木香烃内酯通过调控circ_0000285/miR-409-3p影响胃癌细胞恶性表型★

Costunolide affects the malignant phenotype of gastric cancer cells by regulating circ_0000285/miR-409-3p★

摘要:

目的 探讨木香烃内酯对胃癌细胞恶性表型的影响及可能机制。方法 体外培养胃癌细胞HGC-27，采用不同剂量（2.5、10、40 μmol·L^-1）木香烃内酯干预HGC-27细胞24 h，或转染circ_0000285小干扰RNA至HGC-27细胞后培养24 h。采用40 μmol·L^-1木香烃内酯干预转染circ_0000285过表达载体的HGC-27细胞24 h。CCK-8法和克隆形成实验检测细胞增殖，流式细胞仪检测细胞凋亡，划痕实验和Transwell检测细胞迁移和侵袭，Western blotting检测细胞中Bcl-2、Bax、E-cadherin和N-cadherin蛋白表达，RT-qPCR检测circ_0000285和miR-409-3p表达，双荧光素酶报告基因实验验证circ_0000285和miR-409-3p的调控关系。结果 木香烃内酯可降低HGC-27细胞的OD值、集落形成数、迁移距离、侵袭数，以及Bcl-2、N-cadherin蛋白表达水平，提高细胞凋亡率及Bax、E-cadherin蛋白表达水平，并呈剂量依赖性。木香烃内酯可抑制HGC-27细胞中circ_0000285的表达，促进miR-409-3p表达。干扰circ_0000285表达可降低HGC-27细胞的增殖、迁移能力，并促进细胞凋亡。circ_0000285可靶向结合并负调控miR-409-3p。过表达circ_0000285可逆转木香烃内酯对HGC-27细胞恶性表型的影响。结论 木香烃内酯可能通过调控circ_0000285/miR-409-3p轴抑制胃癌细胞的恶性生物学行为。

Abstract:

Objective To investigate the effect of costunolide on the malignant phenotype of gastric cancer cells and its possible mechanism.Methods Gastric cancer cells HGC-27 were cultured in vitro, and treated with costunolide at different doses (2.5, 10, 40 μmol·L^-1) for 24 h, or transfected with circ_0000285 small interfering RNA and then cultured for 24 h. The 40 μmol·L^-1 costunolide was used to intervene the HGC-27 cells transfected with circ_0000285 overexpression vector for 24 h. CCK-8 method and clone formation test were used to detect the cell proliferation. The flow cytometry was used to detect cell apoptosis. Scratch test and Transwell were used to detect cell migration and invasion. Western blotting was used to detect the protein expressions of Bcl-2, Bax, E-cadherin and N-cadherin in the cells, and RT-qPCR was used to detect the expressions of circ_0000285 and miR-409-3p. The dual luciferase reporter gene experiment was used to verify the regulatory relationship between circ_0000285 and miR-409-3p.Results Costunolide could reduce the OD value of HGC-27cells, the number of colonies formed, the migration distance, the number of invasion, and the protein expression levels of Bcl-2 and N-cadherin, while it could increase the cell apoptosis rate, and the protein expression levels of Bax and E-cadherin in a dose-dependent way. Costunolide could inhibit the expression of circ_0000285, but promote the expression of miR-409-3p in HGC-27cells. Interference with circ_0000285 expression could decrease the proliferation and migration of HGC-27 cells, and promote cell apoptosis. circ_0000285 could target and negatively regulate miR-409-3p. Overexpression of circ_0000285 could reverse the inhibitory effect of costunolide on HGC-27 cell phenotypes.Conclusion Costunolide may inhibit the malignant biological behavior of gastric cancer cells by regulating the circ_0000285/miR-409-3p axis.