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PRUNE2 inhibits progression of colorectal cancer in vitro and in vivo - PubMed

PRUNE2 inhibits progression of colorectal cancer in vitro and in vivo

Ting Li et al. Exp Ther Med. 2022 Feb.

Abstract

Prune homolog 2 with BCH domain (PRUNE2) is associated with prostate cancer, neuroblastoma, glioblastoma and melanoma; however, the function of PRUNE2 in colorectal cancer (CRC) remains unknown. The present study aimed to evaluate the effects of PRUNE2 on the development of CRC. The biological function of PRUNE2 in CRC cell lines was investigated by using Cell Counting Kit-8, colony formation, flow cytometry and Transwell assay. Additionally, a mouse model was established to investigate the effect of PRUNE2 on metastasis of CRC cells. The expression levels of PRUNE2 were lower in CRC compared with adjacent normal tissue and this expression pattern was associated with poor relapse-free survival probability. PRUNE2 overexpression significantly decreased cell proliferation and invasion, increased cell apoptosis and arrested the cell cycle. Consistently, it increased the protein expression levels of pro-apoptosis genes and decreased the expression of antiapoptotic proteins. PRUNE2 knockdown had the opposite effects. Furthermore, PRUNE2 overexpression decreased the tumorigenicity of CRC cells. In conclusion, PRUNE2 decreased cell survival, proliferation, invasion and tumorigenicity and promoted apoptosis, suggesting that PRUNE2 may function as a tumor-suppressive gene in CRC.

Keywords: colorectal cancer; invasion; proliferation; prune homolog 2 with BCH domain; tumorigenicity.

Copyright: © Li et al.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1

PRUNE2 downregulation in human CRC predicts poor recurrence-free survival. (A) Kaplan-Meier survival curves of relapse-free survival probability of patients with CRC with high and low levels of PRUNE2. The log-rank test was used for statistical analysis. (B) Expression levels of PRUNE2 were analyzed in COAD, READ and matched adjacent normal tissue. The expression levels of PRUNE2 were analyzed in COAD based on (C) cancer stage and (D) nodal metastasis status. These data were extracted from the TCGA. (E) PRUNE2 protein expression in CRC and adjacent tissue was using immunohistochemistry (magnification, x200). Scale bar, 100 mm. (F) Quantification of PRUNE2 expression using ImageJ software. (G) PRUNE2 protein expression in CRC and adjacent tissue was detected using western blot assay and (H) quantified. Data are presented as the mean ± SD (n=5). Data were analyzed using Student's t-test. *P<0.05, ***P<0.001 vs. adjacent (normal). PRUNE2, prune homolog 2 with BCH domain; CRC, colorectal cancer; COAD, colon adenocarcinoma; READ, rectal adenocarcinoma; TCGA, The Cancer Genome Atlas.

Figure 2
Figure 2

PRUNE2 expression levels in cell lines and effect on cell viability. (A) mRNA and (B) protein levels in human normal colorectal mucosa cells (FHC) and CRC cell lines (SW620, SW480, HT29, HCT116, LOVO, DLD-1) were determined by reverse transcription-quantitative PCR and western blotting, respectively. *P<0.05, **P<0.01, ***P<0.001 vs. FHC. (C) Transfection efficiency of the pCDNA3.1/PRUNE2 plasmid and shRNA PRUNE2 were detected by western blot assay. (D) Viability of CRC cells was detected by Cell Counting Kit-8 assay. Data are presented as the mean ± SD (n=3). Data were analyzed using one-way ANOVA. **P<0.01, ***P<0.001 vs. normal; ##P<0.01, ###P<0.001. PRUNE2, prune homolog 2 with BCH domain; sh, short hairpin; CRC, colorectal cancer; OD, optical density; ctrl, control; NC, negative control.

Figure 3
Figure 3

Effect of PRUNE2 on colony formation and invasion in CRC cells. (A) Colony formation of CRC cells was assessed by staining with crystal violet. (B) Cell invasion was detected using Transwell assay. Representative images are shown. Magnification, x200. Scale bar, 100 µm. (C) Number of stained colonies in CRC cells. (D) Number of invaded cells was counted. Data are presented as the mean ± SD (n=3). Data were analyzed using one-way ANOVA. **P<0.01, ***P<0.001 vs. normal; ###P<0.001. PRUNE2, prune homolog 2 with BCH domain; CRC, colorectal cancer; ctrl, control; sh, short hairpin; NC, negative control.

Figure 4
Figure 4

Effect of PRUNE2 on CRC cell cycle progression and apoptosis. (A) Flow cytometric analysis of cell cycle progression. (B) Quantitative results of cell cycle distribution. (C) Apoptosis (right quadrants) in CRC cells was detected by flow cytometry with an Annexin V-APC/PI double staining kit. The x-axis represents APC-stained cells; y-axis represents PI-stained cells. (D) Ratio of apoptotic cells among CRC cells. Data are presented as the mean ± SD (n=3). Data were analyzed using one-way ANOVA. *P<0.05, **P<0.01 and ***P<0.001 vs. normal; #P<0.05, ##P<0.01 and ###P<0.001 vs. ctrl; &P<0.05 and &&&P<0.001 vs. shRNA-NC. PRUNE2, prune homolog 2 with BCH domain; CRC, colorectal cancer; ctrl, control; sh, short hairpin; NC, negative control.

Figure 5
Figure 5

Effect of PRUNE2 on expression of apoptotic markers in colorectal cells. (A) Western blot analysis of expression levels of apoptotic (Bax, caspase-3 and caspase-9) and antiapoptotic proteins (Bcl-2 and Cyclin D1) with β-actin as a loading control. (B) Quantitative results of Bcl-2, Bax, caspase-3, caspase-9 and cyclin D1 protein levels relative to β-actin. Data are presented as the mean ± SD (n=3). Data were analyzed using one-way ANOVA. *P<0.05, **P<0.01 and ***P<0.001 vs. normal; #P<0.05, ##P<0.01 and ###P<0.001. PRUNE2, prune homolog 2 with BCH domain; ctrl, control; sh, short hairpin; NC, negative control.

Figure 6
Figure 6

PRUNE2 overexpression inhibits tumorigenic ability of SW620 cells in vivo. (A) Expression levels of PRUNE2 in NC-GFP-SW620 and PRUNE2-GFP-SW620 cells were measured by reverse transcription-quantitative PCR. (B) Representative images of mouse xenograft tumors derived from NC-GFP-SW620 and PRUNE2-GFP-SW620 groups. Subcutaneous tumors were collected 32 days after inoculation. (C) Tumor weight was measured. Data were analyzed using Student's t-test. (D) Growth curves (volume) of xenograft tumors in NC-GFP-SW620 and PRUNE2-GFP-SW620 groups at 0, 3, 6, 9, 12, 15 and 18 days after emergence of the subcutaneous tumors. Data were analyzed using two-way ANOVA. (E) Fluorescence of cell inoculation sites in mice were observed using an In Vivo Bioluminescence imaging system at 5, 10, 15, 20 and 25 days after inoculation. Data are presented as the mean ± SD (n=6). ***P<0.001 vs. NC-GFP-SW620. PRUNE2, prune homolog 2 with BCH domain; NC, negative control; p, pico; sr, steradian.

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Grants and funding

Funding:

The present study was supported by the National Natural Science Foundation of China (grant no. 81860522), Yunnan Health Training Project of High Level Talents (grant no. H-2018039), Joint Foundation of Kunming Medical University and Yunnan Provincial Science and Technology Department (grant nos. 202001AY070001-114 and 2019FE001-121), Internal Division of Yunnan Provincial Health Commission (grant no. 2018NS0263) and Clinical Medical Center of Yunnan Provincial Health Commission (grant no. 2020LCZXKF-XH03).