Stable Overexpression of MZF1 Does Not Alter Proliferation, Viability, or Stemness in HEK293T Cells

Document Type : Research Articles

Authors

Zahra Hosseininia ^{1, 2}

Hesam Dehghani ^{1, 2, 3}

¹ Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran

² Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

³ Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran

https://doi.org/10.22067/jcmr.2025.93826.1113

Abstract

Myeloid Zinc Finger 1 (MZF1) is a member of the SCAN domain-zinc finger (SCAN-ZFP) transcription factor family. With a structure comprising five distinct domains, it regulates various transcriptional processes. Numerous studies suggest that MZF1 has bi-functional roles, acting either as an oncogene or a tumor suppressor. However, its overexpression has also been associated with negligible changes in cellular functions, an aspect that remains underexplored. Additionally, although the HEK293T cell line is widely used to investigate molecular mechanisms, the role of MZF1 in these cells has not been reported. In this study, we assessed the effects of MZF1 overexpression on some cellular functions in these cells. Using transposon-mediated gene transfer, we generated stably expressing cell lines and assessed their cellular functions, including cell proliferation, cell viability, and cell stemness.  Our findings show that following stable expression of MZF1, the number of cells compared to controls did not reveal a significant difference during 6 time-points over 72 hours. Also, the MTT read absorbance, and as a consequence, cell viability did not change in the MZF1 overexpressing cell line compared to control cells (0.49 against 0.48). By performing the colony formation assay, we did not find any significant changes in the number of colonies or the number of cells in each colony. Also, our soft agar assay did not reveal statistically significant changes in the size and number of colonies between the two groups of MZF1-overexpressing cells and control cells. Taken together, our data provide evidence that the stable overexpression of MZF1 does not alter cellular functions of proliferation, viability, and stemness in HEK293T cells.

Keywords

MZF1

HEK293T cell line

cell proliferation

cell viability

cell stemness

---

References

Brix, D. M.,Bundgaard Clemmensen, K. K. and Kallunki, T. (2020). Zinc Finger Transcription Factor MZF1—A Specific Regulator of Cancer Invasion. Cells :1, 223.

Brix, D. M.,Tvingsholm, S. A.,Hansen, M. B.,Clemmensen, K. B.,Ohman, T. and Siino, V., et al. (2019). Release of transcriptional repression via ErbB2-induced, SUMO-directed phosphorylation of myeloid zinc finger-1 serine 27 activates lysosome redistribution and invasion. Oncogene :17, 3170–3184.

Chen, P.-M.,Cheng, Y.-W.,Wang, Y.-C.,Wu, T.-C.,Chen, C.-Y. and Lee, H. (2014). Up-Regulation of FOXM1 by E6 Oncoprotein through the MZF1/NKX2-1 Axis Is Required for Human Papillomavirus–Associated Tumorigenesis. Neoplasia :11, 961–971.

Chen, Y.,Zhang, Z.,Yang, K.,Du, J.,Xu, Y. and Liu, S. (2015). Myeloid zinc-finger 1 (MZF-1) suppresses prostate tumor growth through enforcing ferroportin-conducted iron egress. Oncogene :29, 3839–3847.

Eguchi, T.,Prince, T.,Wegiel, B. and Calderwood, S. K. (2015). Role and Regulation of Myeloid Zinc Finger Protein 1 in Cancer. J of Cellular Biochemistry, 116 :10, 2146–2154.

Eguchi, T.,Prince, T. L.,Tran, M. T.,Sogawa, C.,Lang, B. J. and Calderwood, S. K. (2019). MZF1 and SCAND1 Reciprocally Regulate CDC37 Gene Expression in Prostate Cancer. Cancers :6, 792.

Hoffman, S.,Hromas R,Amemiya, C. and and Mohrenweiser H.W (1996). The location of MZF-1 at telomere of human chromosome 19q makes it vulnerable to degeneration in aging cells. Leuk. Res., 281–283.

Horinaka, M.,Yoshida, T.,Tomosugi, M.,Yasuda, S.,Sowa, Y. and Sakai, T. (2014). Myeloid zinc finger 1 mediates sulindac sulfide-induced upregulation of death receptor 5 of human colon cancer cells. Scientific reports, 6000.

Hromas, R.,Collins, S. J.,Hickstein, D.,Raskind, W.,Deaven, L. L. and O'Hara, P., et al. (1991). A retinoic acid-responsive human zinc finger gene, MZF-1, preferentially expressed in myeloid cells. Journal of Biological Chemistry :22, 14183–14187.

Kan, A.,Liu, S.,He, M.,Wen, D.,Deng, H. and Huang, L., et al. (2024). MZF1 promotes tumour progression and resistance to anti-PD-L1 antibody treatment in hepatocellular carcinoma. JHEP reports : innovation in hepatology :1, 100939.

Lee, C.-J.,Hsu, L.-S.,Yue, C.-H.,Lin, H.,Chiu, Y.-W. and Lin, Y.-Y., et al. (2016). MZF-1/Elk-1 interaction domain as therapeutic target for protein kinase Cα-based triple-negative breast cancer cells. Oncotarget :37, 59845–59859.

Lee, J.-H.,Kim, S.-S.,Lee, H. S.,Hong, S.,Rajasekaran, N. and Wang, L.-H., et al. (2017a). Upregulation of SMAD4 by MZF1 inhibits migration of human gastric cancer cells. International journal of oncology :1, 272–282.

Lee, Y.-K.,Park, U.-H.,Kim, E.-J.,Hwang, J.-T.,Jeong, J.-C. and Um, S.-J. (2017b). Tumor antigen PRAME is up-regulated by MZF1 in cooperation with DNA hypomethylation in melanoma cells. Cancer letters, 144–151.

Li, B.,Li, S.,Shi, X.,Zhang, Y.,Xin, Z. and Yang, Y., et al. (2023). MZF1 (Myeloid Zinc Finger 1) activates transcriptional activity of p53 and suppresses breast cancer cell proliferation via acetyltransferase PCAF (P300/CBP associated factor). Genes & diseases :5, 1775–1778.

Li, J.,Liao, T.,Liu, H.,Yuan, H.,Ouyang, T. and Wang, J., et al. (2021). Hypoxic Glioma Stem Cell-Derived Exosomes Containing Linc01060 Promote Progression of Glioma by Regulating the MZF1/c-Myc/HIF1α Axis. Cancer research :1, 114–128.

Li, S.,He, J.,Zhang, L.,Zhao, Q.,Zhao, S. and Jiang, S. (2024). MitoAMPK inhibits the Warburg effect by MZF1-SIRT6 with glycosis related genes in NSCLC. Journal of cellular and molecular medicine :17, e70053.

Lin, S.,Xu, H.,Qin, L.,Pang, M.,Wang, Z. and Gu, M., et al. (2023). UHRF1/DNMT1-MZF1 axis-modulated intragenic site-specific CpGI methylation confers divergent expression and opposing functions of PRSS3 isoforms in lung cancer. Acta pharmaceutica Sinica. B :5, 2086–2106.

Morris, J. F.,Hromas, R. and Rauscher, F. J. (1994). Characterization of the DNA-Binding Properties of the Myeloid Zinc Finger Protein MZF1: Two Independent DNA-Binding Domains Recognize Two DNA Consensus Sequences with a Common G-Rich Core. Molecular and cellular biology :3, 1786–1795.

Mudduluru, G.,Vajkoczy, P. and Allgayer, H. (2010). Myeloid zinc finger 1 induces migration, invasion, and in vivo metastasis through Axl gene expression in solid cancer. Molecular cancer research : MCR :2, 159–169.

Murai, K.,Murakami, H. and Nagata, S. (1997). A novel form of the myeloid‐specific zinc finger protein (MZF‐2). Genes to Cells :9, 581–591.

Noll, L.,Peterson, F. C.,Hayes, P. L.,Volkman, B. F. and Sander, T. (2008). Heterodimer formation of the myeloid zinc finger 1 SCAN domain and association with promyelocytic leukemia nuclear bodies. Leukemia research :10, 1582–1592.

Ogawa, H.,Murayama, A.,Nagata, S. and Fukunaga, R. (2003). Regulation of Myeloid Zinc Finger Protein 2A Transactivation Activity through Phosphorylation by Mitogen-activated Protein Kinases. Journal of Biological Chemistry, 278 :5, 2921–2927.

Perrotti, D.,Melotti, P.,Skorski, T.,Casella, I.,Peschle, C. and Calabretta, B. (1995). Overexpression of the Zinc Finger Protein MZF1 Inhibits Hematopoietic Development from Embryonic Stem Cells: Correlation with Negative Regulation of CD34 and c- myb Promoter Activity. Molecular and cellular biology :11, 6075–6087.

Peterson, F. C.,Hayes, P. L.,Waltner, J. K.,Heisner, A. K.,Jensen, D. R. and Sander, T. L., et al. (2006). Structure of the SCAN domain from the tumor suppressor protein MZF1. Journal of molecular biology :1, 137–147.

Peterson, M. J. and Morris, J. F. (2000). Human myeloid zinc finger gene MZF produces multiple transcripts and encodes a SCAN box protein. Gene, 254, 105–118.

Qi, Z.,Wang, J.,Li, Y. and Xu, Y. (2022). MZF1 Transcriptionally Activated MicroRNA-328-3p Suppresses the Malignancy of Stomach Adenocarcinoma via Inhibiting CD44. Journal of immunology research, 5819295.

Qian, F.,Li, Q.,Chang, H.,Wei, K.,Chen, X. and Huang, T., et al. (2024). Identification of DNA methylation characteristics associated with metastasis and prognosis in colorectal cancer. BMC medical genomics :1, 127.

Tian, W.,Yu, C.,Xu, X.,Li, J.,Peng, S. and Sun, A., et al. (2025). Myeloid Zinc Finger 1: insights into its oncogenic potential, prognostic value, and impact on immune microenvironment across cancers. Frontiers in immunology, 1591912.

Tsai, L.-H.,Wu, J.-Y.,Cheng, Y.-W.,Chen, C.-Y.,Sheu, G.-T. and Wu, T.-C., et al. (2015). The MZF1/c-MYC axis mediates lung adenocarcinoma progression caused by wild-type lkb1 loss. Oncogene :13, 1641–1649.

Tsai, S.-J.,Hwang, J.-M.,Hsieh, S.-C.,Ying, T.-H. and Hsieh, Y.-H. (2012). Overexpression of myeloid zinc finger 1 suppresses matrix metalloproteinase-2 expression and reduces invasiveness of SiHa human cervical cancer cells. Biochemical and biophysical research communications :2, 462–467.

Vishwamitra, D.,Curry, C. V.,Alkan, S.,Song, Y.-H.,Gallick, G. E. and Kaseb, A. O., et al. (2015). The transcription factors Ik-1 and MZF1 downregulate IGF-IR expression in NPM-ALK⁺ T-cell lymphoma. Molecular cancer, 53.

Wang, C.-H.,Wu, H.-C.,Hsu, C.-W.,Chang, Y.-W.,Ko, C.-Y. and Hsu, T.-I., et al. (2022). Inhibition of MZF1/c-MYC Axis by Cantharidin Impairs Cell Proliferation in Glioblastoma. International journal of molecular sciences :23.

Williams, A. J.,Blacklow, S. C. and Collins, T. (1999). The zinc finger-associated SCAN box is a conserved oligomerization domain. Molecular and cellular biology :12, 8526–8535.

Wu, D.,Tan, H.,Su, W.,Cheng, D.,Wang, G. and Wang, J., et al. (2022). MZF1 mediates oncogene-induced senescence by promoting the transcription of p16INK4A. Oncogene :3, 414–426.

Yue, C.-H.,Huang, C.-Y.,Tsai, J.-H.,Hsu, C.-W.,Hsieh, Y.-H. and Lin, H., et al. (2015). MZF-1/Elk-1 Complex Binds to Protein Kinase Cα Promoter and Is Involved in Hepatocellular Carcinoma. PloS one :5, e0127420.

Yue, C.-H.,Liu, J.-Y.,Chi, C.-S.,Hu, C.-W.,Tan, K.-T. and Huang, F.-M., et al. (2019). Myeloid Zinc Finger 1 (MZF1) Maintains the Mesenchymal Phenotype by Down-regulating IGF1R/p38 MAPK/ERα Signaling Pathway in High-level MZF1-expressing TNBC cells. Anticancer research :8, 4149–4164.

Zhang, Z.,Gao, Q. and Wang, S. (2021). Kinase GSK3β functions as a suppressor in colorectal carcinoma through the FTO-mediated MZF1/c-Myc axis. Journal of cellular and molecular medicine :5, 2655–2665.