抑制BRD4可促进ACC1低表达的食管鳞癌细胞迁移

doi:10.12122/j.issn.1673-4254.2025.10.22

摘要/Abstract

摘要：

目的探究抑制BRD4对乙酰辅酶A羧化酶1（ACC1）低表达食管鳞癌（ESCC）细胞迁移的影响。方法慢病毒转染法构建对照组细胞系shNC和敲低ACC1组细胞系shACC1。分别设置BRD4抑制剂组：shNC-DMSO，shNC-JQ1，shACC1-DMSO，shACC1-JQ1；转染组：shNC-siNC，shNC-siBRD4，shACC1-siNC，shACC1-siBRD4；组蛋白乙酰转移酶抑制剂组：shNC-siNC-DMSO，shACC1-siNC-DMSO，shACC1-siBRD4-DMSO，shACC1-siBRD4-C646；自噬抑制剂组：shNC-JQ1-Veh，shACC1-JQ1-Veh，shACC1-JQ1-3-MA。RT-qPCR实验测定BRD4在ESCC细胞中mRNA水平。CCK-8实验、Transwell迁移实验、Western blotting实验检测抑制BRD4对ACC1低表达细胞的影响。结果 BRD4在shACC1组中表达水平差异无统计学意义；与shNC组对比，shACC1组对JQ1更敏感；1 μmol/L和2 μmol/L JQ1抑制ESCC细胞增殖（P<0.05），0.2 μmol/L和2 μmol/L JQ1促进ESCC细胞迁移（P<0.01）；与shNC-JQ1组对比，shACC1-JQ1组细胞迁移Vimentin（P<0.0001）、Slug（P<0.001、P<0.0001）表达升高，E-cadherin（P<0.0001）表达降低；敲低BRD4促进ESCC细胞迁移，与shNC-siBRD4组对比，shACC1-siBRD4组细胞Vimentin（P<0.0001）、Slug（P<0.0001）表达升高，E-cadherin（P<0.0001）表达降低；与shACC1- siBRD4-DMSO组对比，shACC1-siBRD4-C646组乙酰化水平降低，Vimentin（P<0.0001、P<0.001）、Slug（P<0.0001）表达降低，E-cadherin（P<0.0001、P<0.05）表达升高；与shNC-DMSO组对比，shNC-JQ1组LC3A/BⅡ水平升高（P<0.0001）；与shACC1-DMSO组对比，shACC1-JQ1组LC3A/BⅡ水平升高（P<0.0001）；与shACC1-JQ1-Veh组对比，shACC1-JQ1-3-MA组细胞Vimentin（P<0.0001）、Slug（P<0.0001）表达降低，E-cadherin（P<0.0001、P<0.001）表达升高，LC3A/BⅡ水平降低。结论抑制BRD4通过依赖组蛋白乙酰化机制促进自噬，进而促进上皮-间质转化（EMT），最终增强ACC1低表达所致的ESCC细胞迁移。

关键词: 食管鳞癌, 乙酰辅酶A羧化酶1, BRD4, 迁移

Abstract:

Objective To investigate the effect of BRD4 inhibition on migration of esophageal squamous cell carcinoma (ESCC) cells with low acetyl-CoA carboxylase 1 (ACC1) expression. Methods ESCC cell lines with lentivirus-mediated ACC1 knockdown or transfected with a negative control sequence (shNC) were treated with DMSO, JQ1 (a BRD4 inhibitor), co-transfection with shNC-siBRD4 or siNC with additional DMSO or C646 (an ahistone acetyltransferase inhibitor) treatment, or JQ1combined with 3-MA (an autophagy inhibitor). BRD4 mRNA expression in the cells was detected using RT-qPCR. The changes in cell proliferation, migration, autophagy, and epithelial-mesenchymal transition (EMT) were examined with CCK8 assay, Transwell migration assay, and Western blotting. Results ACC1 knockdown did not significantly affect BRD4 expression in the cells but obviously increased their sensitivity to JQ1. JQ1 treatment at 1 and 2 μmol/L significantly inhibited ESCC cell proliferation, while JQ1 at 0.2 and 2 μmol/L promoted cell migration. The cells with ACC1 knockdown and JQ1 treatment showed increased expresisons of vimentin and Slug and decreased expression of E-cadherin. BRD4 knockdown promoted migration of ESCC cells, and co-transfection with shACC1 and siBRD4 resulted in increased vimentin and Slug expressions and decreased E-cadherin expression in the cells. C646 treatment of the co-transfected cells reduced acetylation levels, decreased vimentin and Slug expressions, and increased E-cadherin expression. Treatment with JQ1 alone obviously increased LC3A/B-II levels in the cells either with or without ACC1 knockdown. In the cells with ACC1 knockdown and JQ1 treatment, additional 3-MA treatment significantly decreased the expressions of vimentin, Slug and LC3A/B-II and increased the expression of E-cadherin. Conclusion BRD4 inhibition promotes autophagy of ESCC cells via a histone acetylation-dependent mechanism, thereby enhancing EMT and ultimately increasing cell migration driven by ACC1 deficiency.

Key words: esophageal squamous cell carcinoma, acetyl-CoA carboxylase 1, BRD4, migration

贾文鑫, 霍书华, 唐家萍, 刘玉珍, 赵宝生. 抑制BRD4可促进ACC1低表达的食管鳞癌细胞迁移[J]. 南方医科大学学报, 2025, 45(10): 2258-2269.

Wenxin JIA, Shuhua HUO, Jiaping TANG, Yuzhen LIU, Baosheng ZHAO. Inhibition of BRD4 promotes migration of esophageal squamous cell carcinoma cells with low ACC1 expression[J]. Journal of Southern Medical University, 2025, 45(10): 2258-2269.

图/表 9

表1 引物序列

Tab.1 Primer sequences for RT-qPCR

Gene	Primer sequence (5'-3')
ACC1-F ACC1-R	ATCCCGTACCTTCTTCTACTG
ACC1-F ACC1-R	CCCAAACATAAGCCTTCACTG
BRD4-F BRD4-R	TGGATGCCGTCAAGCTGAAC
BRD4-F BRD4-R	GTTTCTTCTGTGGGTAGCTCATT
GAPDH-F GAPDH-R	AATGGGCAGCCGTTAGGAAA
GAPDH-F GAPDH-R	GCGCCCAATACGACCAAATC

图1 BRD4在ACC1低表达的ESCC细胞中表达水平无差异

Fig.1 Expression levels of BRD4 mRNA and protein in esophageal squamous cell carcinoma (ESCC) cells with and without ACC1 knockdown. A: RT-qPCR for detecting BRD4 mRNA level. B: Western blotting for determining the protein expression level of BRD4. ****P<0.0001.

图2 抑制BRD4拮抗ESCC细胞增殖

Fig.2 BRD4 inhibitors antagonize the proliferation of ESCC cells. A: Effect of JQ1 on proliferation of KYSE-150/KYSE-450 (shNC, shACC1) cells detected by CCK-8 assay. B: CCK-8 assay for assessing the effect of different concentrations of JQ1 on proliferation of ESCC cells. *P<0.05, **P<0.01, ***P<0.001.

图3 BRD4抑制剂促进ESCC细胞迁移

Fig.3 The promotion of ESCC cell migration by BRD4 inhibitors. Transwell migration assay for evaluating the effect of different concentrations of JQ1 on migration of ESCC cells (Original magnification: ×100). **P<0.01, ****P<0.0001.

图4 抑制BRD4促进ESCC细胞EMT转化

Fig.4 Inhibition of BRD4 promotes EMT in ESCC cells. The results of the Western blotting experiment showed that JQ1 inhibited the expression of E-cadherin protein and promoted the expression of vimentin and Slug. *P<0.05, ***P<0.001, ****P<0.0001.

图5 敲低BRD4促进ESCC细胞迁移

Fig.5 Knockdown of BRD4 promotes migration of ESCC cells. A: Transwell migration assay for assessing the effect of BRD4 knockdown on migration of ESCC cells (×100). B: Western blotting for detecting expression levels of BRD4 and EMT-related proteins. **P<0.01, ***P<0.001, ****P<0.0001.

图6 组蛋白乙酰基转移酶抑制剂拮抗敲低BRD4背景下ACC1低表达细胞迁移

Fig.6 Histone acetyltransferase inhibitors antagonize migration of shACC1 cells after BRD4 knockdown. A: Transwell migration assay for assessing the effect of C646 on migration of ESCC cells with both ACC1 and BRD4 knockdown (×100). B: Western blotting for detecting expression levels of BRD4 and EMT-related proteins in the cells after treatment with C646. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

图7 抑制BRD4通过自噬促进ACC1低表达ESCC细胞迁移

Fig.7 Inhibition of BRD4 promotes migration of ESCC cells with low ACC1 expression through autophagy. A: Western blotting showing that 2 μmol/L JQ1 promotes expressions of LC3A/BII in KYSE-150 and KYSE-450 cells. B: 4 mmol/L 3-MA inhibits migration of the cells with ACC1 knockdown and JQ1 treatment (×100). ***P<0.001, ****P<0.0001.

图8 抑制自噬可拮抗抑制BRD4对ACC1低表达ESCC细胞的EMT转化

Fig.8 Inhibition of autophagy counteracts the effect of BRD4 inhibition on EMT of ESCC cells with low expression of ACC1. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

参考文献 24

[1]	Mao YS, Gao SG, Wang Q, et al. Analysis of a registry database for esophageal cancer from high-volume centers in China[J]. Dis Esophagus, 2020, 33(8): doz091. doi：10.1093/dote/doz091
[2]	Zhang JL, Dong YX, Di SY, et al. Tumor associated macrophages in esophageal squamous carcinoma: Promising therapeutic implications[J]. Biomed Pharmacother, 2023, 167: 115610. doi：10.1016/j.biopha.2023.115610
[3]	钱河, 谷城威, 刘玉珍, 等. 敲低乙酰辅酶A羧化酶1通过上调热休克蛋白27促进食管鳞状细胞癌迁移[J]. 中华肿瘤杂志,2023,45(06): 482-9.
[4]	Guo JW, Zheng QQ, Peng Y. BET proteins: Biological functions and therapeutic interventions[J]. Pharmacol Ther, 2023, 243: 108354. doi：10.1016/j.pharmthera.2023.108354
[5]	Liu L, Lin BC, Yin SS, et al. Arginine methylation of BRD4 by PRMT2/4 governs transcription and DNA repair[J]. Sci Adv, 2022, 8(49): eadd8928. doi：10.1126/sciadv.add8928
[6]	Kotekar A, Singh AK, Devaiah BN. BRD4 and MYC: power couple in transcription and disease[J]. FEBS J, 2023, 290(20): 4820-42. doi：10.1111/febs.16580
[7]	Jin WK, Tan HD, Wu JH, et al. Dual-target inhibitors of bromodomain-containing protein 4 (BRD4) in cancer therapy: Current situation and future directions[J]. Drug Discov Today, 2022, 27(1): 246-56. doi：10.1016/j.drudis.2021.08.007
[8]	Yang WQ, Liang R, Gao MQ, et al. Inhibition of bromodomain-containing protein 4 enhances the migration of esophageal squamous cell carcinoma cells by inducing cell autophagy[J]. World J Gastrointest Oncol, 2022, 14(12): 2340-52. doi：10.4251/wjgo.v14.i12.2340
[9]	Zhu HH, Wei T, Cai Y, et al. Small molecules targeting the specific domains of histone-mark readers in cancer therapy[J]. Molecules, 2020, 25(3): 578. doi：10.3390/molecules25030578
[10]	Le K, Matar H, Gupta M. Bromodomain epigenetic protein promotes metastatic potential in melanoma cells through increased invasiveness and decreased macrophage-mediated phagocytosis[J]. J Invest Dermatol, 2021, 141(2): 454-8.e2. doi：10.1016/j.jid.2020.06.016
[11]	Li LY, Meng Y, Wu XL, et al. Bromodomain-containing protein 4 inhibitor JQ1 promotes melanoma cell apoptosis by regulating mitochondrial dynamics[J]. Cancer Sci, 2021, 112(10): 4013-25. doi：10.1111/cas.15061
[12]	Hu Y, Zhou JQ, Ye F, et al. BRD4 inhibitor inhibits colorectal cancer growth and metastasis[J]. Int J Mol Sci, 2015, 16(1): 1928-48. doi：10.3390/ijms16011928
[13]	Karagiannis D, Wu W, Li A, et al. Metabolic reprogramming by histone deacetylase inhibition preferentially targets NRF2-activated tumors[J]. Cell Rep, 2024, 43(1): 113629. doi：10.1016/j.celrep.2023.113629
[14]	Gibbons HR, Mi DJ, Farley VM, et al. Bromodomain inhibitor JQ1 reversibly blocks IFN-γ production[J]. Sci Rep, 2019, 9(1): 10280. doi：10.1038/s41598-019-46516-x
[15]	吕倩倩, 梁嘉杰, 季红. 基于PROTACs技术的表观遗传抗肿瘤药物研究进展[J]. 化学试剂, 2024, 46(5): 1-9.
[16]	Wu XC, Liu D, Gao XM, et al. Inhibition of BRD4 suppresses cell proliferation and induces apoptosis in renal cell carcinoma[J]. Cell Physiol Biochem, 2017, 41(5): 1947-56. doi：10.1159/000472407
[17]	Kanno T, Kanno Y, LeRoy G, et al. BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones[J]. Nat Struct Mol Biol, 2014, 21(12): 1047-57. doi：10.1038/nsmb.2912
[18]	Rhyasen GW, Yao Y, Zhang JW, et al. BRD4 amplification facilitates an oncogenic gene expression program in high-grade serous ovarian cancer and confers sensitivity to BET inhibitors[J]. PLoS One, 2018, 13(7): e0200826. doi：10.1371/journal.pone.0200826
[19]	Hu JF, Pan D, Li G, et al. Regulation of programmed cell death by Brd4[J]. Cell Death Dis, 2022, 13(12): 1059. doi：10.1038/s41419-022-05505-1
[20]	Zhang JC, Fan XK, Zhou YF, et al. The PRMT5-LSD1 axis confers Slug dual transcriptional activities and promotes breast cancer progression[J]. J Exp Clin Cancer Res, 2022, 41(1): 191. doi：10.1186/s13046-022-02400-7
[21]	Sterneck E, Poria DK, Balamurugan K. Slug and E-cadherin: stealth accomplices[J]? Front Mol Biosci, 2020, 7: 138. doi：10.3389/fmolb.2020.00138
[22]	Shrimp JH, Sorum AW, Garlick JM, et al. Characterizing the covalent targets of a small molecule inhibitor of the lysine acetyltransferase P300[J]. ACS Med Chem Lett, 2015, 7(2): 151-5. doi：10.1021/acsmedchemlett.5b00385
[23]	Zhang GZ, Chen HW, Deng YJ, et al. BRD4 inhibition suppresses senescence and apoptosis of nucleus pulposus cells by inducing autophagy during intervertebral disc degeneration: an in vitro and in vivo study[J]. Oxid Med Cell Longev, 2022, 2022: 9181412. doi：10.1155/2022/9181412
[24]	Strippoli R, Niayesh-Mehr R, Adelipour M, et al. Contribution of autophagy to epithelial mesenchymal transition induction during cancer progression[J]. Cancers (Basel), 2024, 16(4): 807. doi：10.3390/cancers16040807