盐酸石蒜碱通过调控circSEPT9/miR-654-5p影响结直肠癌SW620细胞增殖及凋亡的机制

Effect of Lycorine Hydrochloride on the Proliferation and Apoptosis of Colorectal Cancer SW620 Cells by Regulating circSEPT9/miR-654-5p and the Mechanism

目的探讨盐酸石蒜碱(lycoline hydrochloride, LH)对人结直肠癌细胞SW620细胞增殖、凋亡的影响及其可能的作用机制。方法不同剂量的LH处理SW620细胞(LH-L组、LH-M组、LH-H组),同时将正常培养的SW620细胞记为Con组;实验分组:si-NC组、si-circSEPT9组、LH+pcDNA-circSEPT9组、LH+anti-miR-654-5p组;利用MTT法、平板克隆形成实验、流式细胞术分别检测细胞增殖、克隆形成及凋亡;实时荧光定量PCR(qRT-PCR)法检测circSEPT9、miR-654-5p的表达量;双荧光素酶报告基因实验检测circSEPT9与miR-654-5p的靶向关系;Western blot法检测活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved Caspase-3)、活化的含半胱氨酸的天冬氨酸蛋白水解酶9(cleaved Caspase-9)蛋白表达量。结果与Con组比较,LH-L组、LH-M组、LH-H组细胞增殖抑制率、miR-654-5p的表达量、细胞凋亡率和cleaved Caspase-3、cleaved Caspase-9蛋白水平升高(均P<0.05),细胞克隆形成数减少(均P<0.05),circSEPT9的表达量降低(均P<0.05),且呈剂量依赖性;circSEPT9可靶向调控miR-654-5p的表达;与si-NC组比较,si-circSEPT9组miR-654-5p的表达量、细胞增殖抑制率、细胞凋亡率和cleaved Caspase-3、cleaved Caspase-9蛋白水平升高(均P<0.05),细胞克隆形成数减少(均P<0.05);与LH组比较,LH+pcDNA-circSEPT9组、LH+anti-miR-654-5p组miR-654-5p的表达量、细胞增殖抑制率、细胞凋亡率和cleaved Caspase-3、cleaved Caspase-9蛋白水平降低(均P<0.05),细胞克隆形成数增多(均P<0.05)。结论盐酸石蒜碱可通过调控circSEPT9/miR-654-5p表达而抑制结直肠癌细胞增殖、克隆形成及诱导细胞凋亡。

Objective To explore the effect of lycorine hydrochloride(LH)on the proliferation and apoptosis of human colorectal cancer cells SW620 and its possible mechanism.Methods Different doses of LH were used to treat SW620 cells(LH-L group, LH-M group, LH-H group),and the normally cultured SW620 cells were recorded as contol(Con)group.Experimental groups included si-NC group, si-circSEPT9 group, LH+pcDNA-circSEPT9 group, and LH+anti-miR-654-5 p group.MTT assay, colony-forming assay and flow cytometry were used to detect cell proliferation, colony formation and apoptosis.Real-time quantitative PCR(qRT-PCR)was used to detect the expression of circSEPT9 and miR-654-5 p.The dual-luciferase reporter gene assay was used to detect the targeting relationship between circSEPT9 and miR-654-5 p.Western blotting was used to detect the protein expression of cleaved Caspase-3 and cleaved Caspase-9.Results Compared with the Con group, the cell proliferation inhibition rate, miR-654-5 p expression, apoptosis rate, and the protein levels of cleaved Caspase-3,cleaved Caspase-9 were increased in the LH-L,LH-M,and LH-H groups(all P<0.05),the number of cell colonies decreased significantly(all P<0.05),and the expression of circSEPT9 was decreased(all P<0.05),in a dose-dependent manner.The circSEPT9 could targetedly regulate the expression of miR-654-5 p.Compared with si-NC group, the expression of miR-654-5 p, cell proliferation inhibition rate, apoptosis rate as well as cleaved Caspase-3 and cleaved Caspase-9 protein levels in the si-circSEPT9 group were increased(all P<0.05),while the number of cell colonies was decreased(all P<0.05).Compared with the LH group, the expression of miR-654-5 p, cell proliferation inhibition rate, apoptosis rate as well as cleaved Caspase-3 and cleaved Caspase-9 protein levels in LH+pcDNA-circSEPT9 group and LH+anti-miR-654-5 p group were decreased(all P<0.05),while the number of cell colonies was increased(P<0.05).Conclusion Lycorine hydrochloride could inhibit the proliferation, clone formation and induce apoptosis of colorectal cancer cells by regulating the expression of circSEPT9/miR-654-5 p.