
文章信息
- ECT2基因对宫颈癌细胞增殖的影响及其机制
- Effect of ECT2 Gene on Proliferation of Cervical Cancer Cells and Its Mechanism
- 肿瘤防治研究, 2022, 49(10): 1015-1020
- Cancer Research on Prevention and Treatment, 2022, 49(10): 1015-1020
- http://www.zlfzyj.com/CN/10.3971/j.issn.1000-8578.2022.22.0065
- 收稿日期: 2022-01-18
- 修回日期: 2022-05-04
2. 432100 孝感,武汉科技大学附属孝感医院/孝感市中心医院妇产科;
3. 999025 鹿特丹,荷兰鹿特丹伊拉斯姆斯大学免疫学系
2. Department of Obstetrics and Gynecology, Xiaogan Hospital Affilliated to Wuhan University of Science and Technology, The Central Hospital of Xiaogan, Xiaogan 432100, China;
3. Department of Immunology, Erasmus University Rotterdam, Rotterdam 999025, the Netherlands
宫颈癌是全球女性第四大恶性肿瘤,其中85%的病例发生在发展中国家,尽管实施了有效的筛查和疫苗接种计划,其总发病率有所下降,但在这些国家,宫颈癌的发病率和死亡率仍然较高,且宫颈癌是女性癌症死亡的主要原因之一[1-2]。大多数早期宫颈癌通过手术切除治愈,对于局部晚期宫颈癌,同步放化疗是首选的治疗方法[3-4]。然而,30%的局部晚期宫颈癌患者在根治性同步放化疗后仍会出现复发转移[5-6]。因此,需更深入的研究揭示新的治疗靶点。上皮细胞转化序列2(epithelial cell transformation sequence 2, ECT2)是人类ECT2基因编码的鸟嘌呤核苷酸交换因子,与癌症的发生直接相关[7],在非小细胞肺癌、乳腺癌和结直肠癌等多种肿瘤中发挥促癌作用[7-9]。ECT2对宫颈癌的影响目前尚不清楚,因此,本研究拟观察ECT2对人宫颈癌细胞增殖的影响,并探究其机制,以期为ECT2基因作为宫颈癌治疗的新靶点提供理论依据。
1 材料与方法 1.1 实验材料人宫颈癌细胞系C33A、SiHa和HeLa(湖北省肿瘤生物学行为研究所细胞库提供),DMEM培养基和胎牛血清(美国Hyclone公司),细胞转染试剂Lipo2000(南京诺维赞生物公司),qPCR引物(北京擎科生物科技有限公司),ECT2干扰质粒siRNA和阴性对照质粒(上海吉玛制药有限公司),ECT2过表达慢病毒及空载病毒(广州GeneCopoeia生物公司),Anti-GAPDH、Anti-CDK1、Anti-CyclinB1、羊抗兔、羊抗鼠二抗和兔抗羊二抗(武汉三鹰技术有限公司),Anti-ECT2、Anti-Cdc42和Anti-Rac1(美国Abcam公司),细胞周期检测试剂盒(上海碧云天公司),倒置相差显微镜和荧光显微镜(日本奥林巴斯公司),化学发光成像系统(美国Bio-Rad公司)。
1.2 实验方法 1.2.1 细胞培养及转染将C33A、SiHa和HeLa用含10%FBS的DMEM完全培养基置于5%CO2、37℃恒温培养箱培养。将对数生长期的HeLa细胞按1×105个/孔接种于24孔板内,用ECT2过表达慢病毒转染HeLa细胞,用2 μg/ml的嘌呤霉素进行筛选构建稳定表达实验组(HeLa-ECT2组),用空载体慢病毒转染构建阴性对照组(HeLa-NC组)。将C33a和SiHa细胞按4×105个/孔接种于六孔板内,用ECT2 siRNA质粒转染细胞,构建实验组(SiHa-siRNA组和C33a-siRNA组),用阴性对照质粒构建阴性对照组(SiHa-NC组和C33a-NC组)。
1.2.2 MTT实验取对数生长期的各组细胞接种于96孔板中,加入10 μl MTT试剂,置于恒温培养箱继续孵育4 h,弃上清液,每孔加入150 μl DMSO置摇床上低速振荡10 min,充分溶解结晶,用酶标仪测量490 nm波长处的吸光度值。
1.2.3 流式细胞术用不含EDTA的胰酶消化离心收集细胞至1.5 ml EP管中,无水乙醇固定过夜,1 000 r/min离心5 min,利用流式细胞仪检测碘化丙啶(PI)染色后的细胞悬液。
1.2.4 细胞免疫荧光取各组细胞单细胞悬液于盖玻片上,4%的多聚甲醛固定,细胞膜穿孔后,加入山羊血清封闭,加入羊抗人ECT2抗体(1:500)、兔抗人CDK1抗体(1:100),4℃孵育过夜,加入荧光标记的二抗(1:100)于37℃孵育1 h,加入DAPI染色,最后用荧光显微镜观察。
1.2.5 实时荧光定量PCR提取细胞RNA,用RNA反转录试剂盒合成cDNA,进行荧光定量PCR检测。GAPDH上游引物序列为:5’-CTGTTCGACAGTCAGCCGCATC-3’,下游引物序列为:5’-GCGCCCAATACGACCAAATCCG-3’;ECT2上游引物序列为:5’-TGTAGTCACGGACTTTCAGGA-3’,下游引物序列为:5’-GTACAATACAACGGGCGACAT-3’;Rac1上游引物序列为:5’-ATGTCCGTGCAAAGTGGTATC-3’,下游引物序列为:5’-CTCGGATCGCTTCGTCAAACA-3’;Cdc42上游引物序列为:5’-CCATCGGAATATGTACCGACTG-3’,下游引物序列为:5’-CTCAGCGGTCGTAATCTGTCA-3’;CDK1上游引物序列为:5’-TTGGGGACATTGGTAACAAAGTC-3’,下游引物序列为:5’-ATAGGCTCAGGCGAAAGTTTTT-3’;CyclinB1上游引物序列为:5’-TTGGGGACATTGGTAACAAAGTC-3’,下游引物序列为:5’-ATAGGCTCAGGCGAAAGTTTTT-3’。
1.2.6 Western blot实验提取各组细胞总蛋白,利用BCA法进行蛋白定量,在制好的凝胶板孔中进行电泳,而后依次转膜、5%脱脂牛奶封闭,分别用羊抗人ECT2抗体(1:1 500)、兔抗人CDK1、Cdc42、CyclinB1抗体(1:2 000)和鼠抗人GAPDH、Rac1抗体(1:2 000)4℃孵育过夜,室温下二抗(1:5 000)孵育2 h,最后利用化学发光成像系统进行ECL显影。
1.3 统计学方法用GraphPadPrism9.0软件对数据进行统计分析,两组间比较采用t检验,P < 0.05为差异有统计学意义。
2 结果 2.1 ECT2敲降和过表达效率在宫颈癌细胞中转染siRNA或过表达ECT2后,用qPCR检测ECT2在宫颈癌细胞中敲降和过表达效率,结果表明ECT2在C33a和SiHa细胞的干扰效率分别为70%(P < 0.001)和50%(P < 0.0001),在HeLa细胞中过表达ECT2后,ECT2的表达量为阴性对照组的2倍(P < 0.0001),见图 1。
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ECT2: epithelial cell transformation sequence 2; ***: P < 0.001; ****: P < 0.0001. 图 1 qPCR检测ECT2敲降(A, B)和过表达(C)效率 Figure 1 Construction of ECT2 knockdown(A, B) or overexpression(C) of cervical cancer cell lines detected by qPCR |
MTT检测发现,SiHa-siRNA组细胞增殖速度显著低于SiHa-NC组(P < 0.001),见图 2A,C33a-siRNA组细胞增殖速度显著低于C33a-NC组(P < 0.001),见图 2B,敲低ECT2细胞增殖速度减慢,说明ECT2促进宫颈癌细胞增殖。
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***: P < 0.001. 图 2 MTT法检测ECT2对宫颈癌SiHa细胞(A)和C33a细胞(B)增殖的影响 Figure 2 Effect of ECT2 on viability of SiHa(A) and C33a(B) cell lines detected by MTT method |
流式细胞术结果提示,与SiHa-NC组细胞相比,SiHa-siRNA组细胞G2/M期细胞比例上升而G1期细胞比例下降,见图 3A;与C33a-NC组细胞相比,C33a-siRNA组细胞G2/M期细胞比例上升而G1期细胞比例显著下降,见图 3B;与阴性对照组相比,HeLa-ECT2组细胞更多的由G2/M期进入G1期,见图 3C。因此可知,ECT2可通过调控G2/M期向G1期转化来调控细胞增殖。
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A: flow cytometry was used to detect the cell cycle in the constructed SiHa cell lines with ECT2 knockdown; B: flow cytometry was used to detect the cell cycle in the constructed C33a cell lines with ECT2 knockdown; C: flow cytometry was used to detect the cell cycle in the constructed Hela cell lines with ECT2 overexpression. 图 3 ECT2对宫颈癌细胞周期的影响 Figure 3 Effect of ECT2 on cell cycle of cervical cancer cells |
通过生物信息学通路分析软件(Ingenuity Pathway Analysis, IPA),我们发现ECT2可能与周期蛋白依赖性激酶1(cyclin dependent kinase 1, CDK1)存在相互作用。进一步的细胞免疫荧光结果显示见图 4A、B,CDK1(绿色)与ECT2(红色)共定位,融合色为黄色,ECT2主要分布于细胞质和细胞核。敲降ECT2后,核内标记ECT2和CDK1蛋白的荧光强度均出现下降。而CDK1和磷酸化细胞周期蛋白B1(CyclinB1)共同调节细胞周期G2/M期,qPCR及Western blot结果显示,敲降ECT2后CDK1、CyclinB1的mRNA及蛋白表达水平显著降低,见图 4C、D。
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A: relationship between ECT2 and CDK1 in ECT2 knockdown of Siha cells was detected by cell immunofluorescence co-localization. Scale bar=20μm; B: relationship between ECT2 and CDK1 in ECT2 knockdown of C33a cells was detected by cell immunofluorescence co-localization. Scale bar=20μm; C: effects of ECT2 knockdown on the expression of genes CDK1 and cyclin B1 in SiHa cells were detected by Western blot and qPCR; D: effects of ECT2 knockdown on the expression of genes CDK1 and cyclin B1 in C33a cells were detected by Western blot and qPCR. *: P < 0.05; ***: P < 0.001; ****: P < 0.0001. 图 4 ECT2与CDK1的关系 Figure 4 Relationship between ECT2 and CDK1 |
为研究ECT2是否通过作用于下游Rho GTP酶调控宫颈癌细胞的增殖,我们在宫颈癌细胞中敲降及过表达ECT2后,检测了Rho GTP酶Rac1和Cdc42 mRNA及蛋白水平的变化,qPCR及Western blot结果显示,SiHa和C33a细胞敲降ECT2后,Rac1、Cdc42 mRNA及蛋白水平均较对照组显著降低,见图 5A~B,而HeLa细胞过表达ECT2后,Rac1、Cdc42 mRNA及蛋白水平均较对照组显著增加,见图 5C。
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A: effects of ECT2 knockdown on the expression of genes Rac1 and Cdc42 in SiHa cells were detected by Western blot and qPCR; B: effects of ECT2 knockdown on the expression of genes Rac1 and Cdc42 in C33a cells were detected by Western blot and qPCR; C: effects of ECT2 overexpression on the expression of genes Rac1 and Cdc42 in Hela cells were detected by Western blot and qPCR. *: P < 0.05; ***: P < 0.001; ****: P < 0.0001. 图 5 ECT2对Rac1、Cdc42 mRNA及蛋白表达的影响 Figure 5 Effect of ECT2 on Rac1 and Cdc42 mRNA and protein expression |
ECT2在肺癌、乳腺癌、胃癌和胰腺癌等肿瘤中发挥原癌基因的功能[10-13],其机制主要通过激活RhoA-ERK信号通路,促进VEGF和MMP9的表达,从而促进肿瘤细胞增殖、侵袭、迁移,进而促进肿瘤的发展[14]。此外,ECT2还通过增强有氧糖酵解和抑制NK细胞和T细胞的功能促进M2巨噬细胞的极化[15]。本研究发现,敲降ECT2后,宫颈癌细胞增殖速度降低,且伴随G2期阻滞;而过表达ECT2后,宫颈癌细胞增殖速度增加,同时更多细胞从G2/M转化为G1期。上述结果提示了ECT2可能作为促癌基因促进宫颈癌细胞增殖。
ECT2定位于癌细胞的细胞核和细胞质中,且其致癌能力与Rac1活化相关[16-17]。ECT2通过激活RAC靶向RhoA来调节细胞的分裂,在细胞分裂间期,ECT2/Cdc42通路控制着丝粒处组蛋白变体CENP-A的掺入,而ECT2/Rac1可以促进核糖体DNA(rDNA)转录[18]。位于非小细胞肺癌(NSCLC)细胞核仁的大量ECT2,与核糖体DNA启动子区域上的转录因子上游结合因子1(UBF1)结合,募集并激活小GTP酶Rac1到rDNA,这反过来刺激活性Rac1与核磷蛋白(NPM)的结合,以驱动rDNA转录、转化生长和体内肺肿瘤形成[17, 19]。有研究发现,同为鸟嘌呤核苷酸交换因子的鸟嘌呤核苷酸交换因子T可以通过激活Rac1/Cdc42通路抑制横纹肌肉瘤细胞的凋亡并加速横纹肌肉瘤的生长和肺转移[20]。本研究发现,敲降/过表达ECT2后,Rac1/Cdc42通路核心基因Rac1、Cdc42的mRNA及蛋白水平发生降低/增加,提示ECT2可能通过调控Rac1/Cdc42信号通路进而调控细胞周期。另一方面,ECT2可能通过与CDK1的蛋白互作实现对细胞周期的调控。CyclinB1及其催化伙伴CDK1是调节细胞从G2期到有丝分裂进程的基本激酶,CyclinB1/CDK1磷酸化决定了细胞是否能进入有丝分裂,其特征是核膜破裂、纺锤体形成和染色质凝聚[21]。CyclinB1/CDK1复合物是G2/M期DNA损伤检查点的重要调节剂,华蟾素和高三尖杉酯碱通过负调节CDK1/CyclinB1复合物使细胞周期停滞在G2/M期来抑制恶性黑色素瘤细胞增殖[22-23]。本研究发现,在宫颈癌细胞中敲降ECT2可以使CDK1和CyclinB1的表达降低,过表达ECT2使Rac1、Cdc42 mRNA及蛋白表达显著增加,说明ECT2可能通过正向调节CDK1/CyclinB1调控G2/M期向G1期转化,促进细胞增殖。
综上所述,ECT2作为促癌基因促进宫颈癌细胞恶性转化,可能通过下游Cdc42/Rac1信号通路,同时与CDK1在宫颈癌细胞内共定位调控细胞周期(G2/M期),促进宫颈癌细胞增殖。ECT2基因可能在宫颈癌靶向治疗中具有一定的临床价值。
作者贡献:
陈宇:标本收集、数据统计及论文撰写
宋紫烨:实验操作及数据统计
高阳:论文修改
蔡红兵:实验设计及指导
[1] |
National Comprehensive Cancer Network. Cervical cancer (NCCN guideline version 1.2022)[EB/OL]. https://www.nccn.org/guidelines/category_1.
|
[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] |
Marth C, Landoni F, Mahner S, et al. Cervical cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up[J]. Ann Oncol, 2017, 28(suppl_4): iv72-iv83. |
[4] |
Kim KH, Chang JS, Byun HK, et al. A novel gene signature associated with poor response to chemoradiotherapy in patients with locally advanced cervical cancer[J]. J Gynecol Oncol, 2022, 33(1): e7. DOI:10.3802/jgo.2022.33.e7 |
[5] |
Kim J, Cho Y, Kim N, et al. Magnetic resonance imaging-based validation of the 2018 FIGO staging system in patients treated with definitive radiotherapy for locally advanced cervix cancer[J]. Gynecol Oncol, 2021, 160(3): 735-741. DOI:10.1016/j.ygyno.2020.12.012 |
[6] |
Tan LT, Pötter R, Sturdza A, et al. Change in patterns of failure after image-guided brachytherapy for cervical cancer: analysis from the retroembrace study[J]. Int J Radiat Oncol Biol Phys, 2019, 104(4): 895-902. DOI:10.1016/j.ijrobp.2019.03.038 |
[7] |
Xiu Y, Liu W, Wang T, et al. Overexpression of ECT2 is a strong poor prognostic factor in ER(+) breast cancer[J]. Mol Clin Oncol, 2019, 10(5): 497-505. |
[8] |
Bai X, Yi M, Xia X, et al. Progression and prognostic value of ECT2 in non-small-cell lung cancer and its correlation with PCNA[J]. Cancer Manag Res, 2018, 10: 4039-4050. DOI:10.2147/CMAR.S170033 |
[9] |
Cook DR, Kang M, Martin TD, et al. Aberrant expression and subcellular localization of ECT2 drives colorectal cancer progression and growth[J]. Cancer Res, 2021, 82(1): 90-104. |
[10] |
Chen S, Zhu X, Zheng J, et al. miR-30a-5p regulates viability, migration, and invasion of lung adenocarcinoma cells via targeting ECT2[J]. Comput Math Methods Med, 2021, 2021: 6241469. |
[11] |
Wang HK, Liang JF, Zheng HX, et al. Expression and prognostic significance of ECT2 in invasive breast cancer[J]. J Clin Pathol, 2018, 71(5): 442-445. DOI:10.1136/jclinpath-2017-204569 |
[12] |
Wang HB, Yan HC, Liu Y. Clinical significance of ECT2 expression in tissue and serum of gastric cancer patients[J]. Clin Transl Oncol, 2016, 18(7): 735-742. DOI:10.1007/s12094-015-1428-2 |
[13] |
Liu B, Yang H, Taher L, et al. Identification of Prognostic Biomarkers by Combined mRNA and miRNA Expression Microarray Analysis in Pancreatic Cancer[J]. Transl Oncol, 2018, 11(3): 700-714. DOI:10.1016/j.tranon.2018.03.003 |
[14] |
Sun BY, Wei QQ, Liu CX, et al. ECT2 promotes proliferation and metastasis of esophageal squamous cell carcinoma via the RhoA-ERK signaling pathway[J]. Eur Rev Med Pharmacol Sci, 2020, 24(15): 7991-8000. |
[15] |
Xu D, Wang Y, Wu J, et al. ECT2 overexpression promotes the polarization of tumor-associated macrophages in hepatocellular carcinoma via the ECT2/PLK1/PTEN pathway[J]. Cell Death Dis, 2021, 12(2): 162. DOI:10.1038/s41419-021-03450-z |
[16] |
Chen Z, Liu J, Zhang Y. Role of Epithelial Cell Transforming Sequence 2 (ECT2) in Predicting Prognosis of Osteosarcoma[J]. Med Sci Monit, 2017, 23: 3861-3868. DOI:10.12659/MSM.905951 |
[17] |
Justilien V, Ali SA, Jamieson L, et al. Ect2-Dependent rRNA Synthesis Is Required for KRAS-TRP53-Driven Lung Adenocarcinoma[J]. Cancer Cell, 2017, 31(2): 256-269. DOI:10.1016/j.ccell.2016.12.010 |
[18] |
Cao C, Han P, Liu L, et al. Epithelial cell transforming factor ECT2 is an important regulator of DNA double-strand break repair and genome stability[J]. J BiolChem, 2021, 297(3): 101036. |
[19] |
Justilien V, Lewis KC, Murray NR, et al. Oncogenic Ect2 signaling regulates rRNA synthesis in NSCLC[J]. Small GTPases, 2019, 10(5): 388-394. DOI:10.1080/21541248.2017.1335274 |
[20] |
Liu C, Zhang L, Cui W, et al. Epigenetically upregulated GEFT-derived invasion and metastasis of rhabdomyosarcoma via epithelial mesenchymal transition promoted by the Rac1/Cdc42-PAK signalling pathway[J]. EBioMedicine, 2019, 50: 122-134. DOI:10.1016/j.ebiom.2019.10.060 |
[21] |
Xie B, Wang S, Jiang N, et al. Cyclin B1/CDK1-regulated mitochondrial bioenergetics in cell cycle progression and tumor resistance[J]. Cancer Lett, 2019, 443: 56-66. DOI:10.1016/j.canlet.2018.11.019 |
[22] |
Tang JF, Li GL, Zhang T, et al. Homoharringtonine inhibits melanoma cells proliferation in vitro and vivo by inducing DNA damage, apoptosis, and G2/M cell cycle arrest[J]. Arch Biochem Biophys, 2021, 700: 108774. DOI:10.1016/j.abb.2021.108774 |
[23] |
Pan Z, Zhang X, Yu P, et al. Cinobufagin induces cell cycle arrest at the G2/M phase and promotes apoptosis in malignant melanoma cells[J]. Front Oncol, 2019, 9: 853. DOI:10.3389/fonc.2019.00853 |