Effect of MiR-2779-x on the Growth Characteristics of MDCK Cells and Verification of Target Gene Screening
HUANG Ling-Wei1,2,3,*, YANG Di1,4,*, JIN Li-Wu1,2,3, YANG Ya-Wen1,3,5, WANG Jia-Min1,2,5, QIAO Zi-Lin1,2,5, Ayimuguli ABUDUREYIMU1,2,3,**
1 Biomedical Research Center/Engineering Research Center for Key Technologies and Industrialization of Cell-based Vaccines, Ministry of Education, Northwest Minzu University, Lanzhou 730030, China; 2 Biomedical Research Center/Gansu Tech Innovation Center of Animal Cell, Northwest Minzu University, Lanzhou 730030, China; 3 Life Science and Engineering College, Northwest Minzu University, Lanzhou 730030, China; 4 Department of Experiment & Teaching, Northwest Minzu University, Lanzhou 730030, China; 5 Biomedical Research Center/Key Laboratory of Bioengineering and Technology State Ethnic Affairs Commission, Northwest Minzu University, Lanzhou 730030, China
Abstract：Madin Darby Canine Kidney (MDCK) cell line is one of the most commonly used cell lines to produce Influenza virus vaccine, and miR-2779-x, as a non-coding RNA, can inhibit spontaneous tumor transformation in MDCK cells. In this study, stable cell lines with overexpression and knockdown of miR-2779-x were constructed based on the lentivirus system, and the transfected cells were detected by cell proliferation detection kit CCK-8 (Cell Counting Kit-8), cell apoptosis and cell cycle detection kit, as well as cell scratch test and soft agar cloning test. The results showed that overexpression of miR-2779-x significantly decreased cell proliferation and relative mobility (P<0.01), and increased cell invasion (P<0.01), while knockdown cell lines showed opposite results. The early apoptosis rate of overexpressed cell lines and the late apoptosis rate of knockdown cell lines were significantly increased (P<0.01). miR-2779-x overexpression caused cell block in S phase, and knockdown cell lines were blocked in G0/G1 phase. Measured at 50% tissue culture infective dose (TCID50), the sensitivity of MDCK cells with miR-2779-x lentivirus overexpression and knockdown did not change significantly to Influenza virus A H1N1. The expression changes of miR-2779-x target genes were verified through qRT-PCR and Western blot, and multiple genes related to transformation, tumorigenesis, and growth were found to be negatively regulated by miR-2779-x. It is speculated that the target gene CASP9 (caspase 9) might act as an apoptotic pathway factor and co-regulate MDCK cell proliferation with PI3K/AKT signaling pathway. This study provides a new approach for establishing a novel MDCK cell line, which can provide a better cell matrix for influenza vaccine production.
黄玲巍, 杨迪, 靳莉武, 杨雅雯, 王家敏, 乔自林, 阿依木古丽•阿不都热依木. miR-2779-x对MDCK细胞生长特性的影响及靶基因筛选验证[J]. 农业生物技术学报, 2024, 32(3): 666-678.
HUANG Ling-Wei, YANG Di, JIN Li-Wu, YANG Ya-Wen, WANG Jia-Min, QIAO Zi-Lin, Ayimuguli ABUDUREYIMU. Effect of MiR-2779-x on the Growth Characteristics of MDCK Cells and Verification of Target Gene Screening. 农业生物技术学报, 2024, 32(3): 666-678.
[1] 高文怡, 张天瑞, 姚娟. 2021. 敲低KDM3A抑制MDA-MB-231乳腺癌细胞的侵袭迁移并将细胞周期阻滞于G0/G1期[J]. 细胞与分子免疫学杂志, (9): 808-814. (Gao W Y, Zhang T R, Yao J. 2021. Knockdown of KDM3A inhibits the invasion and migration of MDA-MB-231 breast cancer cells and blocks the cell cycle in G0/G1 phase[J]. Journal of Cellular and Molecular Immunology, (9): 808-814.) [2] 黄玲巍, 张震宇, 王家敏, 等. 2023. microRNA调控肿瘤形成相关的因素及信号通路[J]. 中国细胞生物学学报, 45: 962-973. (Huang L W, Zhang Z Y, Wang J M, et al.2023. microRNA regulation of tumor-related factors and signaling pathways[J]. Chinese Journal of Cell Biology, 45(6): 962-973.) [3] 潘学洪, 王晓东. 2018. 抑制Akt信号通路对病理性瘢痕成纤维细胞生长、细胞周期及凋亡的影响[J]. 郑州大学学报(医学版), 53(2): 229-233. (Pan X H, Wang X D.2018. Effects of inhibition of Akt signaling pathway on the growth, cell cycle and apoptosis of fibroblasts in pathological scars[J]. Journal of Zhengzhou University (Medical Science Edition), 53(2): 229-233.) [4] 石嘉琛. 2022. miR-2779-x对MDCK细胞成瘤性的作用研究[D]. 硕士学位论文, 西北民族大学, 导师: 阿依木古丽, pp. 57-62. (Shi J C.2022. Effect of miR-2779-x on tumorigenicity of MDCK cells[D]. Thesis for M.S., Northwest Minzu University, Supervisor: Ayi M G L, pp. 57-62.) [5] 石嘉琛, 黄玲巍, 田玲,等. 2022. 不同浓度的miR-2779对MDCK细胞Bak1表达的影响[J]. 生物技术, 32(01): 15-20, 40. (Shi J C, Huang L W, Tian L, et al.2022. Effects of different concentrations of miR-2779 on Bak1 expression in MDCK cells[J]. Biotechnology, 32(01): 15-20, 40.) [6] 王文博, 汪清云, 郭卫, 等. 2023. LAMC2通过PI3K/Akt信号通路调控胃癌细胞增殖的研究[J]. 中国比较医学杂志, 33(10): 1-10. (Wang W B, Wang Q Y, Guo W, et al.2023. Regulation of gastric cancer cell proliferation by LAMC2 via PI3K/Akt signaling pathway[J]. Chinese Journal of Comparative Medicine, 33(10): 1-10.) [7] 许靖. 2018. 修饰的MDCK细胞悬浮培养驯化及用于繁殖禽流感病毒的初步探究[D]. 硕士学位论文, 扬州大学, 导师: 刘秀梵, pp. 24-57. (Xu J.2018. Preliminary study on suspension culture domestication of modified MDCK cells and its use in breeding avian influenza virus[D]. Thesis for M.S., Yangzhou University, Supervisor: Lui X F, pp. 24-57.) [8] 杨成成, 南克俊, 秦思达. 2015. 过表达人细胞S期激酶相关蛋白2 (Skp2)促进MCF-7乳腺癌细胞生长及S期细胞增加[J]. 细胞与分子免疫学杂志, 31(10): 1306-1310. (Yang C C, Nan K J, Qin S D2015. Overexpression of human cell S-phase kinase-associated protein 2 (Skp2) promotes the growth of MCF-7 breast cancer cells and the proliferation of S-phase cells[J]. Journal of Cellular and Molecular Immunology, 31(10): 1306-1310.) [9] 杨海智, 马海霞, 杨信东. 2012. 国内关于半数致死量及类似生物效应指标测算方法研究进展[J]. 国外医药(抗生素分册), 33(02): 62-66. (Yang H Z, Ma H X, Yang X D, 2012. Research progress on measurement methods of median lethal dose and similar biological effect indicators in China[J]. Archies of Foreign Pharmaceutical (Antibiotics), 33(02): 62-66.) [10] 张隆陶, 王劲, 张贻庆, 等. 2019. miR-1197负调控Smad3抑制结肠癌细胞SW620的增殖、侵袭、迁移能力及S期细胞周期的聚集相关研究[J]. 中国实验诊断学, 23(05): 874-879. (Zhang L T, Wang J, Zhang Y Q, et al.2019. miR-1197 negatively regulates Smad3 to inhibit the proliferation, invasion, migration and aggregation of S-phase cell cycle of colon cancer cell SW620[J]. Chinese Journal of Experimental Diagnostics, 23(05): 874-879.) [11] 张启龙. 2014. 稳定表达siat7e和st3galI基因MDCK细胞株的建立[D]. 硕士学位论文, 中国兽医药品监察所, 导师: 王栋, pp. 35-44. (Zhang Q L.2014. Establishment of MDCK cell line with stable expression of siat7e and st3galI genes[D]. Thesis for M.S., China Veterinary Drug Supervision Institute, Supervisor: Wang D, pp. 35-44.) [12] 张薇, 李善华. 2020. 下调fascin抑制卵巢癌细胞从G_0/G_1期向S期转变的实验研究[J]. 现代肿瘤医学, 28(03): 397-401. (Zhang W, Li S H.2020. An experimental study on inhibition of fascin in ovarian cancer cells from G_0/G_1 stage to S stage[J]. Modern Oncology Medicine, 28(03): 397-401.) [13] 郑小妹, 林叶飞, 张韶琼, 等. 2023. IGF1调控PI3K/AKT信号通路对卵巢癌细胞增殖和侵袭的影响[J]. 中国计划生育和妇产科, 15(04): 32-37. (Zheng X M, Lin Y F, Zhang S Q, et al.2023. Effects of IGF1 regulation of PI3K/AKT signaling pathway on proliferation and invasion of ovarian cancer cells[J]. Chinese Journal of Family Planning and Obstetrics and Gynecology, 15(04): 32-37.) [14] 周乐, 常晨城, 王宇, 等. 2022. PI3K/Akt/mTOR信号通路调控牛细胞生长作用的研究进展[J]. 饲料研究, 45(09): 138-142. (Zhou L, Chang C C, Wang Y, et al.2022. Advances in studies on the role of PI3K/Akt/mTOR signaling pathway in regulating bovine cell growth[J]. Feed Research, 45(09): 138-142.) [15] 朱紫瑜, 王冠, 庄英萍. 2021. 大规模哺乳动物细胞培养工程的现状与展望[J]. 合成生物学, 2(04): 612-634. (Zhu Z Y, Wang G, Zhuang Y P.2021. Current situation and prospect of large-scale mammalian cell culture projects[J]. Synthetic Biology, 2(04): 612-634.) [16] Aubrit F, Perugi F, Léon A, et al.2015. Cell substrates for the production of viral vaccines[J]. Vaccine, 33(44): 5905-5912. [17] Lanb Y N.2019. Cell-based quadrivalent inactivated influenza virus vaccine (Flucelvax® Tetra/Flucelvax Quadrivalent®): A review in the prevention of influenza[J]. Drugs, 79(12): 1337-1348. [18] Lei C, Yang J, Hu J, et al.2021. On the calculation of TCID(50) for quantitation of virus infectivity[J]. Virologica Sinica, 36(1): 141-144. [19] Montomoli E, Khadang B, Piccirella S, et al.2012. Cell culture-derived influenza vaccines from Vero cells: A new horizon for vaccine production[J]. Expert Review of Vaccines, 11(5): 587-594. [20] Omeir R, Thomas R, Teferedegne B.2015. A novel canine kidney cell line model for the evaluation of neoplastic development: Karyotype evolution associated with spontaneous immortalization and tumorigenicity[J]. Chromosome Research, 23(4): 663-680. [21] Yang H, Li Z, Wang Z, et al.2022. Histocompatibility minor 13 (HM13), targeted by miR-760, exerts oncogenic role in breast cancer by suppressing autophagy and activating PI3K-AKT-mTOR pathway[J]. Cell Death Discovery, 13(8): 728. [22] Ye Q, Phan T, Hu W S, et al.2021. Transcriptomic characterization reveals attributes of high influenza virus productivity in MDCK cells[J]. Viruses, 13(11): 2200.
