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Knockdown of the Shwachman-Diamond syndrome gene, SBDS, induces galectin-1 expression and impairs cell growth

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Abstract

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The depletion of SBDS protein by RNA interference has been shown to cause inhibition of cell proliferation in several cell lines. However, the precise mechanism by which the loss of SBDS leads to inhibition of cell growth remains unknown. To evaluate the impaired growth of SBDS-knockdown cells, we analyzed Epstein-Barr virus-transformed lymphoblast cells (LCLs) derived from two patients with SDS (c. 183_184TA > CT and c. 258 + 2 T > C). After 3 days of culture, the growth of LCL-SDS cell lines was considerably less than that of control donor cells. By annealing control primer-based GeneFishing PCR screening, we found that galectin-1 (Gal-1) mRNA expression was elevated in LCL-SDS cells. Western blot analysis showed that the level of Gal-1 protein expression was also increased in LCL-SDS cells as well as in SBDS-knockdown 32Dcl3 murine myeloid cells. We confirmed that recombinant Gal-1 inhibited the proliferation of both LCL-control and LCL-SDS cells and induced apoptosis (as determined by annexin V-positive staining). These results suggest that the overexpression of Gal-1 contributes to abnormal cell growth in SBDS-deficient cells.

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Data availability

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

The authors declare that they have no conflict of interest. We would like to thank Dr. Tsuneo Imanaka (Hiroshima International University) for useful discussions. This study was supported in part by a Grant-in Aid for Blood Coagulation Abnormalities from the Ministry of Health, Labor, and Welfare of Japan. We thank Dr. Hisashi Kawashima (Tokyo Medical University) for providing the patient samples, and Ms. Chikako Sakai (University of Toyama) for technical assistance. This manuscript was edited by Pacific Edit.

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Authors and Affiliations

  1. Laboratory of Physiological Chemistry, Hiroshima International University, 5-1-1 Hirokoshingai, Kure-Shi, Hiroshima, 737-0112, Japan

    Masafumi Yamaguchi, Yukihiro Sera, Hanae Toga-Yamaguchi & Yusuke Iguchi

  2. Department of Child Health and Development, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, BUnkyo-ku, Tokyo, 113-8519, Japan

    Hirokazu Kanegane

  3. Department of Nursing, Yasuda Women’s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima, 731-0153, Japan

    Kingo Fujimura

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  1. Masafumi Yamaguchi
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Correspondence to Masafumi Yamaguchi.

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12185_2024_3709_MOESM1_ESM.tiff

Supplementary file1 (TIFF 1142 KB) Supplementary Figure 1 rhGal-1 induced aggregation of 32Dcl3 cells. 32Dcl3 were cultured with 5 µM rhGal-1 in IMDM /10% FCS/10% conditioned medium and incubated at 37℃ for 2 days. The images are representation of at least 3 independent experiments

12185_2024_3709_MOESM2_ESM.tiff

Supplementary file2 (TIFF 1142 KB) Supplementary Figure 2 Cell cycle analysis of LCL cells. LCL cells were cultured at a density of 1 × 105 cells/mL in RPMI 1640/10% FCS medium and incubated at 37℃ for 3 days. Cells were fixed with 70% ethanol and stained with propidium iodide (PI). The fluorescence intensity of PI was measured with flow cytometry. Values shown are the averages of three separate experiments ± standard deviation of the mean

12185_2024_3709_MOESM3_ESM.tiff

Supplementary file3 (TIFF 1142 KB) Supplementary Table 1 List of candidate genes that changed the expression in LCL-SDS cells. The expression ratio of identified genes in LCL-SDS/LCL-C was compared relative to GAPDH, which was set as 1

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Yamaguchi, M., Sera, Y., Toga-Yamaguchi, H. et al. Knockdown of the Shwachman-Diamond syndrome gene, SBDS, induces galectin-1 expression and impairs cell growth. Int J Hematol 119, 383–391 (2024). https://doi.org/10.1007/s12185-024-03709-z

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