|
|
Biophysics 2022
SHISAL1基因真核表达质粒构建及其在肝癌细胞中表达
|
Abstract:
本文的目的是克隆人SHISAL1基因和构建其真核表达载体，并在肝细胞癌细胞中表达和分析。以人肝细胞THLE-3总RNA为模板，用RT-PCR获得SHISAL1基因蛋白编码区，通过基因克隆和重组获得真核表达载体pDsRed1-SHISAL1；用脂质体介导法将pDsRed1-SHISAL1导入肝癌细胞HuH-7中，用荧光显微镜和激光共聚焦扫描显微镜观察细胞内红色荧光，用蛋白免疫印迹和免疫荧光染色分析细胞内FZD3的表达情况。结果显示成功克隆人SHISAL1基因蛋白编码区并获得了真核表达质粒pDsRed1-SHISAL1；荧光显微术和蛋白免疫印迹结果显示外源SHISAL1融合蛋白，即SHISAL1-RFP蛋白，在肝癌细胞HuH-7中表达；激光共聚焦扫描显微术和蛋白免疫印迹结果显示SHISAL1可以抑制肝癌细胞HuH-7内源性FZD3表达。本研究成功克隆了SHISAL1基因并在肝癌细胞中表达，并证明SHISAL1影响肝癌细胞内源性FZD3表达。
The aims are to clone human SHISAL1 gene and con-struct its eukaryotic expressional vector, and then to express and analyze the protein in hepatocel-lular carcinoma cells. Using THLE-3 total RNA as a template, the protein coding region of SHISAL1 gene was obtained by RT-PCR, and the eukaryotic expressional vector pDsRed1-SHISAL1 was ob-tained by gene cloning and recombination. The recombinant plasmid was introduced into hepato-cellular carcinoma cell line HuH-7 by liposome method. The intracellular red fluorescence was ob-served by fluorescence microscopy and laser confocal scanning microscopy, and the expression of endogenous FZD3 was analyzed by western blot and immunofluorescence staining. The results showed that the protein coding region of human SHISAL1 gene was cloned successfully and the eu-karyotic expression plasmid pDsRed1-SHISAL1 was obtained. Fluorescence microscopy and western blot showed that SHISAL1 fusion protein, namely SHISAL1-RFP fusion protein, was expressed in HuH-7 cells. Laser confocal scanning microscopy and western blot showed that overexpression of SHISAL1 could inhibit the endogenous expression of FZD3 in HuH-7 cells. Collectively, SHISAL1 gene was successfully cloned and expressed in hepatocellular carcinoma cells, and then it was proved that SHISAL1 could affect the expression of endogenous FZD3 in hepatocellular carcinoma cells.
| [1] | Pei, J. and Grishin, N.V. (2012) Unexpected Diversity in Shisa-Like Proteins Suggests the Importance of Their Roles as Transmembrane Adaptors. Cell Signal, 24, 758-769. https://doi.org/10.1016/j.cellsig.2011.11.011 |
| [2] | 周莉莉, 陈相屹, 余畅, 湛钊, 雷霆雯, 孙达权. 小鼠样人SHisa样蛋白1 (SSL1)多克隆抗体制备及其亚细胞定位[J]. 细胞与分子免疫学杂志, 2019, 35(9): 843-848. |
| [3] | Abdollahi Nejat, M., Klaassen, R.V., Spijker, S. and Smit, A.B. (2021) Auxiliary Subunits of the AMPA Receptor: The Shisa Family of Proteins. Current Opinion in Pharmacology, 58, 52-61. https://doi.org/10.1016/j.coph.2021.03.001 |
| [4] | Onishi, K. and Zou, Y. (2017) Sonic Hedgehog Switches on Wnt/Planar Cell Polarity Signaling in Commissural Axon Growth Cones by Reducing Levels of Shisa2. Elife, 6, e25269. https://doi.org/10.7554/eLife.25269 |
| [5] | Noack, C., Iyer, L.M., Liaw, N.Y., Schoger, E., Khadjeh, S., Wagner, E., Woelfer, M., Zafiriou, M.P., Milting, H., Sossalla, S., Streckfuss-Boemeke, K., Hasenfu？, G., Zimmermann, W.H. and Zelarayán, L.C. (2019) KLF15-Wnt-Dependent Cardiac Reprogramming Up-Regulates SHISA3 in the Mammalian Heart. JACC: Journal of the American College of Cardiology, 74, 1804-1819. https://doi.org/10.1016/j.jacc.2019.07.076 |
| [6] | Liu, Z., Wang, C., Liu, X. and Kuang, S. (2018) Shisa2 Regulates the Fusion of Muscle Progenitors. Stem Cell Research, 31, 31-41. https://doi.org/10.1016/j.scr.2018.07.004 |
| [7] | Kumar, P., Traurig, M. and Baier, L.J. (2020) Identification and Functional Validation of Genetic Variants in Potential miRNA Target Sites of Established BMI Genes. International Journal of Obesity (London), 44, 1191-1195.
https://doi.org/10.1038/s41366-019-0488-8 |
| [8] | Kim, N., Kim, M.J., Sung, P.S., Bae, Y.C., Shin, E.C. and Yoo, J.Y. (2016) Interferon-Inducible Protein SCOTIN Interferes with HCV Replication through the Autolysosomal Degrada-tion of NS5A. Nature Communications, 7, Article No. 10631. https://doi.org/10.1038/ncomms10631 |
| [9] | Schmitz, L.J.M., Klaassen, R.V., Ruiperez-Alonso, M., Zamri, A.E., Stroeder, J., Rao-Ruiz, P., Lodder, J.C., van der Loo, R.J., Mansvelder, H.D., Smit, A.B. and Spijker, S. (2017) The AMPA Receptor-Associated Protein Shisa7 Regulates Hippo-campal Synaptic Function and Contextual Memory. Elife, 6, e24192.
https://doi.org/10.7554/eLife.24192.030 |
| [10] | Klaassen, R.V., Stroeder, J., Coussen, F., Hafner, A.S., Petersen, J.D., Renancio, C., Schmitz, L.J., Normand, E., Lodder, J.C., Rotaru, D.C., Rao-Ruiz, P., Spijker, S., Mansvelder, H.D., Choquet, D. and Smit, A.B. (2016) Shisa6 Traps AMPA Receptors at Postsynaptic Sites and Prevents Their Desensitiza-tion during Synaptic Activity. Nature Communications, 7, Article No. 10682. https://doi.org/10.1038/ncomms10682 |
| [11] | Sabaie, H., Talebi, M., Gharesouarn, J., Asadi, M.R., Jalaiei, A., Arsang-Jang, S., Hussen, B.M., Taheri, M., Jalili Khoshnoud, R. and Rezazadeh, M. (2022) Identification and Analysis of BCAS4/hsa-miR-185-5p/SHISA7 Competing Endogenous RNA Axis in Late-Onset Alzheimer’s Disease Using Bi-oinformatic and Experimental Approaches. Frontiers in Aging Neuroscience, 14, Article ID: 812169. https://doi.org/10.3389/fnagi.2022.812169 |
| [12] | Sabaie, H., Gharesouran, J., Asadi, M.R., Farhang, S., Ahangar, N.K., Brand, S., Arsang-Jang, S., Dastar, S., Taheri, M. and Rezazadeh, M. (2022) Downregulation of miR-185 Is a Common Pathogenic Event in 22q11.2 Deletion Syndrome-Related And Idiopathic Schizophrenia. Metabolic Brain Dis-ease, 37, 1175-1184.
https://doi.org/10.1007/s11011-022-00918-5 |
| [13] | Tokue, M., Ikami, K., Mizuno, S., Takagi, C., Miyagi, A., Taka-da, R., Noda, C., Kitadate, Y., Hara, K., Mizuguchi, H., Sato, T., Taketo, M.M., Sugiyama, F., Ogawa, T., Kobayashi, S., Ueno, N., Takahashi, S., Takada, S. and Yoshida, S. (2017) SHISA6 Confers Resistance to Differentiation-Promoting Wnt/β-Catenin Signaling in Mouse Spermatogenic Stem Cells. Stem Cell Reports, 8, 561-575. https://doi.org/10.1016/j.stemcr.2017.01.006 |
| [14] | Maffei, R., Fiorcari, S., Martinelli, S., Benatti, S., Bulgarelli, J., Rizzotto, L., Debbia, G., Santachiara, R., Rigolin, G.M., Forconi, F., Rossi, D., Laurenti, L., Palumbo, G.A., Vallisa, D., Cuneo, A., Gaidano, G., Luppi, M. and Marasca, R. (2018) Increased SHISA3 Expression Characterizes Chronic Lym-phocytic Leukemia Patients Sensitive to Lenalidomide. Leukemia & Lymphoma, 59, 423-433. https://doi.org/10.1080/10428194.2017.1339872 |
| [15] | Tamura, K., Furihata, M., Satake, H., Hashida, H., Kawada, C., Osakabe, H., Fukuhara, H., Kumagai, N., Iiyama, T., Shuin, T. and Inoue, K. (2017) SHISA2 Enhances the Aggres-sive Phenotype in Prostate Cancer through the Regulation of WNT5A Expression. Oncology Letters, 14, 6650-6658. https://doi.org/10.3892/ol.2017.7099 |
| [16] | Paul, A.M., George, B., Saini, S., Pillai, M.R., Toi, M., Costa, L. and Kumar, R. (2022) Delineation of Pathogenomic Insights of Breast Cancer in Young Women. Cells, 11, 1927. https://doi.org/10.3390/cells11121927 |
| [17] | Liu, L., Lin, J. and He, H. (2019) Identification of Potential Crucial Genes Associated with the Pathogenesis and Prognosis of Endometrial Cancer. Frontiers in Genetics, 10, Article No. 373. https://doi.org/10.3389/fgene.2019.00373 |
| [18] | Chandrashekar, D.S., Bashel, B., Balasubramanya, S.A.H., Creighton, C.J., Rodriguez, I.P., Chakravarthi, B.V.S.K. and Varambally, S. (2017) UALCAN: A Portal for Facilitating Tumor Subgroup Gene Expression and Survival Analyses. Neoplasia, 19, 649-658. https://doi.org/10.1016/j.neo.2017.05.002 |
| [19] | Zhu, G.Q., Wang, Y., Wang, B., Liu, W.R., Dong, S.S., Chen, E.B., Cai, J.L., Wan, J.L., Du, J.X., Song, L.N., Chen, S.P., Yu, L., Zhou, Z.J., Wang, Z., Zhou, J., Shi, Y.H., Fan, J. and Dai, Z. (2022) Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-Activated Hepato-cellular Carcinoma. Cellular and Molecular Gastroenterology and Hepatology, 13, 1413-1447. https://doi.org/10.1016/j.jcmgh.2022.02.006 |
| [20] | Li, C., Nguyen, V., Clark, K.N., Zahed, T., Sharkas, S., Filipp, F.V. and Boiko, A.D. (2019) Down-Regulation of FZD3 Receptor Suppresses Growth and Metastasis of Human Mela-noma Independently of Canonical WNT Signaling. Proceedings of the National Academy of Sciences of the United States of America, 116, 4548-4557.
https://doi.org/10.1073/pnas.1813802116 |
| [21] | Wang, A., Wang, Y., Zhan, J. and Chen, J. (2021) Mi-croRNA-186-5p Inhibits the Metastasis of Cervical Cancer by Targeting FZD3. Journal of BUON, 26, 677-683. |
