Construction and identification of stable PC12 cell lines with high/low expression of miR-122-5p

Tao Daiju; Su Haiyu; Wang Yuqi; Shen Zhiqiang; He Bo

doi:10.12307/2026.082

Abstract：BACKGROUND:MicroRNA-122-5p(miR-122-5p),as a key member of the microRNA(miRNA)family,plays an important role in regulating gene expression and the occurrence and development of various diseases.However,its precise mechanisms remain incompletely understood.The construction of stable miR-122-5p-overexpressing or miR-122-5p-knockdown PC12 cell models may provide a powerful experimental tool for in-depth studies of the exact mechanism of action of miR-122-5p in neurological diseases and the discovery of potential therapeutic targets. OBJECTIVE:To construct a lentiviral vector for high/low expression of rat miR-122-5p and use it to establish a PC12 cell line with stable overexpression of high/low expression of miR-122-5p,which would lay the foundation for further research on the role of miR-122-5p in neurological diseases. METHODS:Synthetic primers were designed according to the miR-122-5p gene sequence,and the resulting gene fragment was amplified via polymerase chain reaction(PCR).A recombinant lentiviral plasmid was constructed via the directional insertion of the target gene into the vector plasmid GV369 digested by AgeI/NheI.Positive clones were screened and sequenced to compare the results.The plasmid vector was cocultured and transfected with the target plasmid vector in 293T cells,and the lentiviral stock solution was obtained for packaging and titer assays.The working concentration of puromycin was determined by culturing PC12 cells in vitro.Lentiviruses were cocultured with PC12 cells separately to determine the transfection efficiency.Stably transfected cells were selected with puromycin,and miR-122-5p expression in the stably transfected cell lines was detected via real-time quantitative PCR. RESULTS AND CONCLUSION:(1)The sequencing sequence was consistent with the target sequence,suggesting that the recombinant lentiviral vector was constructed successfully.The lentiviral titer of high expression was 4×108 TU/mL,and the lentiviral titer of low expression of miR-122-5p was 1×109 TU/mL.(2)The working concentration of puromycin in PC12 cells was 3.5 μg/mL.(3)The optimal conditions for the lentiviral transfection of PC12 cells with high expression of miR-122-5p were HiTransG P transfection enhancement solution and an infection complex value equal to 10;the highest efficiency of low expression of miR-122-5p was achieved at an infection complex value of 50.(4)qRT-PCR revealed a significant increase in miR-122-5p expression in stably transfected cell lines and a significant decrease in miR-122-5p expression in stably transfected cell lines.(5)In this study,a miR-122-5p lentiviral vector was successfully constructed,and a stably transformed PC12 cell line was generated,which provides an experimental basis for further study of the role of miR-122-5p in neurological diseases.