circ_0001368靶向miR-616对肺癌A549细胞增殖、迁移及侵袭的影响

Regulatory effect of circ_0001368 on proliferation,migration and invasion of the lung cancer cell line A549 by targeting miR-616

目的探讨肺癌A549细胞增殖、侵袭及迁移过程中circ_0001368的影响。方法qRT-PCR法检测47例肺癌组织与癌旁组织中circ_0001368、miR-616的表达量;pcDNA、pcDNA-circ_0001368、anti-miR-NC、anti-miR-616、pcDNA-circ_0001368和miR-NC、pcDNA-circ_0001368和miR-616 mimics分别转染至A549细胞;检测细胞增殖、克隆形成、侵袭及迁移;双荧光素酶报告实验检测circ_0001368、miR-616靶向;Western blot检测E-cadherin、N-cadherin蛋白表达量。结果肺癌组织中circ_0001368的表达量低于癌旁组织(P<0.05),miR-616的表达量高于癌旁组织(P<0.05);circ_0001368与miR-616存在结合位点。相较于miR-NC组,miR-616组降低WT-circ_0001368荧光素酶活性(P<0.05)。circ_0001368可负向调控miR-616表达(P<0.05)。相较于anti-miR-NC组,anti-miR-616组N-cadherin蛋白水平、划痕愈合率、细胞活力及侵袭细胞数目与细胞克隆形成数目降低(或减少),E-cadherin蛋白水平升高(P<0.05)。与pcDNA-circ_0001368+miR-NC组比较,pcDNA-circ_0001368+miR-616组细胞活力、N-cadherin蛋白、细胞克隆形成数目和侵袭细胞数目升高(或增多),划痕愈合率和水平升高,E-cadherin蛋白水平降低(P<0.05)。结论circ_0001368过表达能够经过对miR-616表达的靶向调控降低肺癌细胞增殖、克隆形成、侵袭及迁移能力。

Objective To investigate the regulatory effect of circ_0001368 on regulating the proliferation,migration and invasion of the lung cancer cell line A549.Methods Expression levels of circ_0001368 and miR-616 in 47 pairs of lung cancer tissues and paracancerous tissues were detected by quantitative real-time polymerase chain reaction(qRT-PCR).A549 cells were transfected with pcDNA-NC,pcDNA-circ_0001368,anti-miR-NC,anti-miR-616,pcDNA-circ_0001368+miR-NC and pcDNA-circ_0001368+miR-616 mimics,followed by detection of cell proliferation,clone formation,invasion and migration.Dual-luciferase reporter assay was performed to identify the targeting between circ_0001368 and miR-616.Protein expressions of E-cadherin and N-cadherin were detected by Western blot.Results Circ_0001368 was downregulated in lung cancer tissues than that of paracancerous tissues(P<0.05),while miR-616 was overexpressed in lung cancer tissues(P<0.05).A binding site exited in the promoter region of circ_0001368 to that of miR-616.Knockdown of miR-616 significantly decreased luciferase activity in the wild-type plasmid of circ_0001368(P<0.05).Circ_0001368 negatively regulated the expression level of miR-616(P<0.05).Knockdown of miR-616 significantly downregulated N-cadherin,but upregulated E-cadherin,which significantly reduced wound healing rate,cell viability,and the number of invasive cells and cell clones(P<0.05).Compared with those co-transfected with pcDNA-circ_0001368 and miR-NC,co-transfection of pcDNA-circ_0001368 and miR-616 significantly upregulated N-cadherin,downregulated E-cadherin and increased wound healing rate,cell viability,and the number of invasive cells and cell clones(P<0.05).Conclusion Overexpression of circ_0001368 inhibits the proliferation,cell clones,migration and invasion of lung cancer cells by targeting miR-616.