PTEN对FoxM1可变剪接的调控及该过程在肿瘤细胞迁移中的作用

doi:10.3969/j.issn.1674-8115.2023.11.001

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (11): 1339-1347.doi: 10.3969/j.issn.1674-8115.2023.11.001

• 创新团队成果专栏 •

PTEN对FoxM1可变剪接的调控及该过程在肿瘤细胞迁移中的作用

王晓玲¹(), 葛梦凯², 沈少明²()

^1.上海交通大学医学院基础医学公共技术平台，上海 200025
^2.上海交通大学基础医学院细胞病理生理系，分化与凋亡教育部重点实验室，上海 200025

收稿日期:2023-06-29 接受日期:2023-09-15 出版日期:2023-11-28 发布日期:2023-11-28
通讯作者: 沈少明 E-mail:wxl1984.1234@aliyun.com;smshen@shsmu.edu.cn
作者简介:王晓玲（1984—），女，助理实验师，本科；电子信箱：wxl1984.1234@aliyun.com。
基金资助:
上海市高水平地方高校创新团队(SHSMU-ZDCX20211800)

PTEN-regulated alternative splicing of FoxM1 affects tumor cell migration

WANG Xiaoling¹(), GE Mengkai², SHEN Shaoming²()

^1.Common Technology Platform, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
^2.Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China

Received:2023-06-29 Accepted:2023-09-15 Online:2023-11-28 Published:2023-11-28
Contact: SHEN Shaoming E-mail:wxl1984.1234@aliyun.com;smshen@shsmu.edu.cn
Supported by:
Innovative Research Team of High-Level Local Universities in Shanghai(SHSMU-ZDCX20211800)

摘要/Abstract

摘要：

目的·探讨10号染色体磷酸酶和张力同源蛋白（phosphatase and tensin homolog，PTEN）对叉头框转录因子M1（forkhead box M1，FoxM1）可变剪接的调控，以及该调控作用对肿瘤细胞迁移的影响。方法·在人胚肾293T细胞，人前列腺癌DU145细胞，人结肠腺癌RKO细胞，人结肠癌SW480、SW620细胞中用短发夹RNA（short hairpin RNA，shRNA）敲低PTEN的mRNA水平。设计针对FoxM1及其亚型FoxM1B和FoxM1C的特异性引物，用实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）检测FoxM1B和FoxM1C的mRNA表达水平在PTEN敲低前后的差异。在DU145细胞中分别过表达FoxM1B和FoxM1C，利用Transwell细胞迁移实验和划痕实验检测两者对肿瘤细胞迁移的影响。通过免疫荧光和双荧光素酶报告基因检测实验，探索FoxM1B和FoxM1C对肿瘤细胞迁移调控差异的潜在机制。结果·① 将293T、DU145、RKO、SW480和SW620细胞内的PTEN敲低后，qRT-PCR检测发现，与对照细胞相比，在PTEN敲低的细胞中FoxM1B的mRNA均显著增多，而FoxM1C的mRNA表现为减少或不变。PTEN敲低不影响FoxM1的转录水平，而影响FoxM1的可变剪接，促进了FoxM1B亚型的生成。② Transwell细胞迁移实验结果显示，与对照细胞相比，FoxM1B过表达组DU145细胞迁移数量增多（P=0.024），FoxM1C过表达组DU145细胞迁移数量减少（P=0.000）。细胞划痕实验结果显示，过表达FoxM1B的DU145细胞愈合能力显著增强（P=0.001），过表达FoxM1C的DU145细胞愈合能力减弱（P=0.021）。FoxM1B和FoxM1C对肿瘤细胞迁移具有相反的调节作用：FoxM1B促进肿瘤细胞迁移，而FoxM1C则抑制肿瘤细胞迁移。③ FoxM1B和FoxM1C过表达，均未引起β连环蛋白入核。双荧光素酶报告基因检测实验发现FoxM1B和FoxM1C的转录活性没有差别，FoxM1B和FoxM1C在调控肿瘤转移方面的差异不是由β连环蛋白转位介导的。结论·PTEN敲低影响肿瘤细胞内FoxM1的可变剪接，导致促迁移的FoxM1B表达增加，从而促进肿瘤细胞迁移。

关键词: 磷酸酶和张力同源蛋白, 叉头框转录因子M1, 可变剪接, 肿瘤细胞, 迁移

Abstract:

Objective ·To study the effect of phosphatase and tensin homolog on chromosome 10 (PTEN) on alternative splicing of forkhead box M1 (FoxM1), and its impact on tumor cell migration. Methods ·PTEN was knocked down by short hairpin RNA (shRNA) in human embryonic kidney 293T cells, human prostate cancer DU145 cells, human colorectal adenocarcinoma RKO cells, and human colon cancer SW480 and SW620 cells. Specific primers were designed for FoxM1 and its subtypes FoxM1B and FoxM1C, and the mRNA expression levels of FoxM1B and FoxM1C were detected by quantitative real-time PCR (qRT-PCR). FoxM1B and FoxM1C were overexpressed in DU145 cells, and their effects on tumor cell migration were tested by Transwell assay and wound healing assay. Immunofluorescence and dual luciferase reporter gene assay were used to explore the potential mechanism of differential regulation of tumor cell migration by FoxM1B and FoxM1C. Results ·① PTEN was knocked down in 293T, DU145, RKO, SW480, and SW620 cell lines. qRT-PCR results showed that compared with the control cells, the mRNA expression level of FoxM1B significantly increased in PTEN knockdown cells, while the mRNA expression level of FoxM1C decreased or remained unchanged. Knockdown of PTEN did not affect the transcription level of FoxM1, but caused the variable splicing of FoxM1 and promoted the generation of FoxM1B. ② Compared with the control cells, the number of DU145 cells migrating to the below chamber increased in the FoxM1B overexpression group (P=0.024), while the number of migrating DU145 cells in the FoxM1C overexpression group was lower (P=0.000). The healing ability of DU145 cells was significantly enhanced in the FoxM1B overexpression group (P=0.001), while the healing ability of DU145 cells was weakened in the FoxM1C overexpression group (P=0.021). Overall, FoxM1B and FoxM1C had opposite effects on tumor cell migration. FoxM1B promoted tumor cell migration, while FoxM1C inhibited tumor cell migration. ③ Neither FoxM1B nor FoxM1C overexpression could induce β-catenin to enter the nucleus. Dual luciferase reporter gene assay showed no difference in the transcriptional activity of FoxM1B and FoxM1C. The difference between FoxM1B and FoxM1C in the regulation of tumor metastasis was also not mediated by β-catenin translocation. Conclusion ·Knockdown of PTEN regulates the alternative splicing of FoxM1, leading to increasing expression of transcript FoxM1B, which plays a positive role in tumor cell migration.

Key words: phosphatase and tensin homolog (PTEN), forkhead box M1 (FoxM1), alternative splicing, tumor cell, migration

中图分类号:

R363.1

王晓玲, 葛梦凯, 沈少明. PTEN对FoxM1可变剪接的调控及该过程在肿瘤细胞迁移中的作用[J]. 上海交通大学学报（医学版）, 2023, 43(11): 1339-1347.

WANG Xiaoling, GE Mengkai, SHEN Shaoming. PTEN-regulated alternative splicing of FoxM1 affects tumor cell migration[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(11): 1339-1347.

图/表 5

表1 PTEN 敲减序列

Tab 1 PTEN knockdown sequences

shRNA	Sequences (5′→3′)
shEV	CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG
shPTEN#1	GATCTTGACCAATGGCTAAGT
shPTEN#2	CGGGAAGACAAGTTCATGTACTT

表2 qRT-PCR引物序列

Tab 2 Primer sequences for qRT-PCR

Gene	Primer sequence (5′→3′)
FoxM1b-F	AAGCCAGGCTGGAAGAAC
FoxM1b-R	GTTTCTGCTGCTTAAACACC
FoxM1c-F	GAAGAACTCCATCCGCCACA
FoxM1c-R	TGATTCCAAGTGCTCGGGCA
FoxM1-F	GAGCAGAAACGGGAGACCTG
FoxM1-R	ACCTTAACCTGTCGCTGCTC

图1 PTEN 敲低细胞内 FoxM1 mRNA亚型的变化Note： A. Schematic diagram of primer design strategy for mRNA of FoxM1B, FoxM1C and total FoxM1. B. The specificity of the primers was verified by using qRT-PCR in FoxM1B/FoxM1C overexpressing 293T cells. C. Western blotting results showed that PTEN was efficiently knocked down by two pairs of shRNA both in 293T and DU145 cells. D. qRT-PCR results showed that with PTEN knockdown, the expression of FoxM1B mRNA was upregulated in 293T (left) and DU145 (right) cells, whilethe expression of FoxM1C mRNA was downregulated in 293T cells but did not change in DU145 cells. E. qRT-PCR results showed upregulation of FoxM1B mRNA expression, but no significant change in FoxM1C mRNA expression after PTEN being knocked down in RKO (left), SW480 (middle) and SW460 (right) cells. NRD—N-terminal repressor domain; FKH—forkhead/winged-helix domain; TAD—transcativation domain; EV—empty vector.

Fig 1 FoxM1 mRNA isoforms changed in PTEN knockdown cells

图2 FoxM1B和FoxM1C对DU145细胞迁移的影响Note： A. Western blotting was used to detect the overexpression level of FoxM1B and FoxM1C in DU145 cells. B. Transwell migration analysis results of DU145 cells overexpressing FoxM1B and FoxM1C. Left—typical crystal violet staining photos. Bar=100 μm. Right—statistical analysis of cell migration numbers. C. Wound healing assay of DU145 cells transfected with FoxM1B and FoxM1C overexpression lentivirus. Left—photos of cells incubated for 0 and 24 h in the scratch experiment. Bar=100 μm. Right—statistical analysis of the wound healing rate.

Fig 2 Effects of FoxM1B and FoxM1C on DU145 migration

图3 FoxM1B/C转录活性和β-catenin定位鉴定Note： A. Immunofluorescence of 293T cells transfected with Plvx-FOXM1B-Flag and Plvx-FoxM1C-Flag. DAPI represents nucleus, green represents β-catenin, and red represents Flag. Neither FoxM1B nor FoxM1C could cause β-catenin to enter the nucleus. B. Schematic diagram of double luciferase reporter expression system. C. Protein expression of FoxM1B and FoxM1C in FoxM1B/C overexpressing 293T cells transfected with Firefly luciferase and Renilla luciferase reporter gene plasmids. D. Transcriptional activity of FoxM1B and FoxM1C. There was no significant difference in the relative Luc/Ren ratio between FoxM1B and FoxM1C overexpressing 293T cells.

Fig 3 FoxM1B /C transcriptional activities and β-catenin localization

参考文献 30

1	NUSSINOV R, TSAI C J, JANG H. Anticancer drug resistance: an update and perspective[J]. Drug Resist Updat, 2021, 59: 100796.
2	LIN Y X, WANG Y, DING J X, et al. Reactivation of the tumor suppressor PTEN by mRNA nanoparticles enhances antitumor immunity in preclinical models[J]. Sci Transl Med, 2021, 13(599): eaba9772.
3	GAO P, HAO J L, XIE Q W, et al. PELO facilitates PLK1-induced the ubiquitination and degradation of Smad4 and promotes the progression of prostate cancer[J]. Oncogene, 2022, 41(21): 2945-2957.
4	LIU W W, XU L, WANG X, et al. PRDX1 activates autophagy via the PTEN-AKT signaling pathway to protect against cisplatin-induced spiral ganglion neuron damage[J]. Autophagy, 2021, 17(12): 4159-4181.
5	ÁLVAREZ-GARCIA V, TAWIL Y, WISE H M, et al. Mechanisms of PTEN loss in cancer: it′s all about diversity[J]. Semin Cancer Biol, 2019, 59: 66-79.
6	BYRNES K, BLESSINGER S, BAILEY N T, et al. Therapeutic regulation of autophagy in hepatic metabolism[J]. Acta Pharm Sin B, 2022, 12(1): 33-49.
7	FENG J W, DANG Y P, ZHANG W Q, et al. PTEN arginine methylation by PRMT6 suppresses PI3K-AKT signaling and modulates pre-mRNA splicing[J]. Proc Natl Acad Sci U S A, 2019, 116(14): 6868-6877.
8	PENG Q, ZHOU Y J, OYANG L, et al. Impacts and mechanisms of alternative mRNA splicing in cancer metabolism, immune response, and therapeutics[J]. Mol Ther, 2022, 30(3): 1018-1035.
9	FRANKIW L, BALTIMORE D, LI G D. Alternative mRNA splicing in cancer immunotherapy[J]. Nat Rev Immunol, 2019, 19(11): 675-687.
10	WANG K, DAI X Y, YU A, et al. Peptide-based PROTAC degrader of FOXM1 suppresses cancer and decreases GLUT1 and PD-L1 expression[J]. J Exp Clin Cancer Res, 2022, 41(1): 289.
11	VANGENDEREN C, HARKNESS T A A, ARNASON T G. The role of anaphase promoting complex activation, inhibition and substrates in cancer development and progression[J]. Aging, 2020, 12(15): 15818-15855.
12	HU G H, YAN Z W, ZHANG C, et al. FOXM1 promotes hepatocellular carcinoma progression by regulating KIF4A expression[J]. J Exp Clin Cancer Res, 2019, 38(1): 188.
13	NAKAMURA S, HIRANO I, OKINAKA K, et al. The FOXM1 transcriptional factor promotes the proliferation of leukemia cells through modulation of cell cycle progression in acute myeloid leukemia[J]. Carcinogenesis, 2010, 31(11): 2012-2021.
14	RATHER T B, PARVEIZ I, BHAT G A, et al. Evaluation of forkhead box M1 (FOXM1) gene expression in colorectal cancer[J]. Clin Exp Med, 2023, 23(6): 2385-2405.
15	LI S K M, SMITH D K, LEUNG W Y, et al. FoxM1c counteracts oxidative stress-induced senescence and stimulates Bmi-1 expression[J]. J Biol Chem, 2008, 283(24): 16545-16553.
16	NILSSON M B, SUN H Y, ROBICHAUX J, et al. A YAP/FOXM1 axis mediates EMT-associated EGFR inhibitor resistance and increased expression of spindle assembly checkpoint components[J]. Sci Transl Med, 2020, 12(559): eaaz4589.
17	SHEN S M, JI Y, ZHANG C, et al. Nuclear PTEN safeguards pre-mRNA splicing to link Golgi apparatus for its tumor suppressive role[J]. Nat Commun, 2018, 9(1): 2392.
18	MATSUSHITA M, FUJITA K, HAYASHI T, et al. Gut microbiota-derived short-chain fatty acids promote prostate cancer growth via IGF1 signaling[J]. Cancer Res, 2021, 81(15): 4014-4026.
19	KORVER W, ROOSE J, CLEVERS H. The winged-helix transcription factor Trident is expressed in cycling cells[J]. Nucleic Acids Res, 1997, 25(9): 1715-1719.
20	BARGER C J, ZHANG W, HILLMAN J, et al. Genetic determinants of FOXM1 overexpression in epithelial ovarian cancer and functional contribution to cell cycle progression[J]. Oncotarget, 2015, 6(29): 27613-27627.
21	TASSI R A, TODESCHINI P, SIEGEL E R, et al. FOXM1 expression is significantly associated with chemotherapy resistance and adverse prognosis in non-serous epithelial ovarian cancer patients[J]. J Exp Clin Cancer Res, 2017, 36(1): 63.
22	BU H T, TANG S S, LIU G T, et al. In silico, in vitro and in vivo studies: dibutyl phthalate promotes prostate cancer cell proliferation by activating Forkhead Box M1 and remission after Natura-α pretreatment[J]. Toxicology, 2023, 488: 153465.
23	MADHI H, LEE J S, CHOI Y E, et al. FOXM1 inhibition enhances the therapeutic outcome of lung cancer immunotherapy by modulating PD-L1 expression and cell proliferation[J]. Adv Sci (Weinh), 2022, 9(29): e2202702.
24	SHER G, MASOODI T, PATIL K, et al. Dysregulated FOXM1 signaling in the regulation of cancer stem cells[J]. Semin Cancer Biol, 2022, 86(Pt 3): 107-121.
25	KELLEHER F C, O′SULLIVAN H. FOXM1 in sarcoma: role in cell cycle, pluripotency genes and stem cell pathways[J]. Oncotarget, 2016, 7(27): 42792-42804.
26	CHEN W, SHIMANE T, KAWANO S, et al. Human papillomavirus 16 E6 induces FoxM1B in oral keratinocytes through GRHL2[J]. J Dent Res, 2018, 97(7): 795-802.
27	HUANG C, XIE D C, CUI J J, et al. FOXM1c promotes pancreatic cancer epithelial-to-mesenchymal transition and metastasis via upregulation of expression of the urokinase plasminogen activator system[J]. Clin Cancer Res, 2014, 20(6): 1477-1488.
28	LOK G T M, CHAN D W, LIU V W S, et al. Aberrant activation of ERK/FOXM1 signaling cascade triggers the cell migration/invasion in ovarian cancer cells[J]. PLoS One, 2011, 6(8): e23790.
29	ZHOU Y Z, WANG Q, CHU L, et al. FOXM1c promotes oesophageal cancer metastasis by transcriptionally regulating IRF1 expression[J]. Cell Prolif, 2019, 52(2): e12553.
30	TAYLOR B S, SCHULTZ N, HIERONYMUS H, et al. Integrative genomic profiling of human prostate cancer[J]. Cancer Cell, 2010, 18(1): 11-22.

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PTEN对FoxM1可变剪接的调控及该过程在肿瘤细胞迁移中的作用

PTEN-regulated alternative splicing of FoxM1 affects tumor cell migration

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