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RNA interference mediated JAM-A gene silencing promotes human epidermal stem cell proliferation

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Abstract

The objective of the study was to explore the influence of junctional adhesion molecule A (JAM-A) gene decoration on proliferation and differentiation of human epidermal stem cells (hEpSCs). JAM-A gene and JAM-A interference gene lentivirus eukaryotic expression vectors were established. The recombinant lentivirus was introduced into hEpSCs to observe and detect viral transfection by fluorescence microscopy and Western blot, respectively. After confirmation of successful introduction of the target gene, cell growth curves were mapped out by cytometry to detect cell proliferation in different groups. The expression of hEpSCs labeled molecules was detected by immunofluorescence, and cell safety was detected by teratoma test in all groups. (1) Fluorescence microscopy showed that in the JAM-A over-expression (JAM-Aov EpSCs) group, the green fluorescence was mainly distributed in the cell membrane; in the JAM-A interference (JAM-Akd EpSCs) group and blank vector (GFP EpSCs) group, all cell bodies were luminous. Western blot showed that JAM-A protein was up-regulated in JAM-Aov EpSCs and down-regulated in JAM-Akd EpSCs. (2) Growth curves showed that hEpSCs entered the quick-growing phase 4 days after inoculation and reached the platform phase at day 7. JAM-Aov EpSCs proliferated more slowly than GFP EpSCs, while JAM-Akd EpSCs proliferated significantly faster than GFP EpSCs. (3) Immunofluorescence showed that the expression of transient amplification epidermal marker keratin 14, hEpSCs marker keratin I9 and β-integrin was down-regulated in JAM-Akd EpSCs group as compared to that in the GFP EpSCs group, and the expression of epidermal terminal differentiation marker K10 was negative in the JAM-Akd EpSCs group. There was no significant difference in the expression of specific molecules between JAM-Aov EpSCs and hEpSCs. (4) The result of teratoma test was negative in all groups. The proliferative ability of hEpSCs was increased markedly after down-regulation of JAM-A. Cells presented initial differentiation, but retained their stem cell characteristics without evidence of tumorigenesis.

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Acknowledgments

This work was supported by National 973 Project of China (No. 2011CB965101) and China Postdoctoral Science Foundation (No. 2012M512061).

Conflict of interest

The authors have declared that no competing interests exist.

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Authors and Affiliations

  1. Department of Histology and Embryology, Second Military Medical University, Shanghai, 200433, China

    Tong Zhou, Minjuan Wu, Xiaocan Guo & Houqi Liu

  2. Burns Institute of People’s Liberation Army, Changhai Hospital, The Second Military Medical University, Shanghai, 200433, People’s Republic of China

    Minjuan Wu

Authors
  1. Tong Zhou
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  2. Minjuan Wu
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  3. Xiaocan Guo
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  4. Houqi Liu
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Correspondence to Houqi Liu.

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M. Wu, T. Zhou and X. Guo are co-first authors.

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Zhou, T., Wu, M., Guo, X. et al. RNA interference mediated JAM-A gene silencing promotes human epidermal stem cell proliferation. Human Cell 28, 73–80 (2015). https://doi.org/10.1007/s13577-013-0087-2

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  • DOI: https://doi.org/10.1007/s13577-013-0087-2

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