circCALN1/miR-143-3p/BCL-2调控轴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 Influence of circCALN1/miR-143-3p/BCL-2 Regulatory Axis on the Proliferation and Invasion of Nasopharyngeal Carcinoma Cells

该文探讨circCALN1(circular RNA calneuron 1)在鼻咽癌中的表达及调控分子机制。运用生物信息软件预测circCALN1调控的miRNA及miRNA调控的靶基因;运用RT-PCR检测鼻咽癌组织及细胞株中的基因表达水平;常规培养鼻咽癌细胞株(CNE1、HNE1、SUNE-1、5-8F、6-10B),鼻咽永久上皮分化细胞株NP69,人肾小管上皮细胞株293T;设计针对circRCALN1接头处的及BCL-2的siRNA序列,运用脂质体转染细胞株;Transwell检测细胞侵袭能力;MTT检测细胞增殖抑制率;Western blot检测蛋白的相对表达水平;构建双荧光报告基因载体验证基因之间的结合作用。circCALN1、BCL-2在鼻咽癌组织及细胞株的相对表达水平上调,而miR-143-3p表达水平下调;其中,在CNE1、HNE1、SUNE-1、5-8F细胞株中,circCALN1、BCL-2在CNE1细胞株的表达水平最高,而miR-143-3p在CNE1细胞株的表达水平最低;有效降低circCALN1、BCL-2在鼻咽癌细胞株CNE1的表达水平24、48、72 h之后,抑制率分别为(16.3±1.65)%、(29.6±2.6)%、(63±3.6)%;(26.3±2.9)%、(32.6±3.8)%、(59.6±3.9)%;过表达miR-143-3p 24、48、72 h之后,抑制率为(9.6±3.6)%、(13.6±5.6)%、(36.8±2.3)%;过表达miR-143-3p及降低circCALN1、BCL-2的表达,其侵袭细胞个数分别为185±13、200±15、183±18;生物信息学结果提示:miR-143-3p与circCALN1存在结合位点,miR-143-3p与BCL-2存在结合位点。circCALN1、BCL-2-3?UTR双荧光报告基因载体质粒与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,而BCL-2的表达下调。circCALN1/miR-143-3p/BCL-2调控轴影响了鼻咽癌细胞的增殖与侵袭。

This sudy was to investigate the molecular mechanism of circCALN1 expression in NPC (nasopharyngeal carcinoma).The miRNA regulated by circCALN1 and the target gene regulated by miRNA were predicted by the bioinformatics software and the relative gene expression levels in NPC tissues and cell lines were detected by RT-PCR;CNE1,HNE1,SUNE-1,5-8F,6-10B,NP69,293T cell lines were cultured in conventional culture;the siRNA sequences for the junction of circCALN1 and BCL-2 were designed and transfected with liposome;Transwell assay was used to detect cell invasion ability,MTT assay was used to detect cell proliferation inhibition rate,Western blot was used to detect the relative expression of protein,and double fluorescent reporter gene vector was constructed to verify the binding site between genes;the expression of circCALN1,BCL-2 were up-regulated in NPC tissues and cell lines;after 24,48 and 72 h when knock down the expression of circCALN1 and BCL-2,the inhibition rates were (16.3±1.65)%,(29.6±2.6)%,(63±3.6)%,(26.3±2.9)%,(32.6±3.8)% and (59.6±3.9)%,respectively;after overexpression of miR-143-3p for 24,48 and 72 h,the inhibition rates were (9.6±3.6)%,(13.6±5.6)%,(36.8±2.3)%;Overexpression of miR-143-3p and decreased expression of circCALN1and BCL-2,the number of invasive cells were 185±13,200±15,183±18;bioinformatics results showed that there were binding sites between miR-143-3p and circCALN1,and between miR-143-3p and BCL-2.The plasmid of circCALN1,BCL-2-3?UTR double fluorescent reporter gene vector and miR-143-3p mimics were co-transfected into 293T cells,and the fluorescent activity was decreased.The knock down expression of circCALN1 promoted the up-regulation of miR-143-3p,while down regulated expression level of BCL-2.The regulatory axis of circCALN1/miR-143-3p/BCL-2 affects the proliferation and invasion of NPC.