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. 2016 Aug 8;6:30941.
doi: 10.1038/srep30941.

Regulation of SLD5 Gene Expression by miR-370 During Acute Growth of Cancer Cells

Affiliations
Free PMC article

Regulation of SLD5 Gene Expression by miR-370 During Acute Growth of Cancer Cells

Keitaro Yamane et al. Sci Rep. .
Free PMC article

Abstract

SLD5 is a member of the GINS complex, essential for DNA replication in eukaryotes. It has been reported that SLD5 is involved in early embryogenesis in the mouse, and cell cycle progression and genome integrity in Drosophila. SLD5 may be involved in malignant tumor progression, but its relevance in human cancer has not been determined. Here, we found strong SLD5 expression in both human bladder cancer tissues from patients and cell lines. Knockdown of SLD5 using small interfering RNA resulted in reduction of cell growth both in vitro and an in vivo xenograft model. Moreover, we found that high levels of SLD5 in bladder cancer cells result from downregulation of microRNA (miR)-370 that otherwise suppresses its expression. High level expression of DNA-methyltransferase (DNMT) 1 and IL-6 were also observed in bladder cancer cells. Knockdown of IL-6 led to downregulation of DNMT1 and SLD5 expression, suggesting that IL-6-induced overexpression of DNMT1 suppresses miR-370, resulting in high SLD5 expression. Our findings could contribute to understanding tumorigenic processes and progression of human bladder cancer, whereby inhibition of SLD5 could represent a novel strategy to prevent tumor growth.

Conflict of interest statement

N.T. is an employee of GeneStem Co., Ltd. which produces anti-SLD5 antibody on a commercial basis.

Figures

Figure 1
Figure 1. Expression of SLD5 in human bladder cancer tissue.
(A) Representative images of normal bladder tissue and bladder cancer tissue stained with anti-SLD5 antibody. Samples were counterstained with hematoxylin. Brown: SLD5, Blue: nuclei. Scale bars: 100 μm. Graph shows the quantitative evaluation of SLD5-positive cells in random fields. Data show the mean ± SE, *p < 0.05, (n = 3). (B) Immunofluorescence staining of normal and bladder cancer tissues. SLD5 (green), Ki-67 (red), and DAPI (blue). Scale bar: 100 μm. (C) Comparison of mRNA expression levels of SLD5 in normal and cancer cells. Human normal bladder cells (HNBC), human umbilical vein endothelial cells (HUVEC), and human bladder cancer cells (KMBC2, T24) were analyzed by qRT-PCR. Normal cell expression was set at unity. GAPDH was used as an internal control. (D) Comparison of SLD5 protein expression by Western blotting. HUVECs, HNBC and T24 bladder cancer cells (T24) were used. (E) Immunocytochemical analysis of T24 and KMBC2 with anti-SLD5 antibody (green) in cancer cells. Nuclei were stained with DAPI (blue). Scale bars: 100 μm. (F) Comparison of doubling time in HUVECs (Normal) and T24 (Cancer). Bars show the mean ± SE, *p < 0.05.
Figure 2
Figure 2. Relevance of SLD5 expression for cell cycle progression.
Bladder cancer cell lines (T24, KMBC2) were transfected with two types of SLD5-specific siRNA (#1 and #2). (A) Quantitative evaluation of SLD5 mRNA affected by siRNA. Data are mean ± SE, *p < 0.05 (n = 3). (B) Western blotting of SLD5 in control scrambled siRNA (Ctl) and SLD5-specific siRNA #1 and #2 treated cells. (C) Comparison of cell growth after siRNA transfection (T24). Data are mean ± SE, *p < 0.05 (n = 3). (D,E) Cell growth (T24) affected by SLD5 silencing. (D) Uptake of EdU (green). Nuclei were stained with Hoechst 33342 (blue). EdU-positive cells were counted and normalized to nuclear numbers, and are shown graphically. Data are mean ± SE, *p < 0.05 (3 random fields) Scale bars: 100 μm. (E) Ki-67 staining (red). Nuclei were stained by DAPI (blue). Ki-67-positive cells were counted and normalized to the number of all surviving cells. Results are shown as mean ± SE, *p < 0.05 **p < 0.005 (3 random fields). Scale bars: 100 μm. (F) Cell cycle analysis using flow cytometry. After transfection with control or SLD5-specific siRNA, cells were analyzed. The right-hand graph depicts the quantitative evaluation by showing mean ± SE (n = 3).
Figure 3
Figure 3. Silencing of SLD5 attenuates tumor growth.
Bladder cancer cells (T24) were subcutaneously transplanted into nude mice. Control or SLD5-specific siRNA was injected on day10 post-inoculation. (A) Time course of tumor volume dynamics. Data are mean ± SE (n = 3). (B) Tumor weight on day 30 post-inoculation. *p < 0.05 mean ± SE. (C) qRT-PCR analysis for assessment of SLD5 mRNA expression in control and SLD5 siRNA-injected tumors. Data are mean ± SE ***p < 0.0001. (D) Xenograft tumors injected with control or SLD5 siRNA were stained with anti-SLD5 or Ki-67 antibody (brown). Sections were counterstained with hematoxylin. Scale bars: 100 μm.
Figure 4
Figure 4. SLD5 mRNA expression is regulated by miR-370.
(A) Seed sequence of miR-370 (blue) located in the 3′-UTR of SLD5. (B) Expression level of miR-370 in human bladder cancer cells compared to HUVECs (Normal). Data are mean ± SE **p < 0.001 (n = 3). The normal cell was set at unity. U6 is an internal control. (C) Luciferase reporter assay. T24 cells were transfected with pMIR luciferase reporter containing 3′-UTR sequences of SLD5 and miR-370. miR-214 was used as a control. Graph depicts relative luciferase luminescence activity. Control was set at unity. Results are mean ± SE, **p < 0.005 (n = 3). (D) Attenuation of SLD5 expression by miR-370 transfection in T24 cells. The level of miR-370 (left) and SLD5 (right) after transfection of each miR was confirmed by qRT-PCR. Values for mock control cells were set at unity. U6 or GAPDH was used as an internal control. Data are mean ± SE *p < 0.05. **p < 0.001, ***p < 0.0001. (E) Western blotting showing SLD5 expression in miR-370 mimic-transfected T24 cells. (F) Cell growth (T24) affected by miR-370 mimic transfection. Data are mean ± SE *p < 0.05. **p < 0.001. (G) Ki-67 (red) and (H) EdU (green)-positive cells in miRNA-transfected T24 cells. Graphs show quantitative evaluation of Ki-67 or EdU-positive cells. Data are mean ± SE, *p < 0.05,**p < 0.005 (3 random fields). Scale bars: 100 μm. (I) Cell cycle analysis using flow cytometry. Cells were analyzed after transfection with control or miR-370 mimic. The right-hand graph depicts a quantitative evaluation by showing mean ± SE (n = 3).
Figure 5
Figure 5. Tumor growth is affected by miR-370.
(A,B) Bladder cancer cells (T24) were subcutaneously transplanted into nude mice. miR-370 mimics or control miR were injected on day10 post-inoculation. (A) Time course of tumor volume dynamics. Data are mean ± SE (n = 3). (B) Tumor weight on day 30 post-inoculation. *p < 0.05 mean ± SE (C) qRT-PCR analysis for assessment of miR-370 or SLD5 mRNA expression in tumors injected with control or miR-370. Data are mean ± SE, **p < 0.0005, ***p < 0.0001 (n = 3). (D) Xenografted tumors injected with control or miR-370 mimics were stained with anti-SLD5 antibody (brown). Sections were counterstained with hematoxylin. Scale bars: 100 μm.
Figure 6
Figure 6. Regulation of miR-370 expression in tumors.
(A) miR-370 expression following 5-azacitidine treatment of T24 cells. Results are mean ± SE ***p < 0.0005 (n = 3). (B,C) qRT-PCR analysis of DNMT1 (B) and IL-6 (C) mRNA expression in normal bladder cells (Normal) and a cancer cell line (T24; Cancer). Data are mean ± SE, **p < 0.0005, *** < 0.00001 (n = 3). (D) qRT-PCR analysis of DNMT1 mRNA expression in T24 cells after treatment with IL-6. (E–H) qRT-PCR analysis of IL-6 (E), DNMT1 (F), miR-370 (G) and SLD5 (I) expression in T24 cells 48 hours after transfection of IL-6-specific siRNA or control siRNA (ctl). Data are mean ± SE, *p < 0.05, **p < 0.005, ***p < 0.00001 (n = 3). (I) Western blotting for detection of SLD5 in cells as described in (E).

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