Inhibitory Effect of UHRF1 on Invasion and Migration of Colorectal Cancer Cells via WNT/MMP9 Signaling Pathway
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摘要:目的
探索UHRF1基因与结直肠癌(CRC)患者临床病理特性的关系,慢病毒转染过表达和敲减UHRF1对CRC细胞增殖、侵袭及迁移的影响及可能的信号通路。
方法免疫组织化学和RT-PCR法检测112例CRC癌组织与癌旁组织UHRF1的表达。构建慢病毒转染过表达和敲减UHRF1载体,分别转染SW480和HCT116细胞株,RT-PCR和Western blot检测转染前后及IWP-2(WNT拮抗剂)和HLY78(WNT活化剂)干预前后的UHRF1、WNT信号通路关键分子和MMP9的表达。EDU检测细胞增殖能力;Transwell实验检测细胞迁移和侵袭能力。
结果(1)TCGA数据库和临床数据中,癌组织中UHRF1 mRNA和蛋白表达均高于正常组织。UHRF1表达量与TNM分期、N和M分期密切相关。TCGA中UHRF1低表达患者有更长的5年OS和疾病相关存活时间(DSS),UHRF1预测1、3和5年OS的ROC曲线下面积(AUC)分别为0.634、0.652和0.771;在临床数据中3年OS也有相同生存获益,UHRF1高表达是CRC不良预后因素。(2)过表达UHRF1后,SW480中WNT3a、GSK3β和MMP9分子表达明显升高,p-β-catenin表达下降(P < 0.05);敲减UHRF1后,HCT116细胞中WNT3a、GSK3β、MMP9表达下降,而p-β-catenin表达升高(P < 0.05);分别用IWP-2和HLY78进行“拯救”实验,能够获得一致结果。(3)过表达组细胞增殖、迁移和侵袭能力明显高于对照组;IWP-2处理后,细胞增殖、迁移及侵袭能力则受到抑制。敲减实验呈现与过表达实验相反的结果。
结论UHRF1在CRC发生发展中可能具有重要作用,UHRF1高表达可能是CRC不良预后因素,UHRF1可能通过WNT/MMP9信号通路来影响CRC的增殖、迁移和侵袭。
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关键词:
- 结直肠癌 /
- UHRF1 /
- WNT/MMP9信号通路 /
- 增殖 /
- 侵袭
Abstract:ObjectiveTo explore the relationship of UHRF1 with the clinicopathological characteristics of colorectal cancer (CRC) patients, as well as the effects of lentivirus transfection overexpression and knockdown of UHRF1 on the proliferation, invasion, and migration of CRC cells and the possible signaling pathways.
MethodsThe expression of UHRF1 mRNA and protein in CRC tissues and adjacent tissues was detected by immunohistochemical staining and RT-PCR. The effects of the constructed UHRF1 overexpression- and knockdown-group cells on the expression of UHRF1, related molecules in the WNT signaling pathway, and MMPR9 were examined by Western blot and RT-PCR. EDU and Transwell assays were used to detect changes in the proliferation, migration, and invasion of CRC cells.
Results(1) In the TCGA database and clinical data, the mRNA and protein expression levels of UHRF1 in CRC cancer tissues were significantly higher than those in adjacent normal tissues. UHRF1 expression was closely correlated with TNM stage, N stage, and M stage. Patients with low UHRF1 expression in TCGA had better 5-year OS and disease-specific survival. The area under the ROC curve of UHRF1 for predicting 1-, 3-, and 5-year OS were 0.634, 0.652, and 0.771, respectively. The 3-year OS in the clinical data also showed the same survival benefit. UHRF1 overexpression was a poor prognostic factor for CRC patients. (2) After UHRF1 overexpression, the expression of WNT3a, GSK3β, and MMP9 in SW480 cells significantly increased, whereas the expression of p-β-catenin decreased (P < 0.05). After UHRF1 knockdown, the expression of WNT3a, GSK3β, and MMP9 in HCT116 cells decreased, whereas the expression of p-β-catenin increased (P < 0.05). The "rescue" experiment with IWP-2 and HLY78 can produce consistent results. (3) Compared with the control group, the cell proliferation, migration, and invasion abilities of the UHRF1 overexpression group were enhanced. After IWP-2 treatment, the cell proliferation, migration, and invasion abilities were inhibited. Knockdown experiment exhibited the reverse results to overexpression experiment.
ConclusionUHRF1 may play an important role in the occurrence and development of CRC. UHRF1 overexpression may be a poor prognostic factor for CRC patients. UHRF1 may affect the proliferation, migration, and invasion of CRC cells through the WNT/MMP9 signaling pathway.
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Key words:
- Colorectal cancer /
- UHRF1 /
- WNT/MMP9 signaling pathway /
- Proliferation /
- Invasion
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0 引言
胶质母细胞瘤(glioblastoma multiforme,GBM)为恶性程度最高的星形细胞瘤(属WHO分级的Ⅳ级)。临床上,由于该肿瘤呈浸润性快速生长,多数患者,颅内压迫症状严重,手术清除难度大。即便在手术配合放化疗治疗方案下,绝大多数GBM患者仍因肿瘤复发死亡,中位生存时间只有14~18月[1-2]。临床上,无论是原发或继发GBM患者,肿瘤的基因背景与生物学行为都具有不均一性,因此,每个患者都需进行个体化的治疗决策和预后判断。目前,应用于GBM的临床诊断技术和肿瘤监测手段仍比较单一(主要为影像学技术),存在缺陷,不利于GBM的个体化治疗决策,寻找新的更有效的检测手段迫在眉睫。
近年来,随着生物检测技术的发展,GBM患者血液中检测到肿瘤细胞已是不争的事实[3-4],这些与GBM的复发密切相关的细胞称为循环肿瘤细胞(circulating tumor cells, CTCs),它们的检测也被誉为“循环活检”。尽管文献报道不多,CTCs的发现给GBM患者的诊治带来了新的希望,它在GBM的早期诊断、肿瘤复发风险评估和预后评估等方面具有潜在的临床应用价值,使GBM诊治的决策更加“有章可循”。本文就胶质母细胞瘤CTCs的一些研究进展综述如下。
1 胶质母细胞瘤CTCs概述
九十年代初,科学家们用免疫细胞化学和克隆形成实验技术检测到乳腺癌患者外周血中的肿瘤细胞后,CTCs的检测和应用逐渐受到重视[5]。迄今已有多项研究表明,GBM患者与一些肿瘤(乳腺癌、前列腺癌、结直肠癌等)患者一样,血液中可以检测到CTCs[3-4, 6-7]。但在外周血中,CTCs受血流剪应力、失巢凋亡和免疫细胞识别杀伤等因素的影响,绝大多数发生死亡[8],这给CTCs的检测带来了极大的挑战。
有研究认为GBM患者中的CTCs来源于一群具有高度繁殖、自我更新、分化的能力的细胞群体,称为GBM肿瘤干细胞(glioblastoma stem cells, GSC)[8];GSC具有较高的成瘤潜质,且能够通过神经上皮间质转换过程(glio-mesenchymal transition, GMT)[9]逃脱原发的肿瘤组织,由微脉管入侵循环系统,最后在远处或原发部位种植,完成肿瘤的转移和复发。肿瘤患者血液中幸存的CTCs不仅具有增殖、侵袭和迁移能力,而且具有极强的环境适应能力,对于放化疗具有一定的抵抗性,表现出明显的干细胞特性[10]。这种特性可解释GBM各种治疗效果不理想、易复发、预后极差的原因。
2 CTCs与GBM的复发
绝大多数GBM最终会复发。手术治疗很难做到完全清除肿瘤细胞,其目的更多是为了减轻瘤负荷、缓解症状、明确病理。GBM复发机制复杂,除了与肿瘤的分化程度、手术切除范围、放化疗敏感度等因素有关外,与进入循环的肿瘤细胞也有关,Macarthur等[6]在胶母CTCs的检测中发现,在术后化疗的GBM患者血液中,肿瘤细胞较术前明显减少,遗憾的是该研究未能对患者的预后进行统计分析。
GBM复发的机制有多种观点,有研究认为GBM肿瘤干细胞(GSC)是GBM复发的主要原因。GSC相邻的肿瘤细胞会通过旁分泌和细胞接触等方式抑制GSC,使GSC进入休眠状态。在特定的情况下,如手术、化疗、肿瘤细胞脱落入血等因素影响,使静止的GSC与周围肿瘤细胞隔开,增殖能力将会被重新激活。这种静止又被重新激活的GSC按激活部位的不同分为两类:(1)原发灶的GSC;(2)脱落入血的GSC——CTCs。它们都可能是导致GBM复发的主要因素[11-12]。
研究报道虽然显示GBM极少发生其他器官的转移(仅0.4%的GBM患者伴有肝脏、脾脏、肾脏及皮肤的转移)[13-14],但是大多数GBM最终都会复发,其中约2/3复发区域位于原发肿瘤附近(≤2 cm),由原发灶的GSC激活所致。而远处复发(> 2 cm)约占1/6的复发性GBM,发生于对侧大脑半球、其他脑叶或幕下等[15]。一些观点认为,远处复发的GBM并非源于原发肿瘤,而是在放化疗或其他因素下诱导发生,属于新原发肿瘤。而一些研究持有不同的观点,认为GBM的复发起源于原发肿瘤,并提出远处复发的肿瘤与原发肿瘤的基因具有一定的相似性[16]。也就是说,CTCs是导致肿瘤在远处复发的原因,这也是复发肿瘤与原发肿瘤基因相似性的原因,放化疗则可能诱导了CTCs的发生。
3 胶质母细胞瘤CTCs检测技术进展
目前,CellSearch系统是美国食品药品监督管理局唯一批准用于临床检测CTCs的产品(主要针对乳腺癌及前列腺癌)[17],它的应用主要基于CTCs特异的高表达的上皮分子相关抗原(如EpCAM、CK等)。但经过神经上皮间质转换过程(GMT)的胶质母细胞瘤CTCs,EpCAM、CK的表达缺失[18],使CellSearch系统无法应用于胶质母细胞瘤CTCs与外周血细胞的分离,因此识别胶质母细胞瘤CTCs需要新的免疫分子。所以高敏感、高特异的辨识因子和检测手段标准化应用,是当下胶质母细胞瘤CTCs研究重点所在。
如何检测胶质母细胞瘤CTCs,科学家们设计多种方法。Müller等[3]曾采集141例GBM患者血液,与健康者血液对照,通过离心法富集血液中所有细胞,用胶质原纤维酸性蛋白(glial fibrillary acidic protein, GFAP)免疫染色的方法识别CTCs,结果显示GFAP阳性率为20.6%。而这些GFAP阳性的细胞呈现表皮生长因子受体(epidermal growth factor receptor, EGFR)基因的表达明显上调。Sullivan等[4]通过微流体技术——CTC-iChip富集87例GBM患者血液中的细胞,再用一组胶质瘤标志物(SOX2、tubulinbeta-3、EGFR、A2B5和c-Met)进行CTCs的识别,结果在27例GBM患者血液中发现了CTCs,还发现一些与侵袭性相关的基因(如SERPINE1、TGFB1、TGFBR2和vimentin等)在这些CTCs中高表达。
Macarthur等[6]使用物理离心法富集血细胞,然后用含人端粒酶反转录酶启动子和绿色荧光蛋白(green fluorescent protein, GFP)表达盒的腺病毒感染所得细胞,使细胞能在端粒酶反转录酶的作用下激活(GFP telomerase-based assay),结果发现11例患者中有8例(72%)为CTCs阳性,而在8例经放疗的高级别胶质瘤患者血液中仅1例(12%)CTCs阳性。Zhang等[7]用类似的方法(所选择的病毒不同)检测了290例肿瘤患者及178例正常人血液样本,肿瘤病例包括肺癌、结肠癌、胃癌、肝癌、胰腺癌及胶质瘤,其中CTCs阳性患者为219(75.5%)例,而在39胶质瘤患者中,CTCs阳性为23(59%)例。
4 CTCs应用于GBM的展望
CTCs是否存在于早期的GBM患者血液中目前仍有争议。理论上GBM自形成之后,便存在GMT特性,不断的尝试将自身细胞释放入血,并企图在其他环境下进行无性繁殖[19]。因此,CTCs检测理论上能更早于影像学发现GBM。而临床上占位明确的颅内肿瘤患者,很难对肿瘤进行准确的定性分级,影响GBM患者治疗方案的选择,进而影响了GBM患者的总体预后。因此早期的分类定性GBM,相当于术前肿瘤的活体检查,将会给临床的治疗决策和改善患者的预后带来巨大的收益。例如,在以往不同版本的颅内肿瘤治疗指南中,手术切除、联合放射和化学治疗是治疗GBM的总原则,未经手术的经验型化疗及放疗方案,常因错判治疗的敏感度而延误最佳治疗时期,不仅影响治疗效果,而且增加患者经济负担。而术前CTCs的检测可以在基因和细胞层面早期评估肿瘤恶性程度,使临床工作者更迅速地预知放疗和化疗个体方案对患者治疗的敏感度,更利于肿瘤的规范化、个体化治疗,提高患者疗效,延长总体生存时间,并改善生存质量。
5 小结
GBM由于极差的预后而受到广泛的关注,在手术、放化疗多种方案的结合下,预后仍不良,这是因为GBM的恶性程度高和临床上缺乏及时准确的检测手段。近年来,随着生物检测技术的发展,CTCs被证明存在于GBM患者血液中,它具有肿瘤干细胞特性,能够被多种生物标记检测到,但现有的检测技术仍未标准和规范化。
胶质母细胞瘤CTCs的发现让我们看到了新的希望:(1)CTCs有望较影像学更早更及时地发现肿瘤;(2)CTCs有望成为术前准确诊断肿瘤的手段,指导临床治疗工作;(3)CTCs有望成为评估肿瘤复发的关键指标。当下寻找高敏感度的CTCs免疫标志物、优化检测手段是胶质母细胞瘤CTCs检测研究的重难点,目前CTCs的临床价值仍需更大量的研究数据评估。
Competing interests: The authors declare that they have no competing interests.利益冲突声明:所有作者均声明不存在利益冲突。作者贡献:陈志华:研究设计和实验实施、文章撰写郑艳:研究设计和实验实施林素勇:数据分析陈绍勤:文章审阅 -
表 1 UHRF1表达与CRC患者临床病理特征的关系
Table 1 Relationships between UHRF1 expression and clinicopathological characteristics in CRC patients
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[1] Siegel RL, Miller KD, Wagle NS, et al. Cancer statistics, 2023[J]. CA Cancer J Clin, 2023, 73(1): 17-48. doi: 10.3322/caac.21763
[2] 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): 209-249. doi: 10.3322/caac.21660
[3] Munro MJ, Wickremesekera SK, Peng L, et al. Cancer stem cells in colorectal cancer: a review[J]. J Clin Pathol, 2018, 71(2): 110-116. doi: 10.1136/jclinpath-2017-204739
[4] Katoh M, Katoh M. WNT signaling and cancer stemness[J]. Essays Biochem, 2022, 66(4): 319-331. doi: 10.1042/EBC20220016
[5] Birrer DL, Tschuor C, Reiner C, et al. Multimodal treatment strategies for colorectal liver metastases[J]. Swiss Med Wkly, 2021, 151: w20390. doi: 10.4414/smw.2021.20390
[6] Chen S, Chen Z, Lin S, et al. KISS1 methylation and expression as predictors of disease progression in colorectal cancer patients[J]. World J Gastroenterol, 2014, 20(29): 10071-10081. doi: 10.3748/wjg.v20.i29.10071
[7] Hopfner R, Mousli M, Jeltsch JM, et al. ICBP90, a novel human CCAAT binding protein, involved in the regulation of topoisomerase Ⅱalpha expression[J]. Cancer Res, 2000, 60(1): 121-128.
[8] Bronner C, Krifa M, Mousli M. Increasing role of UHRF1 in the reading and inheritance of the epigenetic code as well as in tumorogenesis[J]. Biochem Pharmacol, 2013, 86(12): 1643-1649. doi: 10.1016/j.bcp.2013.10.002
[9] Bronner C, Alhosin M, Hamiche A, et al. Coordinated Dialogue between UHRF1 and DNMT1 to Ensure Faithful Inheritance of Methylated DNA Patterns[J]. Genes (Basel), 2019, 10(1): 65. doi: 10.3390/genes10010065
[10] Hu Q, Qin Y, Ji S, et al. UHRF1 promotes aerobic glycolysis and proliferation via suppression of SIRT4 in pancreatic cancer[J]. Cancer Lett, 2019, 452: 226-236. doi: 10.1016/j.canlet.2019.03.024
[11] Xu X, Ding G, Liu C, et al. Nuclear UHRF1 is a gate-keeper of cellular AMPK activity and function[J]. Cell Res, 2022, 32(1): 54-71. doi: 10.1038/s41422-021-00565-y
[12] Pacaud R, Brocard E, Lalier L, et al. The DNMT1/PCNA/UHRF1 disruption induces tumorigenesis characterized by similar genetic and epigenetic signatures[J]. Sci Rep, 2014, 4: 4230. doi: 10.1038/srep04230
[13] Wang H, Cao D, Wu F. Long noncoding RNA UPAT promoted cell proliferation via increasing UHRF1 expression in non-small cell lung cancer[J]. Oncol Lett, 2018, 16(2): 1491-1498.
[14] Wang F, Yang YZ, Shi CZ, et al. UHRF1 promotes cell growth and metastasis through repression of p16(ink(4)a) in colorectal cancer[J]. Ann Surg Oncol, 2012, 19(8): 2753-2762. doi: 10.1245/s10434-011-2194-1
[15] Kong X, Chen J, Xie W, et al. Defining UHRF1 Domains that Support Maintenance of Human Colon Cancer DNA Methylation and Oncogenic Properties[J]. Cancer Cell, 2019, 35(4): 633-648. e7. doi: 10.1016/j.ccell.2019.03.003
[16] Lin Y, Chen Z, Lin S, et al. MiR-202 inhibits the proliferation and invasion of colorectal cancer by targeting UHRF1[J]. Acta Biochim Biophys Sin (Shanghai), 2019, 51(6): 598-606.
[17] Lin Y, Chen Z, Zheng Y, et al. MiR-506 Targets UHRF1 to Inhibit Colorectal Cancer Proliferation and Invasion via the KISS1/PI3K/NF-κB Signaling Axis[J]. Front Cell Dev Biol, 2019, 7: 266. doi: 10.3389/fcell.2019.00266
[18] Mancini M, Magnani E, Macchi F, et al. The multi-functionality of UHRF1: epigenome maintenance and preservation of genome integrity[J]. Nucleic Acids Res, 2021, 49(11): 6053-6068. doi: 10.1093/nar/gkab293
[19] Ren Y. Regulatory mechanism and biological function of UHRF1-DNMT1-mediated DNA methylation[J]. Funct Integr Genomics, 2022, 22(6): 1113-1126. doi: 10.1007/s10142-022-00918-9
[20] Kim JK, Kan G, Mao Y, et al. UHRF1 downmodulation enhances antitumor effects of histone deacetylase inhibitors in retinoblastoma by augmenting oxidative stress-mediated apoptosis[J]. Mol Oncol, 2019, 14(2): 329-346.
[21] Jiao D, Huan Y, Zheng J, et al. UHRF1 promotes renal cell carcinoma progression through epigenetic regulation of TXNIP[J]. Oncogene, 2019, 38(28): 5686-5699. doi: 10.1038/s41388-019-0822-6
[22] Lu NH, Wei CY, Qi FZ, et al. Hsa-let-7b suppresses cell proliferation by targeting UHRF1 in melanoma[J]. Cancer Invest, 2020, 38(1): 52-60. doi: 10.1080/07357907.2019.1709482
[23] Wang F, Yang Y, Shi C, et al. UHRF1 promotes cell growth and metastasis through repression of p16(ink4a) in colorectal cancer[J]. Ann Surg Oncol, 2012, 19(8): 2753-2762. doi: 10.1245/s10434-011-2194-1
[24] Niinuma T, Kitajima H, Kai M, et al. UHRF1 depletion and HDAC inhibition reactivate epigenetically silenced genes in colorectal cancer cells[J]. Clin Epigenetics, 2019, 11(1): 70. doi: 10.1186/s13148-019-0668-3
[25] Cui X, Cui Y, Du T, et al. SHMT2 Drives the Progression of Colorectal Cancer by Regulating UHRF1 Expression[J]. Can J Gastroenterol Hepatol, 2022, 2022: 3758697.
[26] Patel S, Alam A, Pant R, et al. Wnt Signaling and Its Significance Within the Tumor Microenvironment: Novel Therapeutic Insights[J]. Front Immunol, 2019, 10: 2872.
[27] Zhao H, Ming T, Tang S, et al. Wnt signaling in colorectal cancer: pathogenic role and therapeutic target[J]. Mol Cancer, 2022, 21(1): 144.
[28] Parsons MJ, Tammela T, Dow LE. WNT as a Driver and Dependency in Cancer[J]. Cancer Discov, 2021, 11(10): 2413-2429.
[29] Yu F, Yu C, Li F, et al. Wnt/β-catenin signaling in cancers and targeted therapies[J]. Signal Transduct Target Ther, 2021, 6(1): 307.
[30] Zhou Y, Xu J, Luo H, et al. Wnt signaling pathway in cancer immunotherapy[J]. Cancer Lett, 2022, 525: 84-96.
[31] Han J, Chen X, Xu J, et al. Simultaneous silencing Aurora-A and UHRF1 inhibits colorectal cancer cell growth through regulating expression of DNMT1 and STAT1[J]. Int J Med Sci, 2021, 18(15): 3437-3451.