miR-4727-5p通过调控EPS8L3表达抑制乳腺癌细胞的增殖与迁移

miR-4727-5p inhibits proliferation and migration of breast cancer cells by regulating EPS8L3 expression

目的 探讨微小RNA-4727-5p(miR-4727-5p)靶向表皮生长因子受体激酶底物8样蛋白3(epidermal growth factor receptor kinase substrate 8-like protein 3,EPS8L3)抑制乳腺癌细胞增殖和迁移的分子作用机制。方法 选取2019年2月至2021年9月在南通大学附属肿瘤医院(南通市肿瘤医院)乳腺外科手术切除的38例乳腺癌患者的癌组织和癌旁组织,以及乳腺癌细胞系MB-MDA-468、SKBR3、MCF-7、T-47D及正常乳腺上皮细胞MCF-10A,实时荧光定量PCR(qRT-PCR)法检测乳腺癌组织和细胞系中miR-4727-5p的表达。将重组质粒miR-4727-5p或空载质粒转染至T-47D细胞,分别命名为miR-4727-5p组和对照组(Ctrl组)。用细胞计数试剂盒(cell counting kit-8,CCK-8)和划痕愈合试验分别分析T-47D细胞的增殖和迁移能力。双荧光素酶报告基因实验验证miR-4727-5p与EPS8L3 mRNA非翻译区的靶向关系。qRT-PCR和Western blot检测EPS8L3基因和EGFR信号通路蛋白p-EGFR、p-Raf1、p-MEK1的表达。结果 与癌旁组织比较,乳腺癌组织miR-4727-5p表达显著降低(P<0.01)。与MCF-10A细胞比较,乳腺癌细胞系中miR-4727-5p表达显著降低(P<0.05)。与Ctrl组比较,miR-4727-5p组T-47D细胞增殖能力明显降低(P<0.05),细胞侵袭能力明显降低(P<0.05)。双荧光素酶报告基因实验结果证实,EPS8L3是miR-4727-5p的靶基因。与Ctrl组比较,miR-4727-5p组EPS8L3基因表达水平明显降低(P<0.01),p-EGFR、p-Raf1、p-MEK1蛋白表达水平明显降低。结论 miR-4727-5p通过靶向抑制EPS8L3基因表达,干扰EGFR信号通路转导,从而抑制乳腺癌T-47D细胞的增殖和迁移能力。

Objective To explore the inhibition effect of miR-4727-5 p on breast cancer cell proliferation and migration by targeting epidermal growth factor receptor kinase substrate 8-like protein 3(EPS8 L3) and its molecular mechanism. Methods The tumor and para-cancerous tissues from 38 breast cancer patients with surgical resection in the department of breast surgery in the Affiliated Cancer Hospital of Nantong University(Nantong Cancer Hospital) from February 2019 to September 2021, and the breast cancer cell line MB-MDA-468, SKBR3, MCF-7, T-47 D and normal breast epithelial cells MCF-10 A were selected. Real-time fluorescent quantitative PCR(qRT-PCR) method was used to detect the expression of miR-4727-5 p in breast cancer tissues and cell lines. Recombinant plasmid miR-4727-5 p or empty plasmid was transfected into T-47 D cells respectively, namely as miR-4727-5 p group and control group. Cell counting kit-8(CCK-8) and scratch healing test were used to analyze the proliferation and migration abilities of T-47 D cells. The dual luciferase reporter gene experiment was used to verify the targeting relationship between miR-4727-5 p and the untranslated region of EPS8 L3 mRNA. RT-qPCR and Western blot were used to detect the expression of EPS8 L3 gene and EGFR signaling pathway proteins p-EGFR, p-Raf1, p-MEK1. Results Compared with adjacent tissues, the expression of miR-4727-5 p in breast cancer tissues was significantly reduced(P<0.01). Compared with MCF-10 A cells, the expression of miR-4727-5 p was significantly reduced in breast cancer cell lines(P<0.05). Compared with control group, the proliferation ability of T-47 D cells in miR-4727-5 p group was significantly reduced(P<0.05), and the invasion ability of T-47 D cells in miR-4727-5 p group was significantly reduced(P<0.05). The results of the dual luciferase reporter gene experiment confirmed that EPS8 L3 is the target gene of miR-4727-5 p. Compared with control group, EPS8 L3 gene expression level in miR-4727-5 p group was significantly reduced(P<0.01), and the protein expression levels of p-EGFR, p-Raf1, and p-MEK1 were reduced. Conclusion The miR-4727-5 p interferes with the transduction of EGFR signal pathway by targetedly inhibiting the expression of EPS8 L3 gene, thereby inhibiting the proliferation and migration abilities of breast cancer T-47 D cells.