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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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Related in: MedlinePlus

Stable ectopic miR-31 expression suppresses the anchorage-independent growth and in vivo tumorigenicity of NPC cells. (a) Expression of miR-31 was demonstrated in the stably miR-31-transfected C666-1 cell clones (miR31#1 and miR31#2) by quantitative RT-PCR. The immortalized normal nasopharyngeal epithelial cells NP69 was included as control. (b) In the two stably miR-31-transfected NPC cell clones (miR31#1 and miR31#2), obvious growth inhibition was demonstrated by WST-1 assay. (c) Stable expression of miR-31 inhibits the anchorage-independent growth of C666-1 cells. Obviously reduction in number and size of colonies in the stable miR-31-expressing cells were demonstrated by soft agar assay. (d)In vivo tumorgenic assay in nude mice showed that tumors formed in the sites implanted with C666-1 cells expressing miR-31 (miR-31#1 and miR-31#2) were consistently smaller than those implanted with vector controls. Photographs showing the nude mice (upper row) inoculated with stable clones (vector, miR-31 #1, #2) and tumors extracted (bottom row) on day 38 after inoculation were also shown. Four nude mice were used in the experiment and data was shown with mean ± SEM. Student-t test was used for statistical significance, with a p-value of less than 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).
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Figure 4: Stable ectopic miR-31 expression suppresses the anchorage-independent growth and in vivo tumorigenicity of NPC cells. (a) Expression of miR-31 was demonstrated in the stably miR-31-transfected C666-1 cell clones (miR31#1 and miR31#2) by quantitative RT-PCR. The immortalized normal nasopharyngeal epithelial cells NP69 was included as control. (b) In the two stably miR-31-transfected NPC cell clones (miR31#1 and miR31#2), obvious growth inhibition was demonstrated by WST-1 assay. (c) Stable expression of miR-31 inhibits the anchorage-independent growth of C666-1 cells. Obviously reduction in number and size of colonies in the stable miR-31-expressing cells were demonstrated by soft agar assay. (d)In vivo tumorgenic assay in nude mice showed that tumors formed in the sites implanted with C666-1 cells expressing miR-31 (miR-31#1 and miR-31#2) were consistently smaller than those implanted with vector controls. Photographs showing the nude mice (upper row) inoculated with stable clones (vector, miR-31 #1, #2) and tumors extracted (bottom row) on day 38 after inoculation were also shown. Four nude mice were used in the experiment and data was shown with mean ± SEM. Student-t test was used for statistical significance, with a p-value of less than 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).

Mentions: To further explore whether ectopic expression of miR-31 affects anchorage-independent growth in vitro and tumor growth in vivo, we have established 2 stably transfected C666-1 cell lines (miR-31#1 and miR-31#2) expressing different amount of miR31 (Figure 4a). As shown in Figure 4b, significant suppression in cell proliferation was confirmed in both two stably-miR-31 expressing cells. The stably transfected C666-1 cells with miR-31 showed obvious repression of anchorage-independent growth. The cells expressing miR-31 displayed much fewer and smaller colonies in the soft agar compared with controls (Figure 4c). To investigate the effect of miR-31 on in vivo tumor growth, the stably miR-31-expressing C666-1 cells and controls were subcutaneously injected into nude mice. As shown in Figure 4d, tumor growth by miR-31-expressing cells was significantly inhibited when compared to those transfected with control vector. Notably, the stronger inhibitory effects on in vitro and in vivo tumor growth were found in the miR-31#2 clone which expressed higher level of miR-31. Our study demonstrated a dose-dependent tumor suppressor effect of miR-31 in 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)

Stable ectopic miR-31 expression suppresses the anchorage-independent growth and in vivo tumorigenicity of NPC cells. (a) Expression of miR-31 was demonstrated in the stably miR-31-transfected C666-1 cell clones (miR31#1 and miR31#2) by quantitative RT-PCR. The immortalized normal nasopharyngeal epithelial cells NP69 was included as control. (b) In the two stably miR-31-transfected NPC cell clones (miR31#1 and miR31#2), obvious growth inhibition was demonstrated by WST-1 assay. (c) Stable expression of miR-31 inhibits the anchorage-independent growth of C666-1 cells. Obviously reduction in number and size of colonies in the stable miR-31-expressing cells were demonstrated by soft agar assay. (d)In vivo tumorgenic assay in nude mice showed that tumors formed in the sites implanted with C666-1 cells expressing miR-31 (miR-31#1 and miR-31#2) were consistently smaller than those implanted with vector controls. Photographs showing the nude mice (upper row) inoculated with stable clones (vector, miR-31 #1, #2) and tumors extracted (bottom row) on day 38 after inoculation were also shown. Four nude mice were used in the experiment and data was shown with mean ± SEM. Student-t test was used for statistical significance, with a p-value of less than 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Stable ectopic miR-31 expression suppresses the anchorage-independent growth and in vivo tumorigenicity of NPC cells. (a) Expression of miR-31 was demonstrated in the stably miR-31-transfected C666-1 cell clones (miR31#1 and miR31#2) by quantitative RT-PCR. The immortalized normal nasopharyngeal epithelial cells NP69 was included as control. (b) In the two stably miR-31-transfected NPC cell clones (miR31#1 and miR31#2), obvious growth inhibition was demonstrated by WST-1 assay. (c) Stable expression of miR-31 inhibits the anchorage-independent growth of C666-1 cells. Obviously reduction in number and size of colonies in the stable miR-31-expressing cells were demonstrated by soft agar assay. (d)In vivo tumorgenic assay in nude mice showed that tumors formed in the sites implanted with C666-1 cells expressing miR-31 (miR-31#1 and miR-31#2) were consistently smaller than those implanted with vector controls. Photographs showing the nude mice (upper row) inoculated with stable clones (vector, miR-31 #1, #2) and tumors extracted (bottom row) on day 38 after inoculation were also shown. Four nude mice were used in the experiment and data was shown with mean ± SEM. Student-t test was used for statistical significance, with a p-value of less than 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).
Mentions: To further explore whether ectopic expression of miR-31 affects anchorage-independent growth in vitro and tumor growth in vivo, we have established 2 stably transfected C666-1 cell lines (miR-31#1 and miR-31#2) expressing different amount of miR31 (Figure 4a). As shown in Figure 4b, significant suppression in cell proliferation was confirmed in both two stably-miR-31 expressing cells. The stably transfected C666-1 cells with miR-31 showed obvious repression of anchorage-independent growth. The cells expressing miR-31 displayed much fewer and smaller colonies in the soft agar compared with controls (Figure 4c). To investigate the effect of miR-31 on in vivo tumor growth, the stably miR-31-expressing C666-1 cells and controls were subcutaneously injected into nude mice. As shown in Figure 4d, tumor growth by miR-31-expressing cells was significantly inhibited when compared to those transfected with control vector. Notably, the stronger inhibitory effects on in vitro and in vivo tumor growth were found in the miR-31#2 clone which expressed higher level of miR-31. Our study demonstrated a dose-dependent tumor suppressor effect of miR-31 in 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