2022, Vol. 49
ECT2基因对宫颈癌细胞增殖的影响及其机制
本刊由国家卫生和计划生育委员会主管，湖北省卫生厅、中国抗癌协会、湖北省肿瘤医院主办。
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
引用本文 |
陈宇, 宋紫烨, 高阳, 蔡红兵. ECT2基因对宫颈癌细胞增殖的影响及其机制[J]. 肿瘤防治研究, 2022, 49(10): 1015-1020.
CHEN Yu, SONG Ziye, GAO Yang, CAI Hongbing. Effect of ECT2 Gene on Proliferation of Cervical Cancer Cells and Its Mechanism[J]. Cancer Research on Prevention and Treatment, 2022, 49(10): 1015-1020.
ECT2基因对宫颈癌细胞增殖的影响及其机制
1. 430071 武汉，武汉大学中南医院妇瘤科/湖北省肿瘤医学研究中心/肿瘤生物学行为湖北省重点实验室;
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. 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
摘要: 目的 探讨ECT2基因对宫颈癌细胞增殖的影响及其机制。方法 构建ECT2过表达及敲低的宫颈癌细胞株，MTT法检测细胞增殖能力，流式细胞术检测细胞周期。IPA软件查找ECT2的相互作用蛋白，免疫荧光亚细胞定位验证两者间的关系。qPCR及Western blot检测mRNA及蛋白的表达情况。结果 ECT2可能与CDK1相互作用，促进细胞由G2期进入G1期，促进细胞增殖。ECT2过表达后宫颈癌细胞中Rac1、Cdc42、CDK1、CyclinB1 mRNA及蛋白的表达水平均升高（均P < 0.001）；敲低则作用相反（P < 0.05）。结论 ECT2可能通过下游Cdc42/Rac1信号通路，同时与CDK1相互作用促进细胞周期G2向G1转化，进而促进宫颈癌细胞增殖。
关键词:
宫颈癌 ECT2 增殖 细胞周期 CDK1
Effect of ECT2 Gene on Proliferation of Cervical Cancer Cells and Its Mechanism
1. Department of Gynecological Oncology, Zhongnan Hospital of Wuhan University, Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Wuhan 430071, China;
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
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
Abstract: Objective To study the effect of epithelial cell transformation sequence 2 (ECT2) on the proliferation of cervical cancer cells and its mechanism. Methods We transfected cervical cancer cells HeLa (HeLa-ECT2) with the lentivirus overexpressing ECT2 and the cells SiHa (SiHa-siRNA) and C33a (C33a-siRNA) with the interfering plasmid. MTT assay was performed to detect cell proliferation ability. Flow cytometry was conducted to detect the cell cycle of each group. The IPA database was searched for the interacting proteins of ETC2, and immunofluorescence subcellular localization verified the effect between the two. qPCR and Western blot were carried out to detect the expression of Rac1, Cdc42, CDK1, and Cyclin B1 mRNA and protein in each group of cells. Results ECT2 may interact with CDK1. After ECT2 expression was upregulated, the G2/M phase of HeLa-ECT2 cells accelerated the transformation to G1 phase, cell proliferation ability was enhanced, and the expression levels of Rac1, Cdc42, CDK1, and cyclin B1 mRNA and protein all increased (P < 0.001); the knockdown of ECT2 expression would reverse the effect (P < 0.05). Conclusion ECT2 accelerates G2 phase of cervical cancer cells to G1 phase and promotes cell proliferation by co-localizing with CDK1 through the downstream Cdc42/Rac1 signaling pathway.
Key words:
Cervical cancer ECT2 Proliferation Cell cycle Cyclin-dependent kinase 1
0 引言
2.2 ECT2对宫颈癌细胞增殖的影响
2.3 ECT2对宫颈癌细胞周期的影响
2.4 ECT2与CDK1的关系
2.5 ECT2对Rac1、Cdc42的mRNA及蛋白水平的影响
3 讨论
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
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