Construction of Stable Transfected Cell Line A549 of Non-small Cell Lung Cancer by Overexpressing and Knocking Down LncRNA RP11-521C20.3
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摘要:
目的 构建过表达及敲减LncRNA RP11-521C20.3的非小细胞肺癌A549稳转细胞株。 方法 根据lncRNA RP11-521C20.3基因序列，设计合成引物并进行扩增。将目的基因与Sbf I和EcoRI酶切的载体连接，构建pcSLenti-pA-RP11-521C20.3-CMV-SFH-EGFP-P2A-Puro-WPRE重组质粒，转染293T细胞，包装含lncRNA RP11-521C20.3质粒重组体的慢病毒。构建shRNA（RP11-521C20.3），连接到AgeI和EcoRI双酶切后的pSLenti-U6-shRNA-CMV-EGFP-F2A-Puro-WPRE载体上，经鉴定后转染293T细胞。再利用慢病毒介导，构建重组质粒pcSLenti-pA-RP11-521C20.3-CMV-SFH-EGFP-P2A-Puro-WPRE和pSLenti-U6-shRNA（RP11-521C20.3）-CMV-EGFP-F2A-Puro-WPRE，导入A549细胞。最后采用RT-qPCR技术在基因水平检测lncRNA RP11-521C20.3的表达。 结果 OV-lncRNA RP11-521C20.3组（过表达组）的lncRNA RP11-521C20.3 mRNA表达量高于NC-lncRNA RP11-521C20.3组（过表达对照组）（P < 0.001），差异倍数为（20.43±0.69）。sh-lncRNA RP11-521C20.3组（敲减组）的lncRNA RP11-521C20.3mRNA表达量低于sh-NC组（敲减对照组）（P < 0.001），差异倍数为（0.21±0.08）。 结论 成功构建了lncRNA RP11-521C20.3过表达及敲减稳转细胞株。为后续研究lncRNA RP11-521C20.3在慢性阻塞性肺疾病（COPD）发病机制中的作用奠定重要基础。 -
关键词:
- lncRNA RP11-521C20.3 /
- 非小细胞肺癌 /
- A549 /
- 慢病毒载体
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）. -
Key words:
- lncRNA RP11-521C20.3 /
- Non-small cell lung cancer /
- A549 /
- Lentiviral vector
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图 2 LncRNA RP11-521C20.3敲减表达质粒图谱
Figure 2. LncRNA RP11-521C20.3 knockdown expression plasmid map
图 3 lncRNA RP11-521C20.3过表达重组质粒测序结果
Figure 3. Sequencing results of lncRNA RP11-521C20.3 overexpressed recombinant plasmid
图 4 lncRNA RP11-521C20.3敲减重组质粒测序结果
Figure 4. Sequencing results of lncRNA RP11-521C20.3 knockdown recombinant plasmid
图 5 293T细胞中慢病毒滴度检测荧光图片（100×）
Figure 5. Fluorescent image of lentivirus titer detection in 293T cells （100×）
图 6 LncRNA RP11-521C20.3过表达慢病毒载体转染A549细胞明场图像和荧光图像（100×）
Figure 6. LncRNA RP11-521C20.3 overexpressed lentiviral vector transfected A549 cells with bright field image and fluorescence image （100×）
图 7 RT-qPCR检测A549稳转株中lncRNA RP11-521C20.3表达量
*** P<0.001。
Figure 7. The expression of lncRNA RP11-521C20.3 in stable A549 strain was detected by RT-qPCR
图 8 RP11-521C20.3敲减慢病毒载体转染A549细胞明场图像和荧光图像（100×）
Figure 8. RP11-521C20.3 knockdown virus vector transfected A549 cells bright field image and fluorescence image（100×）
图 9 RT-qPCR检测A549稳转株中lncRNA RP11-521C20.3表达量
***P < 0.001。
Figure 9. The expression of lncRNA RP11-521C20.3 in stable A549 strain detected by RT-qPCR
表 1 LncRNA RP11-521C20.3 PCR扩增引物序列步骤
Table 1. LncRNA RP11-521C20.3 PCR amplification primer sequence
步骤 引物与试剂 扩增引物Forward 5′-CCCTGCTTTGGGGTTGTGA-3′ Reverse 5′-GGAGGCTTTGTGGCTTGCT-3′ PCR扩增体系 5×PrimeSTAR®Buffer 10 µL，dNTP Mixtur 4 µL，正、反向引物各1 µL，模板
DNA < 200 ng，PrimeSTAR® HS DNA Polymerase 0.5 µL，灭菌蒸馏水加至50 µL反应条件 98℃ 10sec，55℃ 5sec，72℃ 1 min/kb，30个循环 扩增产物凝胶电泳 1.5%琼脂糖凝胶电泳 胶回收 TaKaRaMiniBEST Agarose Gel DNA Extraction Kit Ver.3.0 
下载: 导出CSV
表 2 过表达重组载体构建酶切反应体系
Table 2. Enzyme digestion reaction system for construction of overexpressed recombinant vectors
试剂 体积 质粒 2 μg 10 x反应Buffer 5 µL SbfI 1 µL EcoRI 1 µL ddH2O Up to 50 µL
下载: 导出CSV
表 3 过表达重组质粒反应体系
Table 3. Overexpressed recombinant plasmid reaction system
试剂 体积 5x反应Buffer 4 µL 插入片段 2 µL 线性化体 1 µL 无缝克隆酶 2 µL CCH2O Up to 20 µL
下载: 导出CSV
表 4 过表达重组质粒鉴定体系
Table 4. Identification system of overexpressed recombinant plasmid
试剂 体积 Premix Tag 25 µL 模板 1 µL 引物1（20 µM） 1 µL 引物2（20 µM） 1 µL 超纯水 Up to 50 µL
下载: 导出CSV
表 5 慢病毒包装试剂
Table 5. Lentivirus packaging reagent
试剂 体积 载体质粒 20 μg pHelper 1. 0载体质粒 15 μg pHelper2. 0载体质粒 10 μg 转染试剂 Up to 1mL
下载: 导出CSV
表 6 shRNA序列
Table 6. shRNA sequence
名称 序列（5′-3′） Y16185-F CcggCCAGATCTGTTGACCAACTTTCAAGAGAAGTTGGTCAACAGATCTGGTTTTTTg Y16185-R aattcaaaaaaCCAGATCTGTTGACCAACTTCTCTTGAAAGTTGGTCAACAGATCTGG GL427NC2-F CcggCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTTTg GL427NC2-R aattcaaaaaaCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG
下载: 导出CSV
表 7 敲减载体构建酶切反应体系
Table 7. Enzyme digestion reaction system for knockdown vector construction
试剂 体积 质粒 2 μg 10x反应Buffer 5 μL AgeI 1 μL EcoRI 1 μL ddH2O Up to 50 μL
下载: 导出CSV
表 8 敲减重组体PCR鉴定体系
Table 8. Knock-down recombinant PCR identification system
试剂 体积 Premix Tag 2 μg 模板 1 μL 引物1（20 μM） 1 μL 引物2（20 μM） 1 μL ddH2O Up to 50 μL
下载: 导出CSV
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