微小RNA-34a在紫草素抑制肺癌H1299细胞增殖和侵袭中的作用及其机制

Role and mechanism of miR-34a in shikonin inhibiting the proliferation and invasion of lung cancer H1299 cells

目的探讨微小RNA-34a(microRNA-34a,miR-34a)在紫草素抑制肺癌H1299细胞增殖和侵袭中的作用及其机制。方法2019年9月至2020年3月,采用1、2.5和5 mg/L紫草素处理体外培养的肺癌H1299细胞后,采用噻唑蓝(MTT)法检测细胞的存活率;平板克隆实验和Transwell实验分别检测细胞克隆形成能力和侵袭能力;RT-PCR检测细胞中miR-34a的表达情况。采用生物信息软件预测、双萤光素酶报告基因实验检测miR-34a与Yin Yang-1(YY1)的靶向关系。采用脂质体法转染miR-34a抑制剂和pcDNA3.1-YY1-GFP过表达质粒后,RT-PCR检测细胞中miR-34a的表达情况,蛋白质印迹法(western blotting)检测细胞YY1蛋白的表达,MTT法、平板克隆实验和Transwell小室实验观察下调miR-34a和上调YY1表达对紫草素处理的H1299细胞增殖和侵袭的影响。结果与0 mg/L紫草素相比,1、2.5和5 mg/L紫草素能够呈浓度依赖性抑制H1299细胞存活率、克隆细胞数[(127.63±7.38)、(108.85±6.34)、(91.72±6.15)比(156.55±9.06)]和侵袭细胞数[(93.12±5.56)、(75.25±4.68)、(59.85±3.20)比(116.00±7.85)],并促进miR-34a表达。生物信息软件预测到miR-34a与YY1存在互补的结合位点,双萤光素酶报告基因实验证实YY1是miR-34a的靶基因。下调miR-34a可逆转紫草素对H1299细胞增殖和侵袭的抑制作用,并促进YY1蛋白的表达,同时上调YY1表达可逆转紫草素对H1299细胞增殖和侵袭的抑制作用。结论miR-34a在紫草素抑制肺癌H1299细胞增殖和侵袭过程中发挥着积极作用,其作用机制可能与靶向调控YY1表达有关。

Objective To explore the role of miR-34a in the inhibition of shikonin on the proliferation and invasion of lung cancer H1299 cells and its mechanism.Methods Lung cancer H1299 cells in vitro were treated with 1,2.5 and 5 mg/L shikonin from Sep‐tember 2019 to March 2020,and the cell viability was detected by methyl thiazolyl tetrazolium(MTT)method.Formation and invasion of cell cloning were detected by plate cloning assay and Transwell assay;RT-PCR was used to detect the expression of miR-34a.The targeting relationship between miR-34a and Yin Yang-1(YY1)was detected by bioinformation software prediction and dual luciferase reporter gene assay.After transfection of miR-34a inhibitor and pcDNA3.1-YY1-GFP overexpression plasmid by liposome method,the expression of miR-34a was detected by RT-PCR,and the expression of YY1 protein was detected by Western blotting.MTT assay,plate cloning experiments and Transwell chamber experiments were performed to observe the effects of down-regulated miR-34a and up-regu‐lated YY1 expression on the proliferation and invasion of shikonin-treated H1299 cells.Results Compared with 0 mg/L shikonin,1,2.5 and 5 mg/L shikonin could inhibit the survival rate of H1299 cells,the number of cloned cells[(127.63±7.38),(108.85±6.34),(91.72±6.15)vs.(156.55±9.06)]and the number of invasive cells[(93.12±5.56),(75.25±4.68),(59.85±3.20)vs.(116.00±7.85)]in a con‐centration-dependent manner,and promoted the expression of miR-34a.The bioinformation software predicted the presence of a com‐plementary binding site for miR-34a and YY1,and the dual luciferase reporter gene assay confirmed that YY1 was the target gene of miR-34a.Down-regulation of miR-34a reversed the inhibitory effect of shikonin on proliferation and invasion of H1299 cells,and pro‐moted the expression of YY1 protein.Up-regulation of YY1 expression reversed the inhibitory effect of shikonin on the proliferation and invasion of H1299 cells.Conclusions miR-34a plays an active role in the inhibition of proliferation and invasion of lung cancer H1299 cells by shikonin.The mechanism may be related to the targeted regulation of YY1 expression.