地锦草乙醇提取物通过circRHOT1/miR-29a-3p轴抑制结直肠癌SW480细胞的恶性生物学行为

Ethanol extract from Euphorbia humifusa inhibits the malignant biological behaviors of colorectal cancer SW480 cells by regulating the circ RHOT1/miR-29a-3p molecular axis

目的:探讨地锦草乙醇提取物(EEEH)对人结直肠癌SW480细胞生物学行为的影响及其分子机制。方法:体外培养SW480细胞,实验分为Con组、EEEH-L组、EEEH-M组、EEEH-H组、si-NC组、si-circRHOT1组、EEEH-H+pcDNA组、EEEH-H+pcDNA-circRHOT1组,分别以si-NC、si-circRHOT1、pcDNA、pcDNA-circRHOT1转染SW480细胞,采用CCK-8法、细胞克隆形成实验、Transwell实验分别检测转染后各组细胞的增殖、迁移及侵袭能力,qPCR法检测转染后各组SW480细胞circRHOT1和miR-29a-3p的表达,WB法检测各组细胞中MMP-2、MMP-9蛋白的表达。双荧光素酶报告基因实验检测circRHOT1与miR-29a-3p之间的靶向关系。结果:与Con组比较,EEEH-L组、EEEH-M组、EEEH-H组SW480细胞中MMP-2和MMP-9蛋白表达均明显降低(均P<0.05),circRHOT1的表达均降低(均P<0.05)而miR-29a-3p的表达均升高(均P<0.05)且呈剂量依赖性;细胞的存活率、细胞克隆形成数、迁移及侵袭细胞数均减少(均P<0.05)。circRHOT1可靶向负调控miR-29a-3p的表达。敲减circRHOT1可抑制SW480细胞的增殖、迁移及侵袭能力,而过表达circRHOT1则可减弱EEEH对SW480细胞增殖、迁移及侵袭的抑制作用。结论:EEEH可通过调控circRHOT1/miR-29a-3p轴而抑制结直肠癌SW480细胞的增殖、迁移及侵袭能力。

Objective: To explore the effect of ethanol extract from euphorbia humifusa(EEEH) on the biological behaviors of human colorectal cancer SW480 cells and its molecular mechanism. Methods: Human colorectal cancer SW480 cells were cultured in vitro,divided into the following experimental groups: Con group, EEEH-L group, EEEH-M group, EEEH-H group, si-NC group, si-circRHOT1group, EEEH-H+pcDNA group and EEEH-H+pcDNA-circRHOT1 group and transfected with si-NC, si-circRHOT1, pcDNA and PCDNA-circRHOT1 respectively. CCK-8 assay, cell clone formation assay, Transwell assay were used to detect cell proliferation,migration and invasion ability in each group after transfection. qPCR method was employed to detect the expression of circRHOT1 and miR-29a-3p in all the groups of SW480 cells after transfection, and the expressions of MMP-2 and MMP-9 proteins were detected by WB. The dual luciferase reporter experiment was used to detect the targeting relationship between circRHOT1 and miR-29a-3p.Results: Compared with the Con group, the protein expressions of MMP-2 and MMP-9 in SW480 cells in EEEH-L, EEEH-M, and EEEH-H groups were significantly decreased(all P<0.05), the expression of circRHOT1 decreased(all P<0.05) and the expression of miR-29a-3p increased(all P<0.05) in a dose-dependent manner. The cell survival rate, the number of cell clone formation and the number of migration and invasive cells were decreased(all P<0.05). circRHOT1 can target and negatively regulate the expression of miR-29a-3p. Knockdown of circRHOT1 can inhibit the proliferation, migration and invasion of SW480 cells, while overexpression of circRHOT1 can attenuate the inhibitory effects of EEEH on the proliferation, migration and invasion of SW480 cells.Conclusion: EEEH can inhibit the proliferation, migration and invasion of colorectal cancer SW480 cells by regulating the circRHOT1/miR-29a-3p axis.