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miR-31 is consistently inactivated in EBV-associated nasopharyngeal carcinoma and contributes to its tumorigenesis.

Cheung CC, Chung GT, Lun SW, To KF, Choy KW, Lau KM, Siu SP, Guan XY, Ngan RK, Yip TT, Busson P, Tsao SW, Lo KW - Mol. Cancer (2014)

Bottom Line: Among the recurrent genetic alterations defined, loss of 9p21.3 is the most frequent early event in the tumorigenesis of EBV-associated NPC.Dramatic reduction of in vitro anchorage-independent growth and in vivo tumorigenic potential were demonstrated in the stable clones expressing miR-31.The inactivation of miR-31 may contribute to the early development of NPC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomical and Cellular Pathology, State Key Laboratory in Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, People's Republic of China. kwlo@cuhk.edu.hk.

ABSTRACT

Background: As a distinctive type of head and neck cancers, nasopharyngeal carcinoma (NPC) is genesis from the clonal Epstein-Barr virus (EBV)-infected nasopharyngeal epithelial cells accumulated with multiple genetic lesions. Among the recurrent genetic alterations defined, loss of 9p21.3 is the most frequent early event in the tumorigenesis of EBV-associated NPC. In addition to the reported CDKN2A/p16, herein, we elucidated the role of a miRNA, miR-31 within this 9p21.3 region as NPC-associated tumor suppressor.

Methods: The expression and promoter methylation of miR-31 were assessed in a panel of NPC tumor lines and primary tumors. Its in vitro and in vivo tumor suppression function was investigated through the ectopic expression of miR-31 in NPC cells. We also determined the miR-31 targeted genes and its involvement in the growth in NPC.

Results: Downregulation of miR-31 expression was detected in almost all NPC cell line, patient-derived xenografts (PDXs) and primary tumors. Both homozygous deletion and promoter hypermethylation were shown to be major mechanisms for miR-31 silencing in this cancer. Strikingly, loss of miR-31 was also obviously observed in the dysplastic lesions of nasopharynx. Restoration of miR-31 in C666-1 cells inhibited the cell proliferation, colony-forming and migratory capacities. Dramatic reduction of in vitro anchorage-independent growth and in vivo tumorigenic potential were demonstrated in the stable clones expressing miR-31. Furthermore, we proved that miR-31 suppressed the NPC cell growth via targeting FIH1 and MCM2.

Conclusions: The findings provide strong evidence to support miR-31 as a new NPC-associated tumor suppressor on 9p21.3 region. The inactivation of miR-31 may contribute to the early development of NPC.

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Effect of miR-31 expression on cell growth and migration of NPC cells. (a) By WST-1 assay, significant growth inhibition was detected in the C666-1 cell transfected with miR-31 when compared with negative control. Data shown were taken from 5 independent experiments with mean ± SEM. (b) Expression of miR-31 significantly inhibited the colony-forming ability of C666-1 cells (**p < 0.01). Representative photographs of colonies formed in each treatment were shown. Colonies formed were stained in blue by Giemsa stain. (c) Flow cytometry analysis revealed significant reduction of the percentage of cells undergoing S phase in miR-31-transfected C666-1 cells (*p < 0.05). (d) By wound healing assay, significant reduction of the migration ability of miR-31-expressing C666-1 (**P < 0.01). Representative photographs of wound healing progress of C666-1 cells transfected with negative control and miR-31 at 0 hour and 30 hours were shown.
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Figure 3: Effect of miR-31 expression on cell growth and migration of NPC cells. (a) By WST-1 assay, significant growth inhibition was detected in the C666-1 cell transfected with miR-31 when compared with negative control. Data shown were taken from 5 independent experiments with mean ± SEM. (b) Expression of miR-31 significantly inhibited the colony-forming ability of C666-1 cells (**p < 0.01). Representative photographs of colonies formed in each treatment were shown. Colonies formed were stained in blue by Giemsa stain. (c) Flow cytometry analysis revealed significant reduction of the percentage of cells undergoing S phase in miR-31-transfected C666-1 cells (*p < 0.05). (d) By wound healing assay, significant reduction of the migration ability of miR-31-expressing C666-1 (**P < 0.01). Representative photographs of wound healing progress of C666-1 cells transfected with negative control and miR-31 at 0 hour and 30 hours were shown.

Mentions: To explore the tumor suppressor function of miR-31 in NPC cells, the C666-1 cells, in which miR-31 transcripts are downregulated, were transiently transfected with miR-31 mimic or corresponding control. By WST-1 assay, we demonstrated that ectopic expression of miR-31 significantly inhibited the cell proliferation and viability of C666-1 cells (Figure 3a). The miR-31 expression also suppressed the clone formation ability of NPC cells. The number of colonies significantly reduced by 66% in miR-31-transfected C666-1 when compared to that of negative control in the colony formation assay (Figure 3b). By flow cytometry, a significant decrease in the percentage of C666-1 cells undergoing S-phase in cell cycle was detected after transient transfection of miR-31 mimic (Figure 3c). In addition, a decrease of 35% in wound recovery area was measured in miR-31-transfected C666-1 when compared to that of negative control (Figure 3d). This implied that miR-31 expression also inhibited the migratory capacity of NPC cells.


miR-31 is consistently inactivated in EBV-associated nasopharyngeal carcinoma and contributes to its tumorigenesis.

Cheung CC, Chung GT, Lun SW, To KF, Choy KW, Lau KM, Siu SP, Guan XY, Ngan RK, Yip TT, Busson P, Tsao SW, Lo KW - Mol. Cancer (2014)

Effect of miR-31 expression on cell growth and migration of NPC cells. (a) By WST-1 assay, significant growth inhibition was detected in the C666-1 cell transfected with miR-31 when compared with negative control. Data shown were taken from 5 independent experiments with mean ± SEM. (b) Expression of miR-31 significantly inhibited the colony-forming ability of C666-1 cells (**p < 0.01). Representative photographs of colonies formed in each treatment were shown. Colonies formed were stained in blue by Giemsa stain. (c) Flow cytometry analysis revealed significant reduction of the percentage of cells undergoing S phase in miR-31-transfected C666-1 cells (*p < 0.05). (d) By wound healing assay, significant reduction of the migration ability of miR-31-expressing C666-1 (**P < 0.01). Representative photographs of wound healing progress of C666-1 cells transfected with negative control and miR-31 at 0 hour and 30 hours were shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4127521&req=5

Figure 3: Effect of miR-31 expression on cell growth and migration of NPC cells. (a) By WST-1 assay, significant growth inhibition was detected in the C666-1 cell transfected with miR-31 when compared with negative control. Data shown were taken from 5 independent experiments with mean ± SEM. (b) Expression of miR-31 significantly inhibited the colony-forming ability of C666-1 cells (**p < 0.01). Representative photographs of colonies formed in each treatment were shown. Colonies formed were stained in blue by Giemsa stain. (c) Flow cytometry analysis revealed significant reduction of the percentage of cells undergoing S phase in miR-31-transfected C666-1 cells (*p < 0.05). (d) By wound healing assay, significant reduction of the migration ability of miR-31-expressing C666-1 (**P < 0.01). Representative photographs of wound healing progress of C666-1 cells transfected with negative control and miR-31 at 0 hour and 30 hours were shown.
Mentions: To explore the tumor suppressor function of miR-31 in NPC cells, the C666-1 cells, in which miR-31 transcripts are downregulated, were transiently transfected with miR-31 mimic or corresponding control. By WST-1 assay, we demonstrated that ectopic expression of miR-31 significantly inhibited the cell proliferation and viability of C666-1 cells (Figure 3a). The miR-31 expression also suppressed the clone formation ability of NPC cells. The number of colonies significantly reduced by 66% in miR-31-transfected C666-1 when compared to that of negative control in the colony formation assay (Figure 3b). By flow cytometry, a significant decrease in the percentage of C666-1 cells undergoing S-phase in cell cycle was detected after transient transfection of miR-31 mimic (Figure 3c). In addition, a decrease of 35% in wound recovery area was measured in miR-31-transfected C666-1 when compared to that of negative control (Figure 3d). This implied that miR-31 expression also inhibited the migratory capacity of NPC cells.

Bottom Line: Among the recurrent genetic alterations defined, loss of 9p21.3 is the most frequent early event in the tumorigenesis of EBV-associated NPC.Dramatic reduction of in vitro anchorage-independent growth and in vivo tumorigenic potential were demonstrated in the stable clones expressing miR-31.The inactivation of miR-31 may contribute to the early development of NPC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomical and Cellular Pathology, State Key Laboratory in Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, People's Republic of China. kwlo@cuhk.edu.hk.

ABSTRACT

Background: As a distinctive type of head and neck cancers, nasopharyngeal carcinoma (NPC) is genesis from the clonal Epstein-Barr virus (EBV)-infected nasopharyngeal epithelial cells accumulated with multiple genetic lesions. Among the recurrent genetic alterations defined, loss of 9p21.3 is the most frequent early event in the tumorigenesis of EBV-associated NPC. In addition to the reported CDKN2A/p16, herein, we elucidated the role of a miRNA, miR-31 within this 9p21.3 region as NPC-associated tumor suppressor.

Methods: The expression and promoter methylation of miR-31 were assessed in a panel of NPC tumor lines and primary tumors. Its in vitro and in vivo tumor suppression function was investigated through the ectopic expression of miR-31 in NPC cells. We also determined the miR-31 targeted genes and its involvement in the growth in NPC.

Results: Downregulation of miR-31 expression was detected in almost all NPC cell line, patient-derived xenografts (PDXs) and primary tumors. Both homozygous deletion and promoter hypermethylation were shown to be major mechanisms for miR-31 silencing in this cancer. Strikingly, loss of miR-31 was also obviously observed in the dysplastic lesions of nasopharynx. Restoration of miR-31 in C666-1 cells inhibited the cell proliferation, colony-forming and migratory capacities. Dramatic reduction of in vitro anchorage-independent growth and in vivo tumorigenic potential were demonstrated in the stable clones expressing miR-31. Furthermore, we proved that miR-31 suppressed the NPC cell growth via targeting FIH1 and MCM2.

Conclusions: The findings provide strong evidence to support miR-31 as a new NPC-associated tumor suppressor on 9p21.3 region. The inactivation of miR-31 may contribute to the early development of NPC.

Show MeSH
Related in: MedlinePlus