LncRNA-NR_046269对马尔尼菲篮状菌感染人支气管上皮细胞早期CLTC基因表达的影响

Effect of lncRNA-NR_046269 on the expression of CLTC gene in human bronchial epithelial cells infected with Talaromyces marneffei at early stage

目的研究马尔尼菲篮状菌(TM)感染人支气管上皮细胞BEAS-2B早期,长链非编码RNA(lncRNA)-NR_046269对网格蛋白重链基因(CLTC)表达的影响。方法分析前期TM孢子感染BEAS-2B细胞4 h后的lncRNAmRNA基因芯片表达谱。以BEAS-2B细胞为研究对象,分为实验组和空白对照组,实验组用TM孢子感染BEAS-2B细胞4 h,采用实时荧光定量PCR(RT-qPCR)法检测lncRNA-NR_046269、CLTC的相对表达量。通过生物信息学分析lncRNA-NR_046269与CLTC mRNA的相关性,然后使用慢病毒载体转染BEAS-2B细胞,分别过表达和敲低lncRNA-NR_046269(分为过表达组和敲低组),验证lncRNA-NR_046269对CLTC的表达是否具有靶向调控作用。结果与空白对照组相比,实验组lncRNA-NR_046269[(1.05±0.06)vs.(37.17±0.46)]和CLTC[(1.02±0.01)vs.(4.44±0.45)]的表达显著上调,差异均有统计学意义(t=121.49、12.89,P均<0.05),RT-qPCR结果与基因芯片结果一致。生物信息学分析提示NR_046269与CLTC mRNA成显著正相关(PCC=0.987,P<0.05)。成功构建稳定过表达和敲低lncRNA-NR_046269的BEAS-2B细胞株,与阴性对照组(0.865±0.328)相比,过表达组中NR_046269表达上调(10.570±0.250),CLTC的表达随着上调(1.250±0.020),同时敲低组中NR_046269下调(0.020±0.001),CLTC表达亦下调(0.008±0.002),差异均有统计学意义(F=4994.44、3617.74,P均<0.05)。结论TM感染支气管上皮细胞早期lncRNA-NR_046269和CLTC表达显著上调,且lncRNA-NR_046269对CLTC基因有正性调控作用,揭示了TM感染进展中的功能性炎症相关lncRNA及基因,可能作为TM治疗新的潜在靶点。

Objective To investigate the effect of long non-coding RNA(lnc RNA)-NR_046269 on the expression levels ofclathrin heavy chain(CLTC)in human bronchial epithelial cells BEAS-2B response to the early-stage infection ofTalaromyces marneffei(TM).Methods Lnc RNA-m RNA expression profile was obtained after BEAS-2B cells were infectedwith TM spores for 4 h according to the previous gene chip technology.BEAS-2B cells were divided into an experimentalgroup and a blank control group.The experimental group infected BEAS-2B cells with TM spores for 4 h.The m RNAexpression of lnc RNA-NR_046269 and CLTC was detected by real-time fluorescence quantitative PCR(RT-q PCR).Thecorrelation between lnc RNA-NR_046269 and CLTC m RNA was analyzed by bioinformatics,and then BEAS-2B cells weretransfected with a lentiviral vector to overexpress and knockdown lnc RNA-NR_046269 for the verification whether lnc RNA-NR_046269 had a targeting regulatory effect on CLTC expression.Results Compared with the control group,theexpression of lnc RNA-NR_046269[(1.05±0.06)vs.(37.17±0.46)]and CLTC[(1.02±0.01)vs.(4.44±0.45)]weresignificantly up-regulated in the experimental group(t=121.49,12.89,all P<0.05),and the results of RT-q PCR were consistent with those of gene chip.Bioinformatics analysis suggested that NR_046269 was significantly and positively correlated with CLTC m RNA(PCC=0.987,P<0.05).The BEAS-2B cell line with stable overexpression and knockdown of lnc RNA-NR_046269 was successfully constructed,NR_046269 expression was up-regulated in NR_046269 overexpression group(10.570±0.250),CLTC expression was up-regulated(1.250±0.020),and NR_046269 was down-regulated in knockdown group(0.020±0.001);CLTC expression was also down-regulated(0.008±0.002),and the differences were statistically significant(F=4994.44,3617.74,all P<0.05).Conclusions The expression of lnc RNA-NR_046269 and CLTC were significantly up-regulated in the early stage of TM infection in bronchial epithelial cells and lnc RNA-NR_046269had a positive regulatory effect on CLTC gene,revealing functional inflammation-related lnc RNA and genes in TM progression,and could be a new potential targets for TM therapy.