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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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Inactivation of miR-31 in NPC. (a) Homozygous deletions of miR-31, adjacent markers (−2mb, −1mb, +1mb, +2mb, +5mb) and loci (LOC402359, CDKN2A/p16, DMRTA1) in EBV-positive NPC tumor lines were detected by PCR. The location of miR-31 and adjacent markers on chromosome 9p21.3 was shown in the right panel. Homozygous deletions of miR-31 were detected in X1915 and X99186. (b) Methylation status of 5’ CpG islands of miR-31 in NPC tumor lines was examined by bisulfite sequencing. The locations of miR-31 and its host gene LOC554202 were indicated. Dense methylation of 5’CpG islands were detected in the C666-1 cell lines and 3 xenografts (C17, X2117 and X666). No methylation were observed in the normal control, NP69. (c) Detection of promoter hypermethylation of NPC cell lines and primary tumors by MSP. M: methylated allele; U: unmethylated alleles. (d) The restoration of miR-31 transcription in 5’-aza-2’deoxycytidine (5-Aza-dC) treated C666-1 was detected by quantitative RT-PCR analysis. By MSP, unmethylated alleles of miR-31 were found in the 5-Aza-dC-treated C666-1 cells.
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Figure 2: Inactivation of miR-31 in NPC. (a) Homozygous deletions of miR-31, adjacent markers (−2mb, −1mb, +1mb, +2mb, +5mb) and loci (LOC402359, CDKN2A/p16, DMRTA1) in EBV-positive NPC tumor lines were detected by PCR. The location of miR-31 and adjacent markers on chromosome 9p21.3 was shown in the right panel. Homozygous deletions of miR-31 were detected in X1915 and X99186. (b) Methylation status of 5’ CpG islands of miR-31 in NPC tumor lines was examined by bisulfite sequencing. The locations of miR-31 and its host gene LOC554202 were indicated. Dense methylation of 5’CpG islands were detected in the C666-1 cell lines and 3 xenografts (C17, X2117 and X666). No methylation were observed in the normal control, NP69. (c) Detection of promoter hypermethylation of NPC cell lines and primary tumors by MSP. M: methylated allele; U: unmethylated alleles. (d) The restoration of miR-31 transcription in 5’-aza-2’deoxycytidine (5-Aza-dC) treated C666-1 was detected by quantitative RT-PCR analysis. By MSP, unmethylated alleles of miR-31 were found in the 5-Aza-dC-treated C666-1 cells.

Mentions: Homozygous deletion of p16/CDNK2B locus on 9p21.3 was previously reported and identified by aCGH analysis in 3 PDXs (xeno-2117, xeno-1915 and xeno-99186) (Additional file 2: Figure S2) [11,16]. It is suspected that the down-regulation of miR-31 in these tumors is due to complete loss of the miR-31 allele. However, detailed mapping of the deletion regions by multiple PCR analysis demonstrated that miR-31 locus was deleted in only 2 out of 6 xenografts (33.3%; xeno-1915 and xeno-99186) (Figure 2a). The expression of miR-31 was regulated by the promoter of its host gene LOC554202 (Figure 2a). Hypermethylation of the LOC554202-associated 5’CpG islands can lead to the transcription silencing of miR-31[17]. In the 4 NPC tumor lines with miR-31 down-regulation, heavy methylation of the 5’CpG islands was detected by bisulfite sequencing and methylation-specific PCR (Figure 2b and 2c). Notably, promoter hypermethylation of miR-31 was commonly found in the primary tumors (14/16; 87%) (Figure 2b). As shown in Figure 2d, re-expression of miR-31 and unmethylated allele were detected in the C666-1 cells treated with a DNA methylation inhibitor, 5’-aza-2’deoxycytidine (5-Aza-dC). The findings indicated that homozygous deletion and methylation of 5’ CpG islands are the major mechanisms for miR-31 inactivation in NPC.


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)

Inactivation of miR-31 in NPC. (a) Homozygous deletions of miR-31, adjacent markers (−2mb, −1mb, +1mb, +2mb, +5mb) and loci (LOC402359, CDKN2A/p16, DMRTA1) in EBV-positive NPC tumor lines were detected by PCR. The location of miR-31 and adjacent markers on chromosome 9p21.3 was shown in the right panel. Homozygous deletions of miR-31 were detected in X1915 and X99186. (b) Methylation status of 5’ CpG islands of miR-31 in NPC tumor lines was examined by bisulfite sequencing. The locations of miR-31 and its host gene LOC554202 were indicated. Dense methylation of 5’CpG islands were detected in the C666-1 cell lines and 3 xenografts (C17, X2117 and X666). No methylation were observed in the normal control, NP69. (c) Detection of promoter hypermethylation of NPC cell lines and primary tumors by MSP. M: methylated allele; U: unmethylated alleles. (d) The restoration of miR-31 transcription in 5’-aza-2’deoxycytidine (5-Aza-dC) treated C666-1 was detected by quantitative RT-PCR analysis. By MSP, unmethylated alleles of miR-31 were found in the 5-Aza-dC-treated C666-1 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Inactivation of miR-31 in NPC. (a) Homozygous deletions of miR-31, adjacent markers (−2mb, −1mb, +1mb, +2mb, +5mb) and loci (LOC402359, CDKN2A/p16, DMRTA1) in EBV-positive NPC tumor lines were detected by PCR. The location of miR-31 and adjacent markers on chromosome 9p21.3 was shown in the right panel. Homozygous deletions of miR-31 were detected in X1915 and X99186. (b) Methylation status of 5’ CpG islands of miR-31 in NPC tumor lines was examined by bisulfite sequencing. The locations of miR-31 and its host gene LOC554202 were indicated. Dense methylation of 5’CpG islands were detected in the C666-1 cell lines and 3 xenografts (C17, X2117 and X666). No methylation were observed in the normal control, NP69. (c) Detection of promoter hypermethylation of NPC cell lines and primary tumors by MSP. M: methylated allele; U: unmethylated alleles. (d) The restoration of miR-31 transcription in 5’-aza-2’deoxycytidine (5-Aza-dC) treated C666-1 was detected by quantitative RT-PCR analysis. By MSP, unmethylated alleles of miR-31 were found in the 5-Aza-dC-treated C666-1 cells.
Mentions: Homozygous deletion of p16/CDNK2B locus on 9p21.3 was previously reported and identified by aCGH analysis in 3 PDXs (xeno-2117, xeno-1915 and xeno-99186) (Additional file 2: Figure S2) [11,16]. It is suspected that the down-regulation of miR-31 in these tumors is due to complete loss of the miR-31 allele. However, detailed mapping of the deletion regions by multiple PCR analysis demonstrated that miR-31 locus was deleted in only 2 out of 6 xenografts (33.3%; xeno-1915 and xeno-99186) (Figure 2a). The expression of miR-31 was regulated by the promoter of its host gene LOC554202 (Figure 2a). Hypermethylation of the LOC554202-associated 5’CpG islands can lead to the transcription silencing of miR-31[17]. In the 4 NPC tumor lines with miR-31 down-regulation, heavy methylation of the 5’CpG islands was detected by bisulfite sequencing and methylation-specific PCR (Figure 2b and 2c). Notably, promoter hypermethylation of miR-31 was commonly found in the primary tumors (14/16; 87%) (Figure 2b). As shown in Figure 2d, re-expression of miR-31 and unmethylated allele were detected in the C666-1 cells treated with a DNA methylation inhibitor, 5’-aza-2’deoxycytidine (5-Aza-dC). The findings indicated that homozygous deletion and methylation of 5’ CpG islands are the major mechanisms for miR-31 inactivation in NPC.

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