Buccarelli M, Bianchi F, et al. FMRP modulates the Wnt signalling pathway in glioblastoma[J]. Cell Death Dis, 2022, 13(8): 719. DOI: 10.1038/s41419-022-05019-w
6	Lucá R, Averna M, Zalfa F, et al. The fragile X protein binds mRNAs involved in cancer progression and modulates metastasis formation[J]. EMBO Mol Med, 2013, 5(10): 1523-36. DOI: 10.1002/emmm.201302847
7	Zhao XL, Yang J, Zhang J, et al. Inhibitory effect of aptamer-carbon dot nanomaterial-siRNA complex on the metastasis of hepatocellular carcinoma cells by interfering with FMRP[J]. Eur J Pharm Biopharm, 2022, 174: 47-55. DOI: 10.1016/j.ejpb.2022.03.013
8	Shen ZF, Liu BW, Wu BT, et al. FMRP regulates STAT3 mRNA localization to cellular protrusions and local translation to promote hepatocellular carcinoma metastasis[J]. Commun Biol, 2021, 4(1): 540. DOI: 10.1038/s42003-021-02071-8
9	Zeng QQ, Saghafinia S, Chryplewicz A, et al. Aberrant hyperexpression of the RNA binding protein FMRP in tumors mediates immune evasion[J]. Science, 2022, 378(6621): eabl7207. DOI: 10.1126/science.abl7207
10	Hu YH, Gao QZ, Ma S, et al. FMR1 promotes the progression of colorectal cancer cell by stabilizing EGFR mRNA in an m⁶A-dependent manner[J]. Cell Death Dis, 2022, 13(11): 941. DOI: 10.1038/s41419-022-05391-7
11	Di Grazia A, Marafini I, Pedini G, et al. The fragile X mental retardation protein regulates RIPK1 and colorectal cancer resistance to necroptosis[J]. Cell Mol Gastroenterol Hepatol, 2021, 11(2): 639-58. DOI: 10.1016/j.jcmgh.2020.10.009
12	Lei G, Mao C, Yan YL, et al. Ferroptosis, radiotherapy, and combination therapeutic strategies[J]. Protein Cell, 2021, 12(11): 836-57. DOI: 10.1007/s13238-021-00841-y
13	McGillicuddy LT, Fromm JA, Hollstein PE, et al. Proteasomal and genetic inactivation of the NF1 tumor suppressor in gliomagenesis[J]. Cancer Cell, 2009, 16(1): 44-54. DOI: 10.1016/j.ccr.2009.05.009
14	Angius A, Pira G, Scanu AM, et al. MicroRNA-425-5p expression affects BRAF/RAS/MAPK pathways in colorectal cancers[J]. Int J Med Sci, 2019, 16(11): 1480-91. DOI: 10.7150/ijms.35269
15	Al-Obeed O, El-Obeid AS, Matou-Nasri S, et al. Herbal melanin inhibits colorectal cancer cell proliferation by altering redox balance, inducing apoptosis, and modulating MAPK signaling[J]. Cancer Cell Int, 2020, 20: 126. DOI: 10.1186/s12935-020-01206-x
16	O’Donnell WT, Warren ST. A decade of molecular studies of fragile X syndrome[J]. Annu Rev Neurosci, 2002, 25: 315-38. DOI: 10.1146/annurev.neuro.25.112701.142909
17	阳慧芝, 李思锐, 蔡桂月, 等. 脆性X智力低下蛋白在肿瘤致病机制中的研究进展[J]. 皮肤性病诊疗学杂志, 2023, 30(5): 460-4.
18	Lei G, Zhang YL, Koppula P, et al. The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression[J]. Cell Res, 2020, 30(2): 146-62. DOI: 10.1038/s41422-019-0263-3
19	Yang JW, Mo JJ, Dai JJ, et al. Cetuximab promotes RSL3-induced ferroptosis by suppressing the Nrf2/HO-1 signalling pathway in KRAS mutant colorectal cancer[J]. Cell Death Dis, 2021, 12(11): 1079. DOI: 10.1038/s41419-021-04367-3
20	He ZK, Yang JB, Sui CY, et al. FAM98A promotes resistance to 5-fluorouracil in colorectal cancer by suppressing ferroptosis[J]. Arch Biochem Biophys, 2022, 722: 109216. DOI: 10.1016/j.abb.2022.109216
21	Wang R, Xing R, Su Q, et al. Knockdown of SFRS9 inhibits progression of colorectal cancer through triggering ferroptosis mediated by GPX4 reduction[J]. Front Oncol, 2021, 11: 683589. DOI: 10.3389/fonc.2021.683589
22	Roemhild K, von Maltzahn F, Weiskirchen R, et al. Iron metabolism: pathophysiology and pharmacology[J]. Trends Pharmacol Sci, 2021, 42(8): 640-56. DOI: 10.1016/j.tips.2021.05.001
23	Liu XF, Hai Y, Dong JQ, et al. Realgar-induced KRAS mutation lung cancer cell death via KRAS/Raf/MAPK mediates ferroptosis[J]. Int J Oncol, 2022, 61(6): 157. DOI: 10.3892/ijo.2022.5447
24	Sun L, Wang H, Xu D, et al. Lapatinib induces mitochondrial dysfunction to enhance oxidative stress and ferroptosis in doxorubicin-induced cardiomyocytes via inhibition of PI3K/AKT signaling pathway[J]. Bioengineered, 2022, 13(1): 48-60. DOI: 10.1080/21655979.2021.2004980
25	Soleimani A, Rahmani F, Saeedi N, et al. The potential role of regulatory microRNAs of RAS/MAPK signaling pathway in the pathogenesis of colorectal cancer[J]. J Cell Biochem, 2019, 120(12): 19245-53. DOI: 10.1002/jcb.29268
26	Vitiello PP, Cardone C, Martini G, et al. Receptor tyrosine kinase-dependent PI3K activation is an escape mechanism to vertical suppression of the EGFR/RAS/MAPK pathway in KRAS-mutated human colorectal cancer cell lines[J]. J Exp Clin Cancer Res, 2019, 38(1): 41. DOI: 10.1186/s13046-019-1035-0
27	Hong S, Jeon M, Kwon J, et al. Targeting RAF isoforms and tumor microenvironments in RAS or BRAF mutant colorectal cancers with SJ-C1044 for anti-tumor activity[J]. Curr Issues Mol Biol, 2023, 45(7): 5865-78. DOI: 10.3390/cimb45070371
28	Casingal CR, Kikkawa T, Inada H, et al. Identification of FMRP target mRNAs in the developmental brain: FMRP might coordinate Ras/MAPK, Wnt/β‑catenin, and mTOR signaling during corticogenesis[J]. Mol Brain, 2020, 13(1): 167. DOI: 10.1186/s13041-020-00706-1

[1]	李淑贤, 于淑平, 穆亚铭, 王凯, 刘玉, 张美华. 二甲双胍通过抑制铁死亡改善PM2.5导致的胎盘滋养细胞功能损伤[J]. 南方医科大学学报, 2024, 44(3): 437-446.
[2]	孙硕, 黄鑫, 李国东, 张春云, 卢泽梅, 张伟伟, 李泽彦, 杨清竹. 敲低结肠癌转移相关基因1促进RSL3诱导的结直肠癌细胞铁死亡[J]. 南方医科大学学报, 2024, 44(1): 173-178.
[3]	张晓红, 赵品, 蒯建科, 常超, 袁庆. 亚精胺通过抑制细胞凋亡、ROS生成及铁死亡减轻脂多糖诱导的小鼠心肌损伤[J]. 南方医科大学学报, 2024, 44(1): 166-172.
[4]	朱权, 黄柏胜, 位磊艳, 罗奇志. 过表达LncRNA MEG3通过促进铁死亡增强肝癌细胞对顺铂的化疗敏感性[J]. 南方医科大学学报, 2024, 44(1): 17-24.
[5]	黄奕, 林丽珊, 黄浩华, 董航明. VDAC1通过诱导气道上皮细胞铁死亡参与屋尘螨诱导的哮喘小鼠气道炎症[J]. 南方医科大学学报, 2023, 43(8): 1333-1338.
[6]	谢思雨, 李淼生, 江峰乐, 易茜, 杨魏. EHHADH是肝细胞癌脂肪酸代谢通路的关键基因：基于转录组分析[J]. 南方医科大学学报, 2023, 43(5): 680-693.
[7]	李晒, 李丽, 闵思敏, 刘赛赛, 秦志文, 熊志尚, 徐建国, 王博文, 丁渡山, 赵士弟. 大豆异黄酮可减轻大鼠脑缺血/再灌注损伤：基于抑制铁死亡及炎症级联反应[J]. 南方医科大学学报, 2023, 43(2): 323-330.
[8]	李彬, 汪越, 侯凤伟, 杜家如, 童旭辉. 雷帕霉素增强RSL3对睾丸癌细胞I-10的增殖、侵袭与迁移的抑制作用[J]. 南方医科大学学报, 2023, 43(12): 2145-2151.
[9]	李静, 殷丽霞, 张敏, 夏勇生, 左芦根, 刘牧林, 胡建国. 粪便蛋白Luminex液相芯片检测系统构建及其对结直肠肿瘤早期诊断的价值[J]. 南方医科大学学报, 2023, 43(11): 1874-1880.
[10]	胡桐, 勾文峰, 任中昊, 刘改廷, 李祎亮, 左代英, 侯文彬. 淫羊藿素通过调控铁死亡增加鼻咽癌细胞的放射敏感性[J]. 南方医科大学学报, 2023, 43(10): 1665-1673.
[11]	周思聪, 杨威, 曾丽, 曹春浩, 袁速, 荣晓凤. 大黄素对胶原诱导性关节炎大鼠的骨保护作用:基于抑制铁死亡和降解基质金属蛋白酶[J]. 南方医科大学学报, 2023, 43(10): 1776-1781.
[12]	黄庆洋, 纪东东, 田绣云, 马琳艳, 孙小锦. 小檗碱通过激活 Nrf2-HO-1/GPX4 通路抑制小鼠海马神经元HT22细胞的铁死亡[J]. 南方医科大学学报, 2022, 42(6): 937-943.
[13]	杜家如, 李彬, 朱晨露, 韩家乐, 童旭辉. 甘珀酸增强RSL3对耐顺铂睾丸癌细胞增殖、侵袭和迁移的抑制作用[J]. 南方医科大学学报, 2022, 42(3): 405-410.
[14]	黄毓慧, 张共鹏, 梁欢, 曹珍珍, 叶红伟, 高琴. 抑制铁死亡可减轻脓毒症小鼠的心肌损伤：脂钙蛋白-2的作用[J]. 南方医科大学学报, 2022, 42(2): 256-262.
[15]	刘泽鑫, 曹赛, 陈清, 符方勇, 程梅容, 黄显莹. MicroRNA-132通过诱导线粒体氧化应激障碍-铁死亡进程促进动脉粥样硬化[J]. 南方医科大学学报, 2022, 42(1): 143-149.

FMRP通过激活RAS/MAPK信号通路抑制结直肠肿瘤细胞的铁死亡

High expression of fragile X mental retardation protein inhibits ferroptosis of colorectal tumor cells by activating the RAS/MAPK signaling pathway

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 8

参考文献 28

相关文章 15

编辑推荐

Metrics

本文评价