Construction of Stable Transfected Cell Line A549 of Non-small Cell Lung Cancer by Overexpressing and Knocking Down LncRNA RP11-521C20.3

Chuntao LI; Xuemei ZHANG; Yandi SU; Jianqing Zhang

doi:10.12259/j.issn.2095-610X.S20231016

Volume 44 Issue 10

Oct. 2023

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Chuntao LI, Xuemei ZHANG, Yandi SU, Jianqing Zhang. Construction of Stable Transfected Cell Line A549 of Non-small Cell Lung Cancer by Overexpressing and Knocking Down LncRNA RP11-521C20.3[J]. Journal of Kunming Medical University, 2023, 44(10): 1-9. doi: 10.12259/j.issn.2095-610X.S20231016

Citation:

Chuntao LI, Xuemei ZHANG, Yandi SU, Jianqing Zhang. Construction of Stable Transfected Cell Line A549 of Non-small Cell Lung Cancer by Overexpressing and Knocking Down LncRNA RP11-521C20.3[J]. Journal of Kunming Medical University, 2023, 44(10): 1-9. doi: 10.12259/j.issn.2095-610X.S20231016

Citation:

Chuntao LI, Xuemei ZHANG, Yandi SU, Jianqing Zhang. Construction of Stable Transfected Cell Line A549 of Non-small Cell Lung Cancer by Overexpressing and Knocking Down LncRNA RP11-521C20.3[J]. Journal of Kunming Medical University, 2023, 44(10): 1-9. doi: 10.12259/j.issn.2095-610X.S20231016

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Construction of Stable Transfected Cell Line A549 of Non-small Cell Lung Cancer by Overexpressing and Knocking Down LncRNA RP11-521C20.3

doi: 10.12259/j.issn.2095-610X.S20231016

Dept. of Respiratory and Critical Care Medicine，The 1st Affiliated Hospital of Kunming Medical University，Kunming Yunnan 650032，China

Received Date: 2022-12-13
Available Online: 2023-09-12
Publish Date: 2023-10-25

Abstract

Abstract

Objective To construct stable transfected cell lines of non-small cell lung cancer A549 with overexpression and knockdown LncRNA RP11-521C20.3. Methods According to lncRNA RP11-521C20.3 gene sequence, primers were designed and amplified. The target gene was then connected to a vector that had been cleaved with Sbf I and EcoRI enzymes to construct the recombinant plasmid pcSLenti-pA-RP11-521C20.3-CMV-SFH-EGFP-P2A-Puro-WPRE. This plasmid was transfected into 293T cells to package the Lentivirus containing the lncRNA RP11-521C20.3 plasmid. An shRNA （RP11-521C20.3） was constructed and connected to the pSLenti-U6-shRNA-CMV-EGFP-F2A-Puro-WPRE vector after being modified with AgeI and EcoRI enzymes. This vector was then transfected into 293T cells after verification. Recombinant plasmids pcSLenti-pA-RP11-521C20.3-CMV-SFH-EGFP-P2A-Puro-WPRE and pSLenti-U6-shRNA （RP11-521C20.3）-CMV-EGFP-F2A-Puro-WPRE were then constructed using the lentivirus-mediated method and introduced into A549 cells. Finally, RT-qPCR technology was used to detect the expression of lncRNA RP11-521C20.3 at the gene level. Results The expression level of lncRNA RP11-521C20.3 mRNA in the OV-lncRNA RP11-521C20.3 group （overexpression group） was higher than that in the NC-lncRNA RP11-521C20.3 group （overexpression control group）（P < 0.001）, with a fold change of （20.43±0.69）. The expression level of lncRNA RP11-521C20.3 mRNA in the sh-lncRNA RP11-521C20.3 group （knockdown group） was lower than that in the sh-NC group （knockdown control group）（P < 0.001）, with a fold change of （0.21±0.08）. Conclusion In this study, a stable cell line with overexpression and knockdown of lncRNA RP11-521C20.3 was successfully constructed, which laid an important foundation for the subsequent study of the role of lncRNA RP11-521C20.3 in the pathogenesis of chronic obstructive pulmonary disease （COPD）.
- lncRNA RP11-521C20.3,
- Non-small cell lung cancer,
- A549,
- Lentiviral vector

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