长链非编码RNA lnc-GCLC-1稳定沉默的肝细胞株L02的构建与验证

Construction and identification of stable L02 cell lines with down-regulation of long chainnon-coding RNA lnc-GCLC-1

目的利用慢病毒载体构建长链非编码RNA lnc-GCLC-1沉默的稳转L02肝细胞株,为研究lnc-GCLC-1功能提供基础。方法以lnc-GCLC-1为靶点,设计并合成4组双链发卡结构,分别与慢病毒载体psi-LVRH1GP连接构成重组质粒;转化感受态细胞获取大量重组质粒后电泳、测序鉴定;通过瞬时转染筛选出2个有效的短发卡RNA(sh RNA);将重组质粒转染293T细胞后,收集病毒液并感染L02细胞,经嘌呤霉素筛选出稳定转染细胞株。利用荧光显微镜观察细胞绿色荧光情况以检测转染效果,采用Real-time PCR鉴定转染细胞株,并检测lnc-GCLC-1沉默对GCLC m RNA表达的影响。结果电泳及测序结果表明重组慢病毒载体构建成功;sh RNA-2和sh RNA-4是有效的sh RNA;用0.5μg/ml嘌呤霉素成功筛选出稳定低表达L02细胞株;在sh RNA-2和sh RNA-4稳定转染L02细胞株中,lnc-GCLC-1的表达水平均低于阴性载体组(P<0.05),沉默效率分别为21.3%和64.82%;sh RNA-2组和sh RNA-4组GCLC的m RNA表达量均低于阴性载体组(P<0.01)。结论本研究成功构建了lnc-GCLC-1沉默稳转L02细胞株。

Objective To construct the stable L02 cell lines with down-regulation of lnc-GCLC-1 mediated by lentiviral vector for studying the function of lnc-GCLC-1. Methods By targeting lnc-GCLC-1,four double stranded DNA hairpin structures were designed,synthesized and connected with psi-LVRH1 GP to construct the recombinant plasmid. The recombinant plasmids obtained from transformed competent cells were identified by electrophoresis and DNA sequence analysis. Two effective sh RNAs were selected by transient transfection. After 293 T cells were transfected by recombinant plasmids, these recombinant lentivirus were collected and added to L02 cells,then the stably transfected cells were screened by puromycin. The green fluorescence of cells was observed by fluorescence microscope. Real-time PCR was used to identify transfected cell lines,and analyze the effect of lnc-GCLC-1 silence on GCLC expression. Results The result of electrophoresis and DNA sequence analysis showed that the recombinant lentiviral vectors were successfully built.sh RNA-2 and sh RNA-4 were effective sh RNAs.The cells stably transferred by sh RNA were successfully screened with 0.5 μg/ml puromycin. lnc-GCLC-1 expression in the L02 cell lines stably transfected by sh RNA-2 and sh RNA-4 respectively were conspicuously lower than those in the negative control group（P〈0.05）,and the gene silencing effects were 21.3% and 64.82% respectively. GCLC m RNA expression in the sh RNA-2 and sh RNA-4 groups were lower than those in the negative control group（P〈0.01）. Conclusion The stable L02 cell lines with down-regulation of lnc-GCLC-1 are successfully constructed in the present study.