摘要
目的探讨长链非编码(Lnc)RNA NEAT1通过靶向调控miR-331对鼻咽癌细胞辐射敏感性的影响。方法选取2018年1月至2019年1月手术切除鼻咽癌组织及对应的癌旁组织的患者48例样本,qRT-PCR检测Lnc RNA NEAT1在鼻咽癌及相应的癌旁组织、不同的鼻咽癌细胞系中的表达情况;构建Lnc RNA NEAT1沉默细胞系SUNE-1-siNEAT1及阴性对照SUNE-1-siNC,并以SUNE-1作为空白对照;电离辐射后,采用MTT实验、Transwell、免疫荧光标记检测Lnc RNA NEAT1对SUNE-1细胞辐射敏感性、侵袭能力及DNA损伤修复的影响;使用生物信息学网站starBase预测NEAT1可互补结合的微小RNA(microRNA,miRNA),再经双荧光基因报告验证Lnc RNA NEAT1与miR-331的靶向关系。结果qRT-PCR结果显示,鼻咽癌组织中Lnc RNA NEAT1的表达水平高于癌旁正常组织,且差异有统计学意义(P<0.05),Lnc RNA NEAT1在SUNE-1细胞中表达量最高,与其他细胞株相比差异均具有统计学意义(P<0.05);成功构建Lnc RNA NEAT1沉默细胞系后,与SUNE-1组和SUNE-1-siNC组相比,SUNE-1-siNEAT1组细胞辐射后OD492nm值明显下降,细胞侵袭数量明显减少,γH2AX荧光强度明显增加,差异均具有统计学意义(P<0.05),SUNE-1组与SUNE-1-siNC组相比,差异无统计学意义(P>0.05);starBase网站预测结果显示,Lnc RNA NEAT1与miR-331存在互补结合位点,双荧光基因报告结果显示Lnc RNA NEAT1与miR-331可靶向结合。qRT-PCR证实LncRNA NEAT1负向调控miR-331的表达。结论lnc RNA NEAT1在鼻咽癌中呈高表达,沉默Lnc RNA NEAT1表达可能通过上调miR-331水平抑制鼻咽癌细胞增殖、侵袭能力,增加鼻咽癌细胞辐射敏感性。
Objective To investigate the effects of the long chain noncoding(LNC)RNA NEAT1 on the radiosensitivity of nasopharyngeal carcinoma cells by the targeted regulation of miR-331.Methods A total of 48 nasopharyngeal carcinoma tissue specimes and paracincerous tissue specimens from the patients who were treated by surgery in our hospital from January 2018 to January 2019 were enrolled in the study.The expression levels of NEAT1 were detected by qRT-PCR nasopharyngeal carcinoma tissue and paracincerous tissue as well as in different nasopharyngeal carcinoma cell lines.The Lnc RNA NEAT1 silenced cell line SUNE-1-siNEAT1 and the negative control SUNE-1-siNC were constructed,and SUNE-1 was used as the neat blank control.After ionizing radiation,the effects of Lnc RNA NEAT1 on the radiosensitivity,invasive ability and DNA damage repair of SUNE-1 cells were dtected by MTT test,Transwell and immunofluorescence markers,respectively.The NEAT1 was predicted by using the bioinformatics website starBase with complementary neat microRNA(miRNA),and the NEAT1 target relationship with miR-331 was analyzed by using the dual-fluorescence gene report.The qRT-PCR confirmed that the effects of LncRNA NEAT1 in negatively regulating the expression of miR-331.Results The qRT-PCR results show that the expression levels of Lnc RNA NEAT1 in nasopharyngeal carcinoma tissues were significantly higher than those in adjacent normal tissues(P<0.05).The Lnc RNA NEAT1 had the highest expression levels in SUNE-1 cells,as compared with those in the other cell lines(P<0.05).After the successful construction of the Lnc RNA NEAT1 silenced cell line,which was compared with that in SUNE-1 group and SUNE-1-siNC group,the levels of OD492nm value and the number of cell invasiveness in SUNE-1-siNC group were significantly decreased,however,the fluorescence intensity ofγH2AX was significantly increased(P<0.05).And there were no significant differences between SUNE-1 group and SUNE-1-siNC group(P>0.05).The results of the starBase website showed that the Lnc RNA NEAT1 and miR-331 would have complementary binding sites,and the dual-fluorescence gene report showed that the Lnc RNA NEAT1 and miR-331 would be able to bind on target.In addition the qRT-PCR confirmed that LncRNA NEAT1 could negatively regulate the expression of miR-331.Conclusion The LNC RNA NEAT1 is highly expressed in nasopharyngeal carcinoma tissues,and the NEAT1 silencing may inhibit the proliferation and invasion of nasopharyngeal carcinomacells by up-regulating the expression levels of miR-331,and increasing the radiosensitivity of nasopharyngeal carcinoma cells.
作者
梁卫勤
林智
LIANG Weiqin;LIN Zhi(Department of Ophthalmology&Otorhinolaryngology,The Third People’s Hospital of Haikou City,Hainan,Haikou 571100,China)
出处
《河北医药》
CAS
2020年第19期2885-2890,共6页
Hebei Medical Journal
