MELK依赖E2F1调控mTOR信号通路影响TNBC细胞体外增殖、侵袭及迁移的分子机制-现代医学

MELK依赖E2F1调控mTOR信号通路影响TNBC细胞体外增殖、侵袭及迁移的分子机制

单位：河南科技大学第一附属医院河南科技大学临床医学院, 河南洛阳 471003

关键词：三阴性乳腺癌母胚胎亮氨酸拉链激酶 E2F家族转录因子1 哺乳动物雷帕霉素靶蛋白信号通路增殖迁移

分类号：R737.9

出版年·卷·期（页码）：2026·45·第四期（664-675）

摘要：

目的： 探究母胚胎亮氨酸拉链激酶(MELK)对于三阴性乳腺癌(TNBC)恶性进展的作用及其潜在的分子机制。方法： 通过基因表达综合数据库(GEO)获取TNBC队列数据集，利用生物信息学工具筛选出对照组与TNBC患者的差异基因MELK。通过数据库预测出调控MELK表达的转录因子；实时荧光定量逆转录聚合酶链式反应(qRT-PCR)和免疫印迹法(Western blotting)检测人正常乳腺上皮细胞MCF-10A和两种TNBC细胞系HCC-1937和MDA-MB-231中MELK的表达水平；将HCC-1937及MDA-MB-231细胞分为小干扰RNA(siRNA)对照组、siRNA敲降组1、siRNA敲降组2、慢病毒对照组、慢病毒过表达组对MELK基因沉默和慢病毒介导的过表达处理，通过qRT-PCR和Western blotting检测各组mRNA和蛋白表达水平，细胞计数试剂盒-8检测法(CCK-8)、划痕实验和Transwell侵袭实验分别检测细胞增殖、迁移和侵袭能力的影响；将HCC-1937及MDA-MB-231细胞分为上述5组对预测出的转录因子E2F家族转录因子1(E2F1)进行干预，再次通过qRT-PCR和Western blotting检测各组E2F1及MELK的表达水平，并检测细胞的增殖、迁移和侵袭能力。采用Western blotting检测已成功敲低MELK、E2F1的HCC-1937细胞中哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关蛋白的表达水平，探究MELK、E2F1对mTOR信号通路相关蛋白的影响。结果： 本研究筛选出1 300个差异表达基因，其中MELK在TNBC患者组织中表达升高，在TNBC细胞系中分别敲降及过表达MELK，与对照组相比，siRNA敲降组细胞的增殖、迁移及侵袭能力降低，慢病毒过表达组细胞的增殖、迁移及侵袭能力增强；在TNBC细胞系中分别敲降及过表达E2F1，与对照组相比，siRNA敲降组的MELK及E2F1的蛋白相对表达量、mRNA相对表达量、细胞的增殖、迁移及侵袭能力下降，慢病毒过表达组的MELK及E2F1的蛋白相对表达量、mRNA相对表达量、细胞的增殖、迁移及侵袭能力提升；在TNBC细胞中敲降MELK、E2F1的表达，mTOR通路相关蛋白S6K、4E-BP1、mTOR及Akt(ser473)未发现明显改变，MELK及mTOR通路相关蛋白的磷酸化形式p-S6K、p-4E-BP1、p-mTOR及p-Akt(ser473)表达均呈现下降趋势。结论： MELK在TNBC细胞系中表达升高，E2F1调控MELK表达，MELK影响mTOR信号通路进而影响TNBC细胞的增殖、迁移及侵袭。

Objective: To explore the role of maternal embryonic leucine zipper kinase(MELK) in the malignant progression of triple-negative breast cancer(TNBC) and its underlying molecular mechanism. Methods: The Gene Expression Omnibus(GEO) database was used to obtain a TNBC cohort dataset, and the differentially expressed gene MELK between control group and TNBC patients was screened by bioinformatics tools. The transcription factors regulating MELK expression were predicted via database analysis. Quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR) and Western blotting were performed to detect the expression levels of MELK in normal human mammary epithelial cell line MCF-10A and two TNBC cell lines, HCC-1937 and MDA-MB-231. HCC-1937 and MDA-MB-231 cells were assigned to five groups: siRNA control group, siRNA knockdown group 1, siRNA knockdown group 2, lentivirus control group, and lentivirus overexpression group. After MELK gene silencing and lentivirus-mediated overexpression, qRT-PCR and Western blotting were used to detect the mRNA and protein expression levels in each group, while Cell Counting Kit-8(CCK-8), scratch wound healing assay, and Transwell invasion assay were performed to evaluate cell proliferation, migration, and invasion abilities, respectively. Subsequently, HCC-1937 and MDA-MB-231 cells were assigned to the above five groups to intervention with the predicted transcription factor E2F transcription factor 1(E2F1). The expression levels of E2F1 and MELK in each group were detected again by qRT-PCR and Western blotting, and the cell proliferation, migration, and invasion abilities were also examined. Western blotting was used to detect the expression levels of mammalian target of rapamycin(mTOR) signaling pathway-related proteins in HCC-1937 cells with stable knockdown of MELK or E2F1, to explore the effects of MELK and E2F1 on the mTOR signaling pathway-related proteins. Results: In this study, 1 300 differentially expressed genes were screened, among which MELK was upregulated in TNBC tissues. After knockdown and overexpression of MELK in TNBC cell lines, compared with the control group, the proliferation, migration, and invasion abilities of cells in the siRNA knockdown group were significantly reduced, while those in the lentivirus overexpression group were significantly enhanced. After knockdown and overexpression of E2F1 in TNBC cell lines, compared with the control group, the relative mRNA and protein expression levels of both MELK and E2F1, as well as cell proliferation, migration, and invasion abilities, were decreased in the siRNA knockdown group; conversely, these indicators were increased in the lentivirus overexpression group. Furthermore, after knocking down the expression of MELK or E2F1 in TNBC cells, no significant changes were observed in the total protein levels of mTOR pathway-related proteins, including S6K, 4E-BP1, mTOR, or Akt(Ser473). However, the phosphorylation levels of these proteins, namely p-S6K, p-4E-BP1, p-mTOR, and p-Akt(Ser473), were all significantly decreased. Conclusion: MELK expression is elevated in TNBC cell lines, and E2F1 regulates MELK expression. MELK affects the mTOR signaling pathway, which in turn affects the proliferation, migration, and invasion of TNBC cells.

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