Abstract:

Phosphodiesterase 12 （PDE12） is a member of endonuclease/exonuclease/phosphatase （EEP） superfamily. To further investigate the function of PDE12 gene in cells， lentiviral infection technology was used to stably knock down and overexpress the PDE12 in gastric cancer cells and obtain the stable cell models. Firstly， the shRNA sequence targeting the knockdown of PDE12 gene was inserted into the lentiviral interference vector pLVX-shRNA2-Puro， and the full-length cDNA of PDE12 gene was inserted into the lentiviral overexpression vector pCDH-CMV-MCS-EF1-GFP-Puro. After comparing and identifying the sequencing results correctly， the lentivirus was packaged and infected human gastric cancer cell lines MKN-45 and BGC-823. Subsequently， stable cell lines were screened using puromycin， and the knockdown and overexpression efficiency of PDE12 at mRNA and protein levels in stable gastric cancer cell lines were verified by quantitative real-time PCR（qPCR） and Western blot， respectively. In addition， we also constructed a pEGFP-PDE12 eukaryotic expression vector and transfected it into MKN-45 and BGC-823 gastric cancer cells to detect the localization of PDE12 in gastric cancer cells by immunofluorescence analysis. The experimental results indicated that PDE12 expression in MKN-45-shPDE12 and BGC-823-shPDE12 stable cells was significantly reduced， while the expression level of PDE12 in MKN-45-PDE12 and BGC-823-PDE12 stable cells was dramatically increased， suggesting that the successful construction of MKN-45 and BGC-823 stable gastric cancer cell lines with PDE12 knockdown and overexpression. The observation results of confocal microscopy indicated that the green fluorescence expressed by PDE12 was distributed in both the nucleus and cytoplasm. Further mitochondrial staining analysis using MitoTracker Red revealed that the green fluorescence of PDE12 expression was co-localized with the mitochondrial MitoTracker Red fluorescence， suggesting that PDE12 is localized in the mitochondrial of gastric cancer cells. Overall， these research results provide a basis for further in-depth study of the function and mechanism of PDE12 in gastric cancer.

Key words: PDE12, lentivirus, gastric cancer, cellular localization

摘要：

磷酸二酯酶12（phosphodiesterase 12，PDE12）是核酸内切酶/核酸外切酶/磷酸酶（endonuclease/exonuclease/phosphatase，EEP）超家族成员之一。为进一步研究PDE12基因在细胞中的功能，拟使用慢病毒感染技术在胃癌细胞中稳定敲低和过表达PDE12基因并获得相应的稳转细胞模型。首先将靶向敲低PDE12基因的shRNA序列插入慢病毒干扰载体pLVX-shRNA2-Puro，PDE12基因全长cDNA插入慢病毒过表达载体pCDH-CMV-MCS-EF1-GFP-Puro，经测序鉴定正确后包装慢病毒并感染人胃癌细胞MKN-45和BGC-823，然后用嘌呤霉素筛选出稳转细胞系，实时荧光定量PCR (quantitative real-time PCR, qPCR)和Western blot检测PDE12在稳转胃癌细胞系中mRNA和蛋白水平的敲低和过表达效率。此外，还构建了pEGFP-PDE12真核表达载体，转染人胃癌细胞MKN-45和BGC-823，通过免疫荧光实验分析PDE12在胃癌细胞中的定位。结果显示，MKN-45-shPDE12和BGC-823-shPDE12稳转细胞中PDE12的表达量显著降低，MKN-45-PDE12和BGC-823-PDE12稳转细胞中PDE12的表达量显著升高，提示利用慢病毒感染技术成功构建了稳定敲低和过表达PDE12的胃癌细胞系。激光共聚焦显微镜观察显示表达PDE12的绿色荧光在核质中均有分布，进一步通过线粒体MitoTracker Red染色发现PDE12表达的绿色荧光与线粒体MitoTracker Red红色荧光存在共定位，说明PDE12定位在胃癌细胞的线粒体中。这些研究结果可为后续深入研究PDE12在胃癌的功能及作用机制提供了基础。

关键词: PDE12, 慢病毒, 胃癌, 细胞定位