Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (2): 73-79.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0500
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ZHANG Hong-min1(), LONG Wen1, LAO Xiao-qing1, CHEN Wen-yan1, SHANG Xue-mei1, WANG Hong-lian2, WANG Li2, SU Hong-wei2, SHEN Hong-ping2, SHEN Hong-chun1,2()
Received:
2023-05-26
Online:
2024-02-26
Published:
2024-03-13
Contact:
SHEN Hong-chun
E-mail:zhm19970329@163.com;shenhongchun79@163.com
ZHANG Hong-min, LONG Wen, LAO Xiao-qing, CHEN Wen-yan, SHANG Xue-mei, WANG Hong-lian, WANG Li, SU Hong-wei, SHEN Hong-ping, SHEN Hong-chun. Construction of Pmepa1 Knockout TCMK1 Mouse Renal Tubular Epithelial Cell Line Using CRISPR/Cas9 Technology[J]. Biotechnology Bulletin, 2024, 40(2): 73-79.
Fig. 1 Pmepa1 sgRNA targeting location and plasmid construction results A. Schematic representation of the gene structure of the mouse Pmepa1 locus and the structure of the pX333-Pmepa1 vector. B. Sequence diagram of pX333-Pmepa1 vector. C. mCherry fluorescence plot of TCMK1 cells 48 h after transfection with pX333-Pmepa1 plasmid. D. Flow cytometry analysis plot of TCMK1 cells transfected with pX333-Pmepa1 plasmid
Fig. 2 Validation of Pmepa1 gene knockout A. PCR amplification electrophoresis of DNA sequences adjacent to the Cas9 action site of the Pmepa1 gene in 19 monoclonal TCMK1 cell lines. B. Sequence analysis of clone 4(KO-4)and clone 16(KO-16)PMEPA1 gene mutations in TCMK1 cells. C. Western blot for Pmepa1 protein expression
Fig. 3 RT-PCR and Western blot validation of TGF-β signaling activation and fibrotic protein expression after Pmepa1 knockdown A. RT-PCR method was used to detect fibrosis-related gene expression. B. The Western blot method to detecte Smad2/Smad3 pathway-related protein. *P < 0.05, **P < 0.01, ***P < 0.001 vs +TGF-β1/- TGF-β1 group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs +TGF-β1 KO-4, KO-16/+TGF-β1 NC group
[1] |
Akinnibosun OA, Maier MC, Eales J, et al. Telomere therapy for chronic kidney disease[J]. Epigenomics, 2022, 14(17): 1039-1054.
doi: 10.2217/epi-2022-0073 URL |
[2] | Gewin LS. Renal fibrosis: primacy of the proximal tubule[J]. Matrix Biol, 2018, 68-69: 248-262. |
[3] |
Zhang YY, Zhu XY, Huang X, et al. Advances in understanding the effects of erythropoietin on renal fibrosis[J]. Front Med, 2020, 7: 47.
doi: 10.3389/fmed.2020.00047 URL |
[4] |
Humphreys BD. Mechanisms of renal fibrosis[J]. Annu Rev Physiol, 2018, 80: 309-326.
doi: 10.1146/annurev-physiol-022516-034227 pmid: 29068765 |
[5] |
Ruiz-Ortega M, Rayego-Mateos S, Lamas S, et al. Targeting the progression of chronic kidney disease[J]. Nat Rev Nephrol, 2020, 16(5): 269-288.
doi: 10.1038/s41581-019-0248-y pmid: 32060481 |
[6] |
Chen YQ, Chen HY, Tang QQ, et al. Protective effect of quercetin on kidney diseases: from chemistry to herbal medicines[J]. Front Pharmacol, 2022, 13: 968226.
doi: 10.3389/fphar.2022.968226 URL |
[7] |
Watanabe Y, Itoh S, Goto T, et al. TMEPAI, a transmembrane TGF-beta-inducible protein, sequesters Smad proteins from active participation in TGF-beta signaling[J]. Mol Cell, 2010, 37(1): 123-134.
doi: 10.1016/j.molcel.2009.10.028 pmid: 20129061 |
[8] | Li J, Kong WM. PMEPA1 serves as a prognostic biomarker and correlates with immune infiltrates in cervical cancer[J]. J Immunol Res, 2022, 2022: 4510462. |
[9] |
Puteri MU, Watanabe Y, Wardhani BWK, et al. PMEPA1/TMEPAI isoforms function via its PY and Smad-interaction motifs for tumorigenic activities of breast cancer cells[J]. Genes Cells, 2020, 25(6): 375-390.
doi: 10.1111/gtc.12766 pmid: 32181976 |
[10] |
Sharad S, Dobi A, Srivastava S, et al. PMEPA1 gene isoforms: a potential biomarker and therapeutic target in prostate cancer[J]. Biomolecules, 2020, 10(9): 1221.
doi: 10.3390/biom10091221 URL |
[11] |
Sharad S, Sztupinszki ZM, Chen YM, et al. Analysis of PMEPA1 isoforms(a and b)as selective inhibitors of androgen and TGF-β signaling reveals distinct biological and prognostic features in prostate cancer[J]. Cancers, 2019, 11(12): 1995.
doi: 10.3390/cancers11121995 URL |
[12] |
Itoh S, Itoh F. TMEPAI family: involvement in regulation of multiple signalling pathways[J]. J Biochem, 2018, 164(3): 195-204.
doi: 10.1093/jb/mvy059 pmid: 29945215 |
[13] |
Manghwar H, Lindsey K, Zhang XL, et al. CRISPR/cas system: recent advances and future prospects for genome editing[J]. Trends Plant Sci, 2019, 24(12): 1102-1125.
doi: S1360-1385(19)30243-2 pmid: 31727474 |
[14] |
Wang SW, Gao C, Zheng YM, et al. Current applications and future perspective of CRISPR/Cas9 gene editing in cancer[J]. Mol Cancer, 2022, 21(1): 57.
doi: 10.1186/s12943-022-01518-8 |
[15] |
Khouzam JPS, Tivakaran VS. CRISPR-Cas9 applications in cardiovascular disease[J]. Curr Probl Cardiol, 2021, 46(3): 100652.
doi: 10.1016/j.cpcardiol.2020.100652 URL |
[16] |
Xu XY, Liu C, Wang YH, et al. Nanotechnology-based delivery of CRISPR/Cas9 for cancer treatment[J]. Adv Drug Deliv Rev, 2021, 176: 113891.
doi: 10.1016/j.addr.2021.113891 URL |
[17] |
Bao AL, Burritt DJ, Chen HF, et al. The CRISPR/Cas9 system and its applications in crop genome editing[J]. Crit Rev Biotechnol, 2019, 39(3): 321-336.
doi: 10.1080/07388551.2018.1554621 pmid: 30646772 |
[18] | 任云晓, 肖茹丹, 娄晓敏, 等. 基因编辑技术及其在基因治疗中的应用[J]. 遗传, 2019, 41(1): 18-28. |
Ren YX, Xiao RD, Lou XM, et al. Research advance and application in the gene therapy of gene editing technologies[J]. Hereditas, 2019, 41(1): 18-28. | |
[19] | 左其生, 李东, 张亚妮, 等. CRISPR-Cas介导的基因编辑工具[J]. 生物技术通报, 2014(7): 37-43. |
Zuo QS, Li D, Zhang YN, et al. Gene editing tools mediated by CRISPR-cas[J]. Biotechnol Bull, 2014(7): 37-43. | |
[20] | 陈汉宗, 梁颂, 黎新月, 等. 利用CRISPR/Cas9系统构建稳定敲除anxa6基因的Caco-2细胞株[J]. 微生物学报, 2023, 63(3): 1217-1229. |
Chen HZ, Liang S, Li XY, et al. Knockout of human anxa6 gene in Caco-2 cells by CRISPR/Cas9 system[J]. Acta Microbiol Sin, 2023, 63(3): 1217-1229. | |
[21] | 刘铁柱, 李阿茜, 李川, 等. 基于CRISPR/Cas9系统建立SNX11基因敲除A549细胞系[J]. 病毒学报, 2022, 38(3): 652-657. |
Liu TZ, Li AQ, Li C, et al. Construction of A SNX11-knockout A549 cell line based on the CRISPR/Cas9 system[J]. Chin J Virol, 2022, 38(3): 652-657. | |
[22] |
Sharma G, Sharma AR, Bhattacharya M, et al. CRISPR-Cas9: a preclinical and clinical perspective for the treatment of human diseases[J]. Mol Ther, 2021, 29(2): 571-586.
doi: 10.1016/j.ymthe.2020.09.028 pmid: 33238136 |
[23] |
Bai XL, Jing L, Li YC, et al. TMEPAI inhibits TGF-β signaling by promoting lysosome degradation of TGF-β receptor and contributes to lung cancer development[J]. Cell Signal, 2014, 26(9): 2030-2039.
doi: 10.1016/j.cellsig.2014.06.001 pmid: 24933703 |
[24] |
Yoshikawa T, Sanders AR, Esterling LE, et al. Multiple transcriptional variants and RNA editing in C18orf1, a novel gene with LDLRA and transmembrane domains on 18p11.2[J]. Genomics, 1998, 47(2): 246-257.
doi: 10.1006/geno.1997.5118 URL |
[25] |
Nakano N, Maeyama K, Sakata N, et al. C18 ORF1, a novel negative regulator of transforming growth factor-β signaling[J]. J Biol Chem, 2014, 289(18): 12680-12692.
doi: 10.1074/jbc.M114.558981 pmid: 24627487 |
[26] |
Amalia R, Abdelaziz M, Puteri MU, et al. TMEPAI/PMEPA1 inhibits Wnt signaling by regulating β-catenin stability and nuclear accumulation in triple negative breast cancer cells[J]. Cell Signal, 2019, 59: 24-33.
doi: S0898-6568(19)30059-2 pmid: 30890370 |
[27] |
Zhang L, Wang X, Lai C, et al. PMEPA1 induces EMT via a non-canonical TGF-β signalling in colorectal cancer[J]. J Cell Mol Med, 2019, 23(5): 3603-3615.
doi: 10.1111/jcmm.14261 pmid: 30887697 |
[28] |
Funakubo N, Xu XH, Kukita T, et al. Pmepa1 induced by RANKL-p38 MAPK pathway has a novel role in osteoclastogenesis[J]. J Cell Physiol, 2018, 233(4): 3105-3118.
doi: 10.1002/jcp.26147 pmid: 28802000 |
[29] |
HAinmhire EÓ, Quartuccio SM, Cheng W, et al. Mutation or loss of p53 differentially modifies TGFβ action in ovarian cancer[J]. PLoS One, 2014, 9(2): e89553.
doi: 10.1371/journal.pone.0089553 URL |
[30] |
Hagg A, Kharoud S, Goodchild G, et al. TMEPAI/PMEPA1 is a positive regulator of skeletal muscle mass[J]. Front Physiol, 2020, 11: 560225.
doi: 10.3389/fphys.2020.560225 URL |
[31] |
Li H, Chang HM, Shi ZD, et al. ID3 mediates the TGF-β1-induced suppression of matrix metalloproteinase-1 in human granulosa cells[J]. FEBS J, 2019, 286(21): 4310-4327.
doi: 10.1111/febs.14964 pmid: 31215762 |
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