过表达Cry2通过介导c-Myc泛素化降解抑制肝细胞癌细胞增殖并诱导凋亡*

doi:10.3870/j.issn.1672-0741.25.08.035

摘要/Abstract

摘要： 目的探讨过表达隐花色素蛋白2(Cry2)基因对肝细胞癌(HCC)细胞增殖和凋亡的影响及机制。方法采用qRT-PCR和Western blot检测人永生化肝细胞THLE-3及HCC细胞系HepG2、HuH-7、SNU-398、MHCC97-H中的Cry2 mRNA和蛋白表达水平。将Cry2过表达质粒(OE-Cry2)、c-Myc过表达质粒(OE-c-Myc)及其阴性对照(Vector)分别转染至HuH-7细胞中,并联合10 μmol/L蛋白酶体抑制剂(MG132)或100 μg/mL放线菌酮(CHX)干预。qRT-PCR检测各组细胞中Cry2和c-Myc mRNA表达水平;Western blot检测各组细胞中Cry2、c-Myc和cleaved Caspase-3蛋白表达水平;CCK-8检测各组细胞增殖率;流式细胞术检测各组细胞凋亡率;免疫沉淀(IP)实验检测Cry2过表达对c-Myc蛋白泛素化的影响。结果与THLE-3细胞比较,HCC各细胞系中Cry2 mRNA和蛋白表达水平均显著降低(均P＜0.05)。过表达Cry2可降低HuH-7细胞增殖率,诱导细胞凋亡,上调cleaved Caspase-3蛋白表达水平,并通过泛素修饰促进c-Myc蛋白降解。c-Myc过表达可逆转Cry2过表达对HuH-7细胞增殖及凋亡的影响。结论过表达Cry2可抑制HCC细胞增殖并诱导细胞凋亡,其作用机制可能与介导c-Myc泛素化降解有关。

关键词: 肝细胞癌, 增殖, 凋亡, 隐花色素蛋白2, 髓细胞增生原癌基因, 泛素化

Abstract: Objective To investigate the effect and underlying mechanism of cryptochrome 2(Cry2)gene overexpression on the proliferation and apoptosis of hepatocellular carcinoma(HCC)cells.Methods The expression levels of Cry2 mRNA and protein in human immortalized hepatocytes(THLE-3)and HCC cell lines(HepG2,HuH-7,SNU-398,and MHCC97-H)were determined using qRT-PCR and Western blot.Cry2 overexpression plasmid(OE-Cry2),c-Myc overexpression plasmid(OE-c-Myc),and their respective negative controls(Vector)were transfected into HuH-7 cells.Cells were treated with either 10 μmol/L proteasome inhibitor(MG132)or 100 μg/mL cycloheximide(CHX).The mRNA expression levels of Cry2 and c-Myc were assessed by qRT-PCR,while Western blot was used to evaluate the protein expression levels of Cry2,c-Myc,and cleaved Caspase-3.Cell proliferation was measured using CCK-8 assay,and the apoptosis rate was determined by flow cytometry.Additionally,the ubiquitination level of the c-Myc protein following Cry2 overexpression was analyzed using an immunoprecipitation(IP)assay.Results Compared with THLE-3 cells,the expression levels of Cry2 mRNA and protein in all HCC cell lines were significantly reduced(all P<0.05).Cry2 overexpression reduced the proliferation rate,induced apoptosis,upregulated cleaved Caspase-3 expression,and promoted ubiquitin-mediated degradation of c-Myc in HuH-7 cells.However,c-Myc overexpression reversed the effects of Cry2 overexpression on both cell proliferation and apoptosis in HuH-7 cells.Conclusion Overexpression of Cry2 may inhibit the proliferation and induce apoptosis of HCC cells.Its mechanism of action may be related to mediating c-Myc ubiquitination and degradation.

Key words: hepatocellular carcinoma, proliferation, apoptosis, cryptochrome 2, myelocytomatosis oncogene, ubiquitination

中图分类号:

R735.7

熊杰, 刘扬, 张喜华, 何前进. 过表达Cry2通过介导c-Myc泛素化降解抑制肝细胞癌细胞增殖并诱导凋亡^*[J]. 华中科技大学学报（医学版）, 2026, 55(4): 519-525.

Xiong Jie, Liu Yang, Zhang Xihua, et al. Overexpression of Cry2 Suppresses Proliferation and Induces Apoptosis in Hepatocellular Carcinoma Cells by Mediating Ubiquitination and Degradation of c-Myc[J]. Acta Medicinae Universitatis Scientiae et Technologiae Huazhong, 2026, 55(4): 519-525.

参考文献

[1] Sung H,Ferlay J,Siegel R L,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):209-249.
[2] Llovet J M,Brú C,Bruix J.Prognosis of hepatocellular carcinoma:The BCLC staging classification[J].Semin Liver Dis,1999,19(3):329-338.
[3] Chan Y T,Zhang C,Wu J,et al.Biomarkers for diagnosis and therapeutic options in hepatocellular carcinoma[J].Mol Cancer,2024,23(1):189.
[4] Dhanasekaran R,Deutzmann A,Mahauad-Fernandez W D,et al.The MYC oncogene-the grand orchestrator of cancer growth and immune evasion[J].Nat Rev Clin Oncol,2022,19(1):23-36.
[5] Moon H,Park H,Ro S W.c-Myc-driven hepatocarcinogenesis[J].Anticancer Res,2021,41(10):4937-4946.
[6] Zhou J N,Zhang B,Wang H Y,et al.A functional screening identifies a new organic selenium compound targeting cancer stem cells:Role of c-Myc transcription activity inhibition in liver cancer[J].Adv Sci(Weinh),2022,9(22):e2201166.
[7] Hasakova K,Vician M,Reis R,et al.The expression of clock genes Cry1 and Cry2 in human colorectal cancer and tumor adjacent tissues correlates differently dependent on tumor location[J].Neoplasma,2018,65(6):986-992.
[8] Mazzoccoli G,Colangelo T,Panza A,et al.Deregulated expression of cryptochrome genes in human colorectal cancer[J].Mol Cancer,2016,15:6.
[9] 李超,袁鹏,颜昭勇,等.CRY2分子对肝癌细胞生长和转移的影响[J].现代生物医学进展,2017,17(36):7030-7035,7084.
Li C,Yuan P,Yan Z Y,et al.Effects of CRY2 on the growth and metastasis of hepatocellular carcinoma cells[J].Progress in Modern Biomedicine,2017,17(36):7030-7035,7084.
[10] Zhang Z,Liang Z,Gao W,et al.Identification of circadian clock genes as regulators of immune infiltration in hepatocellular carcinoma[J].J Cancer,2022,13(11):3199-3208.
[11] Huber A L,Papp S J,Chan A B,et al.CRY2 and FBXL3 cooperatively degrade c-Myc[J].Mol Cell,2016,64(4):774-789.
[12] Huyen V T,Echizen K,Yamagishi R,et al.Regular exercise suppresses steatosis-associated liver cancer development by degrading E2F1 and c-Myc via circadian gene upregulation[J].Genes Cells,2024,29(11):1012-1025.
[13] Lin M,Fang Z,Lin X,et al.TRIM55 inhibits colorectal cancer development via enhancing protein degradation of c-Myc[J].Cancer Med,2023,12(12):13511-13521.
[14] Lin C,Feng S,DeOliveira C C,et al.Cryptochrome-timeless structure reveals circadian clock timing mechanisms[J].Nature,2023,617(7959):194-199.
[15] Xia K,Li S,Yang Y,et al.Cryptochrome 2 acetylation attenuates its antiproliferative effect in breast cancer[J].Cell Death Dis,2023,14(4):250.
[16] Yu Y,Li Y,Zhou L,et al.Cryptochrome 2(CRY2)suppresses proliferation and migration and regulates clock gene network in osteosarcoma cells[J].Med Sci Monit,2018,24:3856-3862.
[17] Huisman S A,Ahmadi A R,Ijzermans J N M,et al.Disruption of clock gene expression in human colorectal liver metastases[J].Tumour Biol,2016,37(10):13973-13981.
[18] 许静,孙诚谊,喻超,等.CRY2过表达对肝癌细胞增殖及生物钟基因、蛋白表达的影响[J].山东医药,2019,59(8):5-8.
Xu J,Sun C Y,Yu C,et al.Effects of CRY2 overexpression on cell proliferation,clock gene and protein expression of hepatocellular carcinoma cells[J].Shandong Medical Journal,2019,59(8):5-8.
[19] Zhang Z,Liang Z,Gao W,et al.Identification of circadian clock genes as regulators of immune infiltration in hepatocellular carcinoma[J].J Cancer,2022,13(11):3199-3208.
[20] Land H,Parada L F,Weinberg R A.Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes[J].Nature,1983,304(5927):596-602.
[21] Zhu Y,Zhou B,Hu X,et al.LncRNA LINC00942 promotes chemoresistance in gastric cancer by suppressing MSI2 degradation to enhance c-Myc mRNA stability[J].Clin Transl Med,2022,12(1):e703.
[22] Mastronikolis N,Ragos V,Kyrodimos E,et al.Mechanisms of c-Myc oncogenic activity in head and neck squamous cell carcinoma[J].J BUON,2019,24(6):2242-2244.
[23] Cao Z,Jing Y,Cheng C,et al.EIF2Ss,a novel c-Myc-correlated gene family,is associated with poor prognosis and immune infiltration in pancreatic adenocarcinoma[J].Front Biosci(Landmark Ed),2024,29(3):119.
[24] Hoffman B,Liebermann D A.Apoptotic signaling by c-Myc[J].Oncogene,2008,27(50):6462-6472.
[25] Chan A B,Parico G C G,Fribourgh J L,et al.Cry2 missense mutations suppress P53 and enhance cell growth[J].Proc Natl Acad Sci U S A,2021,118(27):e2101416118.
[26] Wu L,Cheng B,Liu Q,et al.Cry2 suppresses trophoblast migration and invasion in recurrent spontaneous abortion[J].J Biochem,2020,167(1):79-87.
[27] 赵行宇,杨昆,宋梓桐,等.胡桃醌通过促进c-Myc泛素化降解抑制宫颈癌细胞的生长和促进凋亡[J].南方医科大学学报,2022,42(7):1026-1031.
Zhao X Y,Yang K,Song Z T,et al.Juglone induces proliferation inhibition and apoptosis of cervical cancer cells via promoting c-Myc ubiquitination[J].Journal of Southern Medical University,2022,42(7):1026-1031.
[28] Dhanasekaran R,Park J,Yevtodiyenko A,et al.Myc ASO impedes tumorigenesis and elicits oncogene addiction in autochthonous transgenic mouse models of HCC and RCC[J].Mol Ther Nucleic Acids,2020,21:850-859.
[29] Lin C J,Nasr Z,Premsrirut P K,et al.Targeting synthetic lethal interactions between Myc and the eIF4F complex impedes tumorigenesis[J].Cell Rep,2012,1(4):325-333.
[30] Wang H,Yang W,Qin Q,et al.E3 ubiquitin ligase MAGI3 degrades c-Myc and acts as a predictor for chemotherapy response in colorectal cancer[J].Mol Cancer,2022,21(1):151.
[31] Chen S,Zhao L,Liu J,et al.Inhibition of KIF20A enhances the immunotherapeutic effect of hepatocellular carcinoma by enhancing c-Myc ubiquitination[J].Cancer Lett,2024,598:217105.
[32] Huber A L,Papp S J,Chan A B,et al.Cry2 and FBXL3 cooperatively degrade c-Myc[J].Mol Cell,2016,64(4):774-789.
[33] Guo J,Hao J,Jiang H,et al.Proteasome activator subunit 3 promotes pancreatic cancer growth via c-Myc-glycolysis signaling axis[J].Cancer Lett,2017,386:161-167.
[34] Wang J,Yang Y,Shao F,et al.Acetate reprogrammes tumour metabolism and promotes PD-L1 expression and immune evasion by upregulating c-Myc[J].Nat Metab,2024,6(5):914-932.

过表达Cry2通过介导c-Myc泛素化降解抑制肝细胞癌细胞增殖并诱导凋亡^*

Overexpression of Cry2 Suppresses Proliferation and Induces Apoptosis in Hepatocellular Carcinoma Cells by Mediating Ubiquitination and Degradation of c-Myc

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