circCALN1影响鼻咽癌细胞增殖与侵袭的分子机制研究

Study on the molecular mechanism of the effect of circCALN1 on the proliferation and invasion of NPC cells

目的探讨circCALN1(circular RNA calneuron 1)在鼻咽癌组织及细胞株中的相对表达水平与对鼻咽癌细胞株增殖及侵袭的影响,进一步探讨潜在的分子机制。方法运用RT-PCR检测鼻咽癌组织及细胞株中的circCALN1相对表达水平;常规培养鼻咽癌细胞株(CNE1、HNE1、SUNE-1、5-8F、6-10B、NP69、293T);设计针对circCALN1接头处的siRNA序列;运用脂质体转染细胞株;Transwell检测细胞侵袭能力;运用生物信息学软件预测circCALN1结合的miRNA;构建双荧光报告基因载体验证基因之间的结合作用。结果circCALN1在鼻咽癌组织及细胞株的相对表达水平上调。有效降低circCALN1在鼻咽癌细胞株的表达水平之后,能有效抑制CNE1细胞株增殖与侵袭,其抑制率分别为:24 h(15.2±0.9)%、48 h(27.3±2.3)%、72 h(56.7±3.9)%;其侵袭细胞个数为(65±6.9)。生物信息学结果提示:miR-143-3p与circCALN1存在结合位点。circCALN1双荧光报告基因载体与miR-143-3p mimics共转梁293T细胞,荧光活性降低。降低circCALN1的表达促进miR-143-3p的表达上调。结论circCALN1负性调控miR-143-3p的表达影响鼻咽癌细胞增殖与侵袭。

Objective To investigate the relative expression level of circular RNA calneuron 1(circCALN1)in nasopharyngeal carcinoma(NPC)and its influence on the proliferation and invasion of NPC cell lines,and to explore the potential molecular mechanism.Methods RT-PCR was used to detect the relative expression level of circCALN1 in NPC tissues and cell lines;CNE1,HNE1,SUNE-1,5-8F,6-10B,NP69,293T cell lines were cultured in conventional culture;siRNA sequence was designed for the junction of circCALN1;cell lines were transfected with liposomes 2000;cell invasiveness was detected by Transwell assay;miRNA combined with circCALN1 was predicted by bioinformatics software;the binding site between genes was verified by double fluorescence reporter vector.Results The relative expression level of circCALN1 in NPC tissues and cell lines was up-regulated.After effectively knock-down the expression level of circCALN1 in NPC cell lines,the proliferation and invasion of CNE1 cell lines were effectively inhibited.The inhibition rates were:24 hours(15.2±0.9)%,48 hours(27.3±2.3)%,72 hours(56.7±3.9)%,and the number of invasive cells was(65±6.9).Bioinformatics results indicated that there was a binding site between miR-143-3p and circCALN1.The double fluorescent reporter gene vector of circCALN1 and miR-143-3p mimics were co-transfected to the 293T cells,and the fluorescent activity decreased.Decreased expression of circCALN1 promoted up-regulation of miR-143-3p.Conclusion circCALN1 negatively regulates the expression of miR-143-3p and affects the proliferation and invasion of NPC cells.