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. 2016 Feb 1;11(2):e0148223.
doi: 10.1371/journal.pone.0148223. eCollection 2016.

Nuclear Multidrug Resistance-Related Protein 1 Is Highly Associated with Better Prognosis of Human Mucoepidermoid Carcinoma through the Suppression of Cell Proliferation, Migration and Invasion

Bo-Lei Cai  1 , Yan Li  2 , Liang-Liang Shen  3 , Jin-Long Zhao  1 , Yuan Liu  4 , Jun-Zheng Wu  5 , Yan-Pu Liu  1 , Bo Yu  1
Affiliations

Nuclear Multidrug Resistance-Related Protein 1 Is Highly Associated with Better Prognosis of Human Mucoepidermoid Carcinoma through the Suppression of Cell Proliferation, Migration and Invasion

Bo-Lei Cai et al. PLoS One. .

Abstract

Objectives: Multidrug resistance-related protein 1 (MRP1) overexpression is a well acknowledged predictor of poor response to chemotherapy, but MRP1 also correlated to better prognosis in some reports, especially for patients not pretreated with chemotherapy. In our previous study, we found nuclear translocation of MRP1 in mucoepidermoid carcinoma (MEC) for the first time. The purpose of this study was to further investigate the function of nuclear MRP1 in MEC.

Materials and methods: Human MEC tissue samples of 125 patients were selected and stained using immunohistochemistry. The expression level of total MRP1/nuclear MRP1 of each sample was evaluated by expression index (EI) which was scored using both qualitative and quantitative analysis. The correlations between the clinicopathologic parameters and the EI of nuclear MRP1 were analyzed using Spearman's rank correlation analysis, respectively. The effects of RNAi-mediated downregulation of nuclear MRP1 on MEC cells were assessed using flow cytometric analysis, MTT assay, plate colony formation assay, transwell invasion assay and monolayer wound healing assay.

Results: In this study, we found the EI of nuclear MRP1 was negatively correlated to the pathologic grading (r = -0.498, P<0.01)/clinical staging (r = -0.41, P<0.01)/tumor stage (r = -0.28, P = 0.02)/nodal stage (r = -0.29, P<0.01) of MEC patients. The RNAi-mediated downregulation of nuclear MRP1 further proved that the downregulation of nuclear MRP1 could increase the cell replication, growth speed, colony formation efficiency, migration and invasion ability of MEC cells.

Conclusion: Our results suggested that nuclear MRP1 is highly associated with better prognosis of human mucoepidermoid carcinoma and further study of its function mechanism would provide clues in developing new treatment modalities of MEC.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Nuclear MRP1 expression was negatively correlated to the pathologic grading of MEC.
(A): a. No MRP1 was detected in normal salivary specimens; b. the expression of MRP1 in MEC adjacent tissues is slightly higher than normal tissues but still negligible, MRP1 was only lightly stained in the cells of striated duct; c. MRP1 was strongly expressed in the nuclei and lightly expressed in the cytoplasm in low-grade MEC; d. In moderate-grade MEC tissues, MRP1 was mostly distributed in the cytoplasm and rarely expressed in the nuclei of the cells; e. In high-grade MEC tissues, MRP1 was negligible. (B): Compared with moderate-grade or low-grade MEC, total MRP1 expression of high-grade MEC was significantly lower. But the difference of total MRP1 expression between moderate-grade MEC and low-grade MEC was not significant. The nuclear MRP1 expression significantly decreased as the pathologic grade increased. The differences of nuclear MRP1 expression among different pathologic grades were all significant (P<0.001). ***P<0.001.
Fig 2
Fig 2. Nuclear MRP1 expression was negatively correlated to the clinical staging of MEC.
(A) a. The difference of total MRP1 expression among different tumor stages (P = 0.18) was not significant; b. The expression of nuclear MRP1 decreased as the T stage upgraded. Furthermore, nuclear MRP1 expression in T1 was significantly higher than that in T4. (B) a. The total MRP1 expression among different nodal stages was not significantly different (P = 0.13); b. The nuclear MRP1 expression in N0 was significantly higher than that in N1 (P<0.05) and N2 (P<0.01). (C) Total MRP1 expression between stage 1 and stage 4 (P<0.01), between stage 2 and stage 4 (P<0.05) were significantly different. Nuclear MRP1 expression among different clinical stages was significantly different (P<0.001). Nuclear MRP1 expression between stage 1 and stage 4 (P<0.001), between stage 1 and stage 3 (P<0.01), between stage 2 and stage 4 (P<0.01) were all significantly different. *P<0.05, **P<0.01, ***P<0.001.
Fig 3
Fig 3. Down-regulation of nuclear MRP1 motivated the growth of MEC cells.
The expression of nuclear MRP1 is down-regulated by short-hairpin RNA (shRNA). The multidrug-resistant MC3/5FU cells were transfected with plasmids containing an MRP1 specific shRNA and a non-specific control shRNA, the resulting clones were MC3/5FU—S (short for S) and MC3/5FU-NS (short for NS). (A) Flow cytometry was used to determine the altered cell cycle of MEC cells as nuclear MRP1 decreased. Compared with MC3/5FU cells (49.9±1.01%) and MC3/5FU-NS cells (48.76±0.63%), the percentage of MC3/5FU-S cells in G0/G1 phase significantly decreased to 44.03±1.04% (P<0.01, n = 4). Compared with MC3/5FU (15.25±1.25%) and MC3/5FU-NS (16.61±1.04%), the percentage of MC3/5FU-S cells in G2/M phase was also significantly decreased to 11.20±0.77% (P<0.05, n = 4). Also compared with MC3/5FU (34.70±1.19%) and MC3/5FU-NS (34.26±0.63%), the cell number of MC3/5FU-S cells in S phase significantly increased to 43.63±1.49% (P<0.01, n = 4). (B) The growth curves showed that the viability of MC3/5FU-S cells was obviously higher than that of MC3/5FU cells and MC3/5FU-NS cells during the exponential growth phase between day 2 and day 7. (C) Compared with MC3/5FU (78.67±5.49) and MC3/5FU-NS cells (72.67±2.91), plate colony formation assay showed that colony number of MC3/5FU-S (165.0±9.07) cells substantially increased (P<0.01, n = 3). (D) After 14 days of culturing, the colonies of MEC cells were fixed then stained. The staining showed that not only the number of MC3/5FU-S colonies was obviously more substantial than other groups but also the size of the MC3/5FU-S colonies was larger in relation to other groups. **P<0.01, ***P<0.001.
Fig 4
Fig 4. Down-regulation of nuclear MRP1 increased the migration and invasion of MC3/5FU cells.
(A) The cells that migrated to the lower surface of the filter were stained with Giemsa. The Transwell invasion assay showed that more MC3/5FU-S cells invaded through the filter than the other two groups. (B) The average number of cells in 6 high power fields was 22.0±2.80 for the MC3/5FU-S cells, which is significantly higher than the MC3/5FU cells (11.25±1.49, P = 0.015, n = 3) and the MC3/5FU-NS cells (0.50±1.19, P<0.01, n = 3). No significant difference was found between MC3/5FU cells and MC3/5FU-NS cells. (C)The migration distance in the wound was calculated by the formula below: cell free area at 0 hour—cell free area at 24 hour. (D) The migration distance of MC3/5FU-S cells was 257.0±21.44μm, which is significantly higher than that of MC3/5FU cells (78.09±5.29μm, P<0.01, n = 3) and the MC3/5FU-NS cells (2.29±8.84μm, P<0.01, n = 3). No significant difference was found between MC3/5FU cells and MC3/5FU-NS cells.
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This work was supported by National Natural Science Research Program of China (81502338) to Dr. Bo-Lei Cai; and National Natural Science Research Program of China (31501036) to Dr. Yan Li. The authors also gratefully acknowledge the financial support from China Scholarship Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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