沉默长链非编码RNA HCG18通过miRNA-107/PAG1轴逆转喉癌细胞放射抗拒机制研究

Mechanism of silencing LncRNA HCG18 reversing radiation resistance of laryngeal cancer cells via miR-107/PAG1 axis

目的分析长链非编码RNA(long non-coding RNA,lncRNA)HCG18对放疗抗拒喉癌的放疗敏感性的影响及作用机制。方法流式细胞术检测喉癌细胞系经过放射后的细胞凋亡率,应用荧光定量PCR(qRT-PCR)分别检测lncRNA HCG18、微小RNA-107(miR-107)和PAG1在喉癌细胞中的相对表达含量,分别于喉癌细胞中敲除和过表达HCG18、miR-107和PAG1,用克隆形成等检测其对喉癌细胞放疗敏感性的影响。利用实时荧光qRT-PCR和蛋白质印迹法检测HCG18对miR-107及PAG1表达的影响,并运用荧光素酶报告实验分析HCG18/miR-107/PAG1之间的调控作用。结果Tu212max细胞系细胞凋亡率低于Tu212和Tu212min细胞系,将其选定为放疗抗拒的喉癌细胞系。HCG18和PAG1在喉癌组织和Tu212max中表达上调,miR-107在喉癌组织和Tu212max中表达下调(t=22.60、18.15、14.38,P均<0.05)。在Tu212max中沉默HCG18和PAG1的表达能够抑制Tu212max克隆(P均<0.05)、提高凋亡率(t=35.55、24.25、51.44、49.75,P均<0.05)。双荧光素酶报告基因实验证实HCG18可负向调控miR-107的表达(t=32.46,P<0.05),miR-107可以负向调控其下游靶基因PAG1的表达水平(t=34.71,P<0.05),HCG18通过调控miR-107而并正向调控PAG1的表达水平。HCG18对Tu212ma x放射敏感性的影响又依赖于m i R-107。结论沉默HCG18表达,促进miR-107上调,而抑制PAG1的表达,最终提高了放疗抗拒喉癌细胞的放射敏感性。

OBJECTIVE To analyze the effect of long-chain noncoding RNA HCG18(LncRNA HCG18)on the radiosensitivity of laryngeal cancer resistant to radiotherapy and its specific mechanism.METHODS The apoptosis rate of laryngeal cancer cell line after radiotherapy was detected by flow cytometry,and LncRNA HCG18 and microrna-107(miR-107)were detected by fluorescence quantitative PCR(qRT-PCR)and PAG1 in laryngeal cancer cells;HCG18,miR-107 and PAG1 were knocked out and overexpressed in laryngeal cancer cells respectively.Their effects on the radiosensitivity of laryngeal cancer cells were detected by clone formation.qRT-PCR and Western blot were used to detect the effect of HCG18 on the expression of miR-107 and PAG1,and Luciferase Report experiment was used to analyze the regulatory effect between HCG18/miR-107/PAG1.RESULTS The apoptosis rate of Tu212max cell line was lower than the other two groups(Tu212min and Tu212)of cell lines,which were selected as radiotherapy-resistant laryngeal cancer cell lines.HCG18 and PAG1 expression was upregulated in laryngeal cancer tissues and Tu212max,and miR-107 expression was downregulated in laryngeal cancer tissues and Tu212max(t=22.60,18.15,14.38,respectively;all P<0.05).Silencing the expression of HCG18 and PAG1 in Tu212max inhibited Tu212max clones(P<0.05)and increased the apoptosis rate(t=35.55,24.25,51.44,49.75,P<0.05,respectively).The dual-luciferase reporter gene assay confirmed that HCG18 negatively regulated the expression of miR-107(t=32.46,P<0.05)and miR-107 negatively regulated the expression level of its downstream target gene PAG1(t=34.71,P<0.05),and HCG18 positively regulated the expression level of PAG1 by regulating miR-107.The effect of HCG18 on Tu212max radiosensitivity was again dependent on miR-107.CONCLUSION Silencing the expression of HCG18 promotes the up regulation of miR-107 and inhibits the expression of PAG1,and finally improves the radiosensitivity of radiotherapy resistant laryngeal cancer cells.