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. 2019 Aug 6;16(8):1132-1141.
doi: 10.7150/ijms.34386. eCollection 2019.

Ribosomal Protein L15 is involved in Colon Carcinogenesis

Zhixiong Dong  1   2   3 , Hongyu Jiang  2   3 , Shuangshuang Liang  2   4 , Yajie Wang  2   3 , Wei Jiang  3   5 , Changjun Zhu  2   3
Affiliations

Ribosomal Protein L15 is involved in Colon Carcinogenesis

Zhixiong Dong et al. Int J Med Sci. .

Abstract

Ribosomal biogenesis is responsible for protein synthesis in all eukaryotic cells. Perturbation of ribosomal biogenesis processes can cause dysfunctions of protein synthesis and varieties of human diseases. In this study, we examine the role of RPL15, a large ribosomal subunit protein, in human colon carcinogenesis. Our results reveal that RPL15 is remarkably upregulated in human primary colon cancer tissues and cultured cell lines when compared with paired non-cancerous tissues and non-transformed epithelium cells. Elevated expression of RPL15 in colon cancer tissues is closely correlated with clinicopathological characteristics in patients. We determine the effects of RPL15 on nucleolar maintenance, ribosomal biogenesis and cell proliferation in human cells. We show that RPL15 is required for maintenance of nucleolar structure and formation of pre-60S subunits in the nucleoli. Depletion of RPL15 causes ribosomal stress, resulting in a G1-G1/S cell cycle arrest in non-transformed human epithelium cells, but apoptosis in colon cancer cells. Together, these results indicate that RPL15 is involved in human colon carcinogenesis and might be a potential clinical biomarker and/or target for colon cancer therapy.

Keywords: RPL RPL15; apoptosis; apoptosis 15; colon cancer; nucleolus; ribosome biogenesis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Characterization of ribosomal protein RPL15. (A) Immunofluorescence analysis of HeLa cells with rabbit α-RPL15 and mouse α-Bip, anti-α-tubulin, α-nucleolin, α-fibrillarin or α-UBF. DNA was visualized by DAPI staining. R values were obtained as described (see Materials and Methods). Scale bars, 10 μm. (B) Immunofluorescence analysis of HeLa cells with rabbit α-RPL15 and mouse α-RPL11 or mouse α-RPS6 and DAPI. Scale bars, 10 μm. R values were obtained as described in (A). (C) Nuclear or cytoplasmic lysates from HeLa cells were extracted and subjected to immunoblotting by indicated antibodies. The density of each protein in each component was quantitated against the level of WCL (1/5 of total). WCL: whole cell lysate. (D) Histograms represented average proportion of RPL15 or RPL11 in Nuclear or cytoplasmic component relative to WCL in (C). All experiments were performed in triplicate. Data are shown as mean ± standard deviations.
Figure 2
Figure 2
The effects of RPL15 depletion on maintaining of nucleolar structure. (A) HeLa cells were transfected with nonsense (NS) siRNA or RPL15 siRNA for 48 h and cell lysates were immunoblotted with α-RPL15 and anti-α-Tubulin antibody. (B) HeLa cells grown on coverslips were transfected with NS siRNA or RPL15 siRNA for 48 h, fixed and immunostained with α-RPL15 and α-nucleolin, DNA was visualized by DAPI staining. Scale bars, 10 μm. (C-D) Immunofluorescent area and intensity of nucleolin or DAPI in (B) in 20 cells were determined by Image-Pro Plus 7.0. The ratio of nucleolin area to DAPI against controls (C) and immunofluorescent density (IOD/area) (D) were calculated. (E) Cells treated as (B) were fixed and immunostained with α-RPL15 and α-fibrillarin, DNA was visualized by DAPI staining. Scale bars, 10 μm. (F-G) Immunofluorescent area and intensity of fibrillarin or DAPI in (E) in 20 cells were determined. The ratio of fibrillarin area to DAPI against controls (F) and immunofluorescent density (IOD/area) (G) were calculated. (H) Cells treated as (B) were fixed and immunostained with α-RPL15 and α-UBF, DNA was visualized by DAPI staining. Scale bars, 10 μm. (I-J) Immunofluorescent area and intensity of UBF or DAPI in (H) in 20 cells were determined. The ratio of UBF area to DAPI against controls (I) and immunofluorescent density (IOD/area) (J) were calculated. Results represent means ± standard deviations of five independent experiments *P<0.05; ** P<0.01; *** P<0.001.
Figure 3
Figure 3
Ablation of RPL15 compromises ribosome biogenesis of pre-60S particles. (A) HeLa cells were transfected with nonsense (NS) siRNA or RPL15 siRNA for 48 h and cell lysates were immunoblotted with α-RPL15 and anti-α-Tubulin antibody. (B) Cells were treated as in (A), and nuclear extracts were prepared from HeLa cells and fractionized on 10% to 40% sucrose density gradient. The absorbance at 260 nm (A260) of each fraction was profiled and the positions of pre-ribosomal subunits were indicated.
Figure 4
Figure 4
Expression of RPL15 is upregulated in colon cancer. (A) The expression level of RPL15 was detected by immunoblotting in four colon cancer cell lines and a non-transformed epithelial cell line (RPE1). The density of each protein in each plane was quantitated. (B) quantification of the relative protein level of RPL15 in four colon cancer cell lines and non-transformed epithelial RPE1 cell. Data represent means ± standard deviations of three independent experiments. (C) RPL15 immunostaining in TMAs are shown. Note: top panel, magnification ×100; bottom panel, magnification ×200.
Figure 5
Figure 5
The effects of RPL15 ablation on cell proliferation and viability in various human cells. (A-B) RPE1 (A) and HCT116 (B) were transfected with NS siRNA or RPL15 siRNA and cell proliferation was determined by MTT assay at indicated times. Results represent means ± standard deviations of five independent experiments. (C) Cells transfected with NS siRNA or RPL15 siRNA for 48 h were labeled with 10 mM BrdU (1 h). The cells were fixed and immunostained with mouse α-BrdU. DNA was visualized by DAPI staining. Histograms represented the mean percentage of BrdU incorporation in indicated human cells depleted of RPL15. *P<0.05 and **P<0.01. (D) Cells were transfected with NS siRNA or RPL15 siRNA for 48 h. The cells were fixed and stained with Annexin V and PI. DNA was visualized by DAPI staining. Scale bars, 10 μm. (E) Histograms represented the mean percentage of Annexin V-positive cell in indicated human cells depleted of RPL15. ***P<0.001. (F) Immunoblotting to detect the effect of RPL15 depletion on cell cycle or apoptosis related proteins in indicated human cells. Cells were transfected with NS siRNA or RPL15 siRNA for 48 h. Whole cell lysates were immunoblotted with indicated antibodies.
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References

    1. Boisvert FM, van Koningsbruggen S, Navascues J. et al. The multifunctional nucleolus. Nat Rev Mol Cell Biol. 2007;8:574–85. - PubMed
    1. Aseev LV, Boni IV. [Extraribosomal functions of bacterial ribosomal proteins] Mol Biol (Mosk) 2011;45:805–16. - PubMed
    1. Warner JR, McIntosh KB. How common are extraribosomal functions of ribosomal proteins? Mol Cell. 2009;34:3–11. - PMC - PubMed
    1. McCann KL, Baserga SJ. Genetics. Mysterious ribosomopathies. Science. 2013;341:849–50. - PMC - PubMed
    1. Narla A, Ebert BL. Ribosomopathies: human disorders of ribosome dysfunction. Blood. 2010;115:3196–205. - PMC - PubMed