环状RNA同源域相互作用蛋白激酶3(circHIPK3)通过吸附miR-124-3p促进神经胶质瘤细胞增殖及转移

Circular RNA homeodomain-interacting protein kinase 3 (circHIPK3) promotes growth and metastasis of glioma cells by sponging miR-124-3p

目的探究环状RNA同源域相互作用蛋白激酶3(circHIPK3)通过海绵吸附微小RNA-124-3p(miR-124-3p)对神经胶质瘤细胞增殖和转移的影响。方法按照LipofectamineTM 3000试剂说明书将circHIPK3短发夹RNA(sh-circHIPK3)、pcDNA3.1-circHIPK3、miR-124-3p模拟物(miR-124-3p mimics)和pcDNA3.1-WEE1转染T98G细胞;实时荧光定量PCR检测circHIPK3和miR-124-3p在神经胶质瘤组织和细胞系中的表达情况;CCK-8法检测T98G细胞增殖情况;TranswellTM法检测T98G细胞侵袭能力;双荧光素酶报告基因验证miR-124-3p与circHIPK3和丝氨酸/苏氨酸激酶WEE1的靶向关系;Western blot法检测WEE1和上皮间质转化(EMT)相关蛋白[上皮钙黏素(E-cadherin)、神经钙黏素(N-cadherin)和波形蛋白(vimentin)]的表达情况;此外,circHIPK3和WEE1竞争性结合miR-124-3p后,采用CCK-8法检测T98G细胞增殖,TranswellTM检测T98G细胞侵袭。结果circHIPK3在神经胶质瘤组织和细胞中高表达,敲减circHIPK3抑制T98G细胞增殖和侵袭能力,并抑制其EMT;双荧光素酶报告基因结果证实miR-124-3p是circHIPK3的靶基因,WEE1是miR-124-3p的靶基因;同时过表达miR-124-3p与circHIPK3或WEE1,或共转染sh-circHIPK3和pcDNA3.1-WEE1可恢复过表达miR-124-3p对T98G细胞增殖、侵袭和EMT的抑制作用。结论circRNA-HIPK3与WEE1海绵吸附miR-124-3p促进神经胶质瘤细胞增殖、侵袭和EMT。

Objective To investigate the effect of circular RNA homeodomain-interacting protein kinase 3(circHIPK3)on the proliferation and metastasis of glioma cells via sponging miR-124-3p.Methods T98G cells were transfected with circHIPK3 short hairpin RNA(sh-circHIPK3),pcDNA3.1-circHIPK3,miR-124-3p mimics or pcDNA3.1-WEE1 using LipofectamineTM 3000 reagent following the manufacturer’s instructions.Real-time quantitative PCR was performed to evaluate the expression of circHIPK3 and miR-124-3p in glioma tissues and cell lines.CCK-8 assay was employed to assess the proliferation of T98G cells.TranswellTM assay was applied to validate the invasion of T98G cells.The targeting relationship among miR-124-3p,circHIPK3 and serine/threonine kinase WEE1 were verified by dual-luciferase reporter gene assay.The expression of WEE1 and epithelial mesenchymal transition(EMT)-related factors(E-cadherin,N-cadherin and vimentin)were measured by Western blot analysis.In addition,after the competitive binding of circHIPK3 and WEE1 to miR-124-3p,the proliferation of T98G cells was detected by CCK-8 assay;the invasion of T98G cells was evaluated by TranswellTM assay.Results The circHIPK3 was upregulated in glioma tissues and cell lines.Knockdown of circHIPK3 repressed the proliferation,invasion and EMT of T98G cells.Dual-luciferase reporter gene assay confirmed that miR-124-3p was the target gene of circHIPK3,while WEE1 was the target gene of miR-124-3p.The miR-124-3p was over-expressed simultaneously with circHIPK3 or WEE1.Co-transfected sh-circHIPK3 and pcDNA3.1-WEE1 restored the inhibitory effect of miR-124-3p overexpression on the proliferation,invasion and EMT of T98G cells.Conclusion The circRNA-HIPK3 and WEE1 can promote the proliferation,invasion and EMT of glioma cells by sponging miR-124-3p.