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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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miR-31 suppresses FIH1 and MCM2 expression in NPC cells. (a) Protein expression of FIH1 and MCM2 proteins were reduced in the C666-1 cells transfected with miR-31 when compared with controls (Right panel). By qRT-PCR, no significant changes of FIH1 and MCM2 transcripts were found in the miR-31-transfected C666-1 cells (Left panel). (b) Luciferase reporter assay showing the effects of miR-31 on 3’ untranslated region (3’UTR) of FIH1 and MCM2 mRNA. Luciferase activity was normalized by the renilla luciferase control. The binding of miR-31 on 3’UTR of FIH1 and MCM2 significantly decreased the luciferase activity. As a control, reporter vector carrying miR-31 complementary sequence in the 3’ UTR were also constructed (miR-31) which upon binding showed a near complete abolishment of miR-31 luciferase activity. Three independent experiments with mean ± SEM. Student-t test was used for statistical significance, with a p-value of less than 0.05 was considered significant (**p < 0.01, ***p < 0.001).
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Figure 5: miR-31 suppresses FIH1 and MCM2 expression in NPC cells. (a) Protein expression of FIH1 and MCM2 proteins were reduced in the C666-1 cells transfected with miR-31 when compared with controls (Right panel). By qRT-PCR, no significant changes of FIH1 and MCM2 transcripts were found in the miR-31-transfected C666-1 cells (Left panel). (b) Luciferase reporter assay showing the effects of miR-31 on 3’ untranslated region (3’UTR) of FIH1 and MCM2 mRNA. Luciferase activity was normalized by the renilla luciferase control. The binding of miR-31 on 3’UTR of FIH1 and MCM2 significantly decreased the luciferase activity. As a control, reporter vector carrying miR-31 complementary sequence in the 3’ UTR were also constructed (miR-31) which upon binding showed a near complete abolishment of miR-31 luciferase activity. Three independent experiments with mean ± SEM. Student-t test was used for statistical significance, with a p-value of less than 0.05 was considered significant (**p < 0.01, ***p < 0.001).

Mentions: To investigate the mechanism by which miR-31 suppressed the tumor cell growth in NPC, we validated a number of candidate targets of miR-31 which are reported previously or predicated by the TargetScan and miRanda database. We found that miR-31 expressed did not inhibit the expression of NIK, E2F2, RDX, RhoA, MCM7 in C666-1 cells (Additional file 3: Figure S3). Only FIH1 and MCM2 expression was obviously repressed by the miR-31 in NPC cells (Figure 5a). Overexpression of these two proteins was commonly found in the NPC tumor lines (Additional file 4: Figure S4). By luciferase reporter assay, FIH1 and MCM2 were further confirmed to be direct targets of miR-31 in C666-1. The binding of miR-31 to the 3’ UTR of these genes markedly inhibited luciferase activity (Figure 5b). As shown in Figure 5, miR-31 highly suppressed the expression of MCM2 and FIH1 in NPC cells. The finding confirmed FIH1 and MCM2 are direct targets of miR-31 in NPC. MCM2 is a well-known component of the minichromosome maintenance (MCM) proteins 2–7 complex which plays crucial roles in DNA replication licensing. The important role of MCM2 in tumorigenesis has also been demonstrated in our previous report [18]. In this study, we also knocked down the expression of MCM2 in NPC C666-1 cells by siRNAs (Figure 6a). Significant growth inhibition of the C666-1 cells with MCM2 depletion was observed (Figure 6b). It indicated that miR-31 may modulate NPC cell growth via repressing MCM2 expression. To further explore whether FIH1 is the target associated with the tumor suppressor function of miR-31, we knocked down the expression of FIH1 by siRNAs in C666-1 cells and assessed its effects on growth inhibition (Figure 7a). As shown in Figure 7b, by WST-1 assay, the proliferation of C666-1 cells was significantly inhibited by the treatment of siRNAs targeting FIH1. Furthermore, we also found that FIH1 knockdown enhanced Ser15 phosphorylation of p53 and up-regulated p21 expression (Figure 7c). The finding confirmed FIH1 function in the suppression of p53 activity as reported previously [19]. Since a majority of NPC contains the wild-type p53, down-regulation of miR-31 is believed to be an important mechanism for impairing p53 tumor suppressor function in this EBV-associated cancer.


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)

miR-31 suppresses FIH1 and MCM2 expression in NPC cells. (a) Protein expression of FIH1 and MCM2 proteins were reduced in the C666-1 cells transfected with miR-31 when compared with controls (Right panel). By qRT-PCR, no significant changes of FIH1 and MCM2 transcripts were found in the miR-31-transfected C666-1 cells (Left panel). (b) Luciferase reporter assay showing the effects of miR-31 on 3’ untranslated region (3’UTR) of FIH1 and MCM2 mRNA. Luciferase activity was normalized by the renilla luciferase control. The binding of miR-31 on 3’UTR of FIH1 and MCM2 significantly decreased the luciferase activity. As a control, reporter vector carrying miR-31 complementary sequence in the 3’ UTR were also constructed (miR-31) which upon binding showed a near complete abolishment of miR-31 luciferase activity. Three independent experiments with mean ± SEM. Student-t test was used for statistical significance, with a p-value of less than 0.05 was considered significant (**p < 0.01, ***p < 0.001).
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Related In: Results  -  Collection

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Figure 5: miR-31 suppresses FIH1 and MCM2 expression in NPC cells. (a) Protein expression of FIH1 and MCM2 proteins were reduced in the C666-1 cells transfected with miR-31 when compared with controls (Right panel). By qRT-PCR, no significant changes of FIH1 and MCM2 transcripts were found in the miR-31-transfected C666-1 cells (Left panel). (b) Luciferase reporter assay showing the effects of miR-31 on 3’ untranslated region (3’UTR) of FIH1 and MCM2 mRNA. Luciferase activity was normalized by the renilla luciferase control. The binding of miR-31 on 3’UTR of FIH1 and MCM2 significantly decreased the luciferase activity. As a control, reporter vector carrying miR-31 complementary sequence in the 3’ UTR were also constructed (miR-31) which upon binding showed a near complete abolishment of miR-31 luciferase activity. Three independent experiments with mean ± SEM. Student-t test was used for statistical significance, with a p-value of less than 0.05 was considered significant (**p < 0.01, ***p < 0.001).
Mentions: To investigate the mechanism by which miR-31 suppressed the tumor cell growth in NPC, we validated a number of candidate targets of miR-31 which are reported previously or predicated by the TargetScan and miRanda database. We found that miR-31 expressed did not inhibit the expression of NIK, E2F2, RDX, RhoA, MCM7 in C666-1 cells (Additional file 3: Figure S3). Only FIH1 and MCM2 expression was obviously repressed by the miR-31 in NPC cells (Figure 5a). Overexpression of these two proteins was commonly found in the NPC tumor lines (Additional file 4: Figure S4). By luciferase reporter assay, FIH1 and MCM2 were further confirmed to be direct targets of miR-31 in C666-1. The binding of miR-31 to the 3’ UTR of these genes markedly inhibited luciferase activity (Figure 5b). As shown in Figure 5, miR-31 highly suppressed the expression of MCM2 and FIH1 in NPC cells. The finding confirmed FIH1 and MCM2 are direct targets of miR-31 in NPC. MCM2 is a well-known component of the minichromosome maintenance (MCM) proteins 2–7 complex which plays crucial roles in DNA replication licensing. The important role of MCM2 in tumorigenesis has also been demonstrated in our previous report [18]. In this study, we also knocked down the expression of MCM2 in NPC C666-1 cells by siRNAs (Figure 6a). Significant growth inhibition of the C666-1 cells with MCM2 depletion was observed (Figure 6b). It indicated that miR-31 may modulate NPC cell growth via repressing MCM2 expression. To further explore whether FIH1 is the target associated with the tumor suppressor function of miR-31, we knocked down the expression of FIH1 by siRNAs in C666-1 cells and assessed its effects on growth inhibition (Figure 7a). As shown in Figure 7b, by WST-1 assay, the proliferation of C666-1 cells was significantly inhibited by the treatment of siRNAs targeting FIH1. Furthermore, we also found that FIH1 knockdown enhanced Ser15 phosphorylation of p53 and up-regulated p21 expression (Figure 7c). The finding confirmed FIH1 function in the suppression of p53 activity as reported previously [19]. Since a majority of NPC contains the wild-type p53, down-regulation of miR-31 is believed to be an important mechanism for impairing p53 tumor suppressor function in this EBV-associated cancer.

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