慢病毒介导的PDE12稳定敲低和过表达胃癌细胞模型的构建及其细胞定位分析

doi:10.19586/j.2095-2341.2024.0156

生物技术进展 ›› 2025, Vol. 15 ›› Issue (2): 355-363.DOI: 10.19586/j.2095-2341.2024.0156

• 研究论文 • 上一篇

慢病毒介导的PDE12稳定敲低和过表达胃癌细胞模型的构建及其细胞定位分析

刘乙锦(), 许文扬, 魏锦骥, 宋欢喜, 李佳慧, 张丽娜()

北京工业大学化学与生命科学学院，北京 100124

收稿日期:2024-09-25 接受日期:2025-02-12 出版日期:2025-03-25 发布日期:2025-04-29
通讯作者: 张丽娜
作者简介:刘乙锦 E-mail: yijin0101@163.com；
基金资助:
国家自然科学基金-面上项目(82172969);北京市自然科学基金-面上项目(5202001);北京工业大学第24届星火基金项目(XH-2023-04-09)

Establishment of Stable Gastric Cancer Cell Model with PDE12 Knockdown and Overexpression Mediated by Lentivirus and its Cellular Localization Analysis

Yijin LIU(), Wenyang XU, Jinji WEI, Huanxi SONG, Jiahui LI, Lina ZHANG()

College of Chemistry and Life Science，Beijing University of Technology，Beijing 100124，China

Received:2024-09-25 Accepted:2025-02-12 Online:2025-03-25 Published:2025-04-29
Contact: Lina ZHANG

摘要/Abstract

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

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

中图分类号:

Q291

刘乙锦, 许文扬, 魏锦骥, 宋欢喜, 李佳慧, 张丽娜. 慢病毒介导的PDE12稳定敲低和过表达胃癌细胞模型的构建及其细胞定位分析[J]. 生物技术进展, 2025, 15(2): 355-363.

Yijin LIU, Wenyang XU, Jinji WEI, Huanxi SONG, Jiahui LI, Lina ZHANG. Establishment of Stable Gastric Cancer Cell Model with PDE12 Knockdown and Overexpression Mediated by Lentivirus and its Cellular Localization Analysis[J]. Current Biotechnology, 2025, 15(2): 355-363.

图/表 7

图1 慢病毒介导的PDE12敲低与过表达载体的构建图A：PDE12敲低的慢病毒载体构建图；B：PDE12过表达的慢病毒载体构建图

Fig.1 Construction diagram of lentivirus mediated PDE12 knockdown and overexpression vectors

图2 敲低和过表达PDE12基因的慢病毒载体构建结果A：敲低PDE12基因的慢病毒载体pLVX-shPDE12测序比对结果；B：PDE12基因PCR扩增产物的琼脂糖凝胶电泳结果，M—DNA maker；C：过表达PDE12基因的慢病毒载体pCDH-PDE12菌落PCR琼脂糖凝胶电泳鉴定结果,M—DNA maker，泳道1~4：挑取的4个单克隆菌落；D：过表达PDE12基因的慢病毒载体pCDH-PDE12测序比对结果

Fig. 2 Results of pLVX-shPDE12 and pCDH-PDE12 lentiviral vectors with PDE12 gene knockdown and overexpression

图3 PDE12基因敲低和过表达的MKN-45和BGC-823稳转细胞系荧光结果图A: 敲低PDE12的MKN-45和BGC-823稳转细胞荧光图；B: 过表达PDE12的MKN-45和BGC-823稳转细胞荧光图；标尺=200 μm

Fig. 3 Fluorescence results of MKN-45 and BGC-823 stable cell lines with PDE12 knockdown and overexpression

图4 PDE12在胃癌稳转细胞系mRNA水平上的表达差异A：PDE12在稳转细胞系中干扰效率的mRNA水平； B：PDE12在稳转细胞系中过表达效率的mRNA水平

Fig. 4 Differential expression of PDE12 at mRNA level in stale gastric cancer cell lines

图5 PDE12在胃癌稳转细胞系蛋白水平上的表达差异A：PDE12在胃癌稳转细胞系中干扰效率的蛋白水平检测；B：PDE12在胃癌稳转细胞系中过表达效率的蛋白水平检测

Fig. 5 Differential expression of PDE12 at the protein level in stable gastric cancer cell lines

图6 PDE12在MKN-45和BGC-823胃癌细胞中的定位分析A：免疫荧光分析PDE12在MKN-45细胞中的定位；B：免疫荧光分析PDE12在BGC-823细胞中的定位；标尺=10 μm

Fig. 6 Cellular localization analysis of PDE12 in MKN-45 and BGC-823 gastric cancer cells

图7 PDE12在胃癌细胞中与线粒体的共定位分析注：标尺=10 μm。

Fig. 7 Co-localization analysis of PDE12 with mitochondria in gastric cancer cells

参考文献 21

1	OMORI K, KOTERA J. Overview of PDEs and their regulation[J]. Circ. Res., 2007, 100(3): 309-327.
2	ORHAN I E, RAUF A, SALEEM M, et al.. Natural molecules as talented inhibitors of nucleotide pyrophosphatases/phosphodiesterases (PDEs)[J]. Curr. Top. Med. Chem., 2022, 22(3): 209-228.
3	MAURICE D H, KE H, AHMAD F, et al.. Advances in targeting cyclic nucleotide phosphodiesterases[J]. Nat. Rev. Drug Discov., 2014, 13(4): 290-314.
4	DANG T A, SCHUNKERT H, KESSLER T. cGMP signaling in cardiovascular diseases: linking genotype and phenotype[J]. J. Cardiovasc. Pharmacol., 2020, 75(6): 516-525.
5	SAMIDURAI A, XI L, DAS A, et al.. Role of phosphodiesterase 1 in the pathophysiology of diseases and potential therapeutic opportunities[J/OL]. Pharmacol. Ther., 2021, 226: 107858[2025-02-21]. .
6	SCHWENKGRUB J, ZAREMBA M, MIROWSKA-GUZEL D, et al.. Ibudilast: a non-selective phosphodiesterase inhibitor in brain disorders[J/OL]. Postępy Hig. Med. Dośw., 2017, 71(1):137-148.
7	BAILLIE G S, TEJEDA G S, KELLY M P. Therapeutic targeting of 3',5'-cyclic nucleotide phosphodiesterases: inhibition and beyond[J]. Nat. Rev. Drug Discov., 2019, 18(10): 770-796.
8	SCHAFER P. Apremilast mechanism of action and application to psoriasis and psoriatic arthritis[J]. Biochem. Pharmacol., 2012, 83(12): 1583-1590.
9	李思杨. 磷酸二酯酶12在结肠癌发生发展中的作用与机制[D]. 厦门: 厦门大学, 2020.
10	陈观. 磷酸二酯酶12下调对胃癌的增殖、迁移以及侵袭的影响[D]. 福州: 福建医科大学, 2020.
11	POULSEN J B, ANDERSEN K R, KJÆR K H, et al.. Human 2'-phosphodiesterase localizes to the mitochondrial matrix with a putative function in mitochondrial RNA turnover[J]. Nucleic Acids Res., 2011, 39(9): 3754-3770.
12	ESTRELLA M A, DU J, KORENNYKH A. Crystal structure of human nocturnin catalytic domain[J/OL]. Sci. Rep., 2018, 8(1): 16294[2025-02-21]. .
13	KUBOTA K, NAKAHARA K, OHTSUKA T, et al.. Identification of 2'-phosphodiesterase, which plays a role in the 2-5A system regulated by interferon[J]. J. Biol. Chem., 2004, 279(36): 37832-37841.
14	PEARCE S F, RORBACH J, VAN HAUTE L, et al.. Maturation of selected human mitochondrial tRNAs requires deadenylation[J/OL]. eLife, 2017, 6: e27596[2025-02-21]. .
15	RORBACH J, NICHOLLS T J J, MINCZUK M. PDE12 removes mitochondrial RNA poly(A) tails and controls translation in human mitochondria[J]. Nucleic Acids Res., 2011, 39(17): 7750-7763.
16	SILVERMAN R H. Implications for RNase L in prostate cancer biology[J]. Biochemistry, 2003, 42(7): 1805-1812.
17	孙霄麟, 张迪迪, 王馨雅, 等. TOE1敲低和过表达稳转细胞系的构建及其在胃癌细胞中的定位分析[J]. 生物技术进展, 2023, 13(2): 273-281.
	SUN X L, ZHANG D D, WANG X Y, et al.. Construction of stable cell lines with TOE1 knockdown and overexpression and analysis on its cellular localization in gastric cancer cells[J]. Curr. Biotechnol., 2023, 13(2): 273-281.
18	BRAY F, LAVERSANNE M, SUNG H, et al.. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J. Clin., 2024, 74(3): 229-263.
19	CHANDRA R, BALACHANDAR N, WANG S, et al.. The changing face of gastric cancer: epidemiologic trends and advances in novel therapies[J]. Cancer Gene Ther., 2021, 28: 390-399.
20	RUSCH L, ZHOU A, SILVERMAN R H. Caspase-dependent apoptosis by 2', 5'-oligoadenylate activation of RNase L is enhanced by IFN-beta[J]. J. Interferon Cytokine Res., 2000, 20(12): 1091-1100.
21	SONG X H, LIAO X Y, ZHENG X Y, et al.. Human Ccr4 and Caf1 deadenylases regulate proliferation and tumorigenicity of human gastric cancer cells via modulating cell cycle progression[J/OL]. Cancers, 2021, 13(4): 834[2025-02-21]. .

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慢病毒介导的PDE12稳定敲低和过表达胃癌细胞模型的构建及其细胞定位分析

Establishment of Stable Gastric Cancer Cell Model with PDE12 Knockdown and Overexpression Mediated by Lentivirus and its Cellular Localization Analysis

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