circCLK3/miR-216a-5p对乳腺癌MDA-MB-453细胞影响分析

Effects of circCLK3/miR-216a-5p on the proliferation,apoptosis,migration and invasion of breast cancer MDA-MB-453cells

目的 探讨circCLK3/miR-216a-5p分子轴对乳腺癌MDA-MB-453细胞增殖、凋亡、迁移和侵袭的影响及其可能作用机制。方法 收集2020-02-01-2022-05-31天津医科大学肿瘤医院63例经病理确诊为乳腺癌的患者癌及癌旁组织(距癌周≥5 cm)。采用实时荧光定量PCR(qRT-PCR)检测乳腺癌患者癌及癌旁组织标本与MDA-MB-453细胞中circCLK3和miR-216a-5p的表达量;体外培养人乳腺癌细胞MDA-MB-453,将si-NC、si-circCLK3、miR-NC和miR-216a-5p分别转染至MDA-MB-453细胞,将si-circCLK3+anti-miR-NC和si-circCLK3+anti-miR-216a-5p分别共转染至MDA-MB-453细胞;双荧光素酶报告实验检测circCLK3与miR-216a-5p的靶向关系;MTT法、平板克隆形成实验、流式细胞术与Transwell实验分别检测细胞增殖、克隆形成、凋亡、迁移及侵袭能力;蛋白质印迹法检测MDA-MB-453细胞中Bax和Bcl-2蛋白表达量。结果 与癌旁组织(0.96±0.13)相比,乳腺癌组织(2.37±0.38)中circCLK3的表达量升高,t=27.866,P<0.001;miR-216a-5p的表达量(1.03±0.14 vs 0.43±0.08)降低,t=29.535,P<0.001。与si-NC组比较,转染si-circCLK3上调miR-216a-5p的表达(t=29.684,P<0.001),抑制了细胞生长以及侵袭和迁移能力,t值分别为20.743、22.395和27.915,均P<0.001,增加了细胞凋亡,t=37.221,P<0.001。与miR-NC组比较,转染miR-216a-5p抑制了细胞生长以及侵袭和迁移能力,t值分别为21.266、28.590和27.756,均P<0.001,增加了细胞凋亡,t=36.884,P<0.001。与si-circCLK3+anti-miR-NC组相比,si-circCLK3+anti-miR-216a-5p组细胞克隆形成能力、迁移及侵袭能力均增强,t值分别为10.447、20.211和18.429,均P<0.001。结论 circCLK3敲除通过上调miR-216a-5p表达而抑制乳腺癌细胞生长、迁移及侵袭。

Objective To explore the effects of circCLK3/miR-216a-5p molecular axis on the proliferative,apoptotic,migratory and invasive abilities of breast cancer MDA-MB-453cells and their possible mechanisms.Methods We collected the cancerous and paracancerous tissues of 63patients diagnosed as breast cancer by pathology in the Tianjin Medical University Cancer Institute and Hospital from February 1,2020to May 31,2022.The expressions of circCLK3and miR-216a-5p were detected by qRT-PCR;human breast cancer cells MDA-MB-453were cultured in vitro,and si-NC,sicircCLK3,miR-NC,miR-216a-5p were transfected into MDA-MB-453cells,respectively.Meanwhile,si-circCLK3and anti-miR-NC,si-circCLK3and anti-miR-216a-5p were co-transfected into MDA-MB-453cells,respectively.The dual luciferase reporter experiment was used to detect the targeting relationship between circCLK3and miR-216a-5p.MTT,colony formation,flow cytometry,and transwell assays were applied to examine cell proliferative,apoptotic,migratory,and invasive abilities.Western blot assay was used to detect Bax and Bcl-2protein expression.Results Compared with paracancerous tissues(0.96±0.13),the expression of circCLK3was elevated in breast cancer tissues(2.37±0.38),t=27.866,P<0.001,and the expression of miR-216a-5p was decreased(1.03±0.14 vs 0.43±0.08),t=29.535,P<0.001;Transfection with si-circCLK3up-regulated miR-216a-5p expression compared with si-NC group(t=29.684,P<0.001),inhibited the clone number of cells(t=20.743,P<0.001),invasion(t=22.395,P<0.001),and migration ability(t=27.915,P<0.001),and increased apoptosis(t=37.221,P<0.001);compared with the miR-NC group,transfection with miR-216a-5p resulted in inhibition of cell clone number(t=21.266,P<0.001),invasion(t=28.590,P<0.001),migration ability(t=27.756,P<0.001)and increased apoptosis(t=36.884,P<0.001).Compared with the si-circCLK3+anti-miR-NC group,the clonogenesis,migration and invasion of cells in the si-circCLK3+anti-miR-216a-5p co-transfection group were enhancedthe t-values were 10.447,20.211and 18.429,respectively,both P<0.001.Conclusion circCLK3knockdown inhibited breast cancer cell growth,migration,and invasion through upregulation of miR-216a-5p expression.