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Differential expression of HPV16 L2 gene in cervical cancers harboring episomal HPV16 genomes: influence of synonymous and non-coding region variations.

Mandal P, Bhattacharjee B, Das Ghosh D, Mondal NR, Roy Chowdhury R, Roy S, Sengupta S - PLoS ONE (2013)

Bottom Line: L2 (mRNA and protein) expression was evident only among cases with episomal viral genomes and L2 mRNA expression correlated significantly with E2 gene copy numbers suggesting expression from all episomal genomes.Among such cases, Asian American (AA) isolates portrayed all of the humanized codons (100%; 4-6/sample) recorded within L2, which was significantly higher (p = 2.02E-7) compared to the European (E) isolates (22.8%; none or 1-2/sample).This resulted in loss of 9 out of 14 miRNA binding sites (hsa-miR-548 family), despite the significant overexpression of miR548a-5p and miR548d-5p among such cases (28.64 and 36.25 folds, respectively), in comparison to HPV negative control samples.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal, India.

ABSTRACT
We tested the hypothesis that (i) synonymous variations within the coding regions, and (ii) variations within the non-coding regions of HPV, influence cervical cancer (CaCx) pathogenesis under the impact of intact HPV16 genomes. Whole genome sequence analysis of HPV16 isolates within 70 CaCx cases and 25 non-malignant samples revealed that synonymous variations were significantly higher within the E6 (p = 0.014), E5 (p = 0.001) and L2 (p = 0.0002) genes of HPV16 isolates within cases, compared to isolates within non-malignant samples. All of the 25 (100%) humanized codons identified within L2 ORF of the samples analyzed, were harbored by CaCx cases, while 8 out of 25 (32%) were harbored by HPV16 positive non-malignant samples (p = 3.87105E-07). L2 (mRNA and protein) expression was evident only among cases with episomal viral genomes and L2 mRNA expression correlated significantly with E2 gene copy numbers suggesting expression from all episomal genomes. Among such cases, Asian American (AA) isolates portrayed all of the humanized codons (100%; 4-6/sample) recorded within L2, which was significantly higher (p = 2.02E-7) compared to the European (E) isolates (22.8%; none or 1-2/sample). Additionally, majority of E variant isolates within cases (54/57; 94.7%) portrayed a variation (T4228C) within the short non-coding region (NCR2) between E5 and L2 genes, which portrays a weak promoter activity specific for L2 mRNA expression. This resulted in loss of 9 out of 14 miRNA binding sites (hsa-miR-548 family), despite the significant overexpression of miR548a-5p and miR548d-5p among such cases (28.64 and 36.25 folds, respectively), in comparison to HPV negative control samples. The findings exemplify the biological relevance of sequence variations in HPV16 genomes and highlight that episomal HPV16 in CaCx cases employ multiple mechanisms to sustain L2 expression, thereby justifying the potential role of L2 in such cancers, as opposed to those harboring viral integration.

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miRNA binding sites and variant nucleotide position within NCR2 of E2 intact/episomal (episomal or concomitant) HPV16 European (E) variant isolate within CaCx cases.(A) Depicts the NCR2 (nucleotide positions 4139–4236) located within 5′ UTR of L2 gene, with a single nucleotide polymorphism (SNP) at position 4228 (T to C). (B) RegRNA software based identification of fourteen miRNA binding sites within NCR2 with loss of binding sites corresponding to nine miRNAs (*) of the hsa-miR-548family due to the SNP (T4228C).
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pone-0065647-g006: miRNA binding sites and variant nucleotide position within NCR2 of E2 intact/episomal (episomal or concomitant) HPV16 European (E) variant isolate within CaCx cases.(A) Depicts the NCR2 (nucleotide positions 4139–4236) located within 5′ UTR of L2 gene, with a single nucleotide polymorphism (SNP) at position 4228 (T to C). (B) RegRNA software based identification of fourteen miRNA binding sites within NCR2 with loss of binding sites corresponding to nine miRNAs (*) of the hsa-miR-548family due to the SNP (T4228C).

Mentions: A short non-coding region (NCR2) commonly exists between the E5 and L2 open reading frames of HPVs. NCR2 is characterized by a weak promoter activity that is tightly regulated by keratinocyte differentiation and used only for transcripts encoding the minor capsid protein L2 of HPV16 [26]. Another study reported 13 transcripts of HPV16 in cervical epithelial cell line W12 (harboring episomal HPV16 genomes), of which, 6 transcripts were found to encompass the NCR2 and L2 [32]. In view of the fact that the CaCx cases harboring episomal HPV16 genomes also expressed the L2 gene, we focussed on deciphering the factors that could be associated with L2 expression in such CaCx cases. By using RegRNA (www.regrna.mbc.nctu.edu.tw/) software, we identified binding sites in the NCR2 (nt 4139–4234) of HPV16 intact isolates, corresponding to 14 human miRNAs (hsa-miR-3148,hsa-miR-3174,hsa-miR-3613-3p,hsa-miR-3916,hsa-miR-495,hsa-miR-548a-5p, hsa-miR-548b-5p, hsa-miR-548c-5p, hsa-miR-548d-5p, hsa-miR-548h-5p, hsa-miR-548i-5p, hsa-miR-548j-5p, hsa-miR-548w-5p, hsa-miR-548y-5p) (Figure 6). Such miRNA binding sites were selected on the basis of minimum free energy (MFE≤7) and hybridization score (≥140) (Table S3) as per standards normally used for formation of miRNA:mRNA hybrid. Our resequenced data revealed the occurrence of a SNP (T4228C) (Figure S1) in the NCR2 of E variant intact isolates only, which could lead to loss of 9 miRNA binding sites in the corresponding transcripts (hsa-miR-548a-5p, hsa-miR-548b-5p, hsa-miR-548c-5p, hsa-miR-548d-5p, hsa-miR-548h-5p, hsa-miR-548i-5p, hsa-miR-548j-5p, hsa-miR-548w-5p, hsa-miR-548y-5p) (Figure 6), all of which belonged to the hsa-miR-548 family of miRNAs. Interestingly, proportion of E2 intact CaCx cases (54/70, 77%) harboring SNPs in the miRNA binding sites within the NCR2 was significantly higher (p = 0.007) compared to that of non-malignant samples (12/25, 48%). Within E2 intact CaCx cases, it was also observed that none of AA the variants (0/13, 0%) harbored a SNP in the miRNA binding sites in the NCR2. Thus, no loss of miRNA binding sites in the NCR2 was observed in AA variants.


Differential expression of HPV16 L2 gene in cervical cancers harboring episomal HPV16 genomes: influence of synonymous and non-coding region variations.

Mandal P, Bhattacharjee B, Das Ghosh D, Mondal NR, Roy Chowdhury R, Roy S, Sengupta S - PLoS ONE (2013)

miRNA binding sites and variant nucleotide position within NCR2 of E2 intact/episomal (episomal or concomitant) HPV16 European (E) variant isolate within CaCx cases.(A) Depicts the NCR2 (nucleotide positions 4139–4236) located within 5′ UTR of L2 gene, with a single nucleotide polymorphism (SNP) at position 4228 (T to C). (B) RegRNA software based identification of fourteen miRNA binding sites within NCR2 with loss of binding sites corresponding to nine miRNAs (*) of the hsa-miR-548family due to the SNP (T4228C).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675152&req=5

pone-0065647-g006: miRNA binding sites and variant nucleotide position within NCR2 of E2 intact/episomal (episomal or concomitant) HPV16 European (E) variant isolate within CaCx cases.(A) Depicts the NCR2 (nucleotide positions 4139–4236) located within 5′ UTR of L2 gene, with a single nucleotide polymorphism (SNP) at position 4228 (T to C). (B) RegRNA software based identification of fourteen miRNA binding sites within NCR2 with loss of binding sites corresponding to nine miRNAs (*) of the hsa-miR-548family due to the SNP (T4228C).
Mentions: A short non-coding region (NCR2) commonly exists between the E5 and L2 open reading frames of HPVs. NCR2 is characterized by a weak promoter activity that is tightly regulated by keratinocyte differentiation and used only for transcripts encoding the minor capsid protein L2 of HPV16 [26]. Another study reported 13 transcripts of HPV16 in cervical epithelial cell line W12 (harboring episomal HPV16 genomes), of which, 6 transcripts were found to encompass the NCR2 and L2 [32]. In view of the fact that the CaCx cases harboring episomal HPV16 genomes also expressed the L2 gene, we focussed on deciphering the factors that could be associated with L2 expression in such CaCx cases. By using RegRNA (www.regrna.mbc.nctu.edu.tw/) software, we identified binding sites in the NCR2 (nt 4139–4234) of HPV16 intact isolates, corresponding to 14 human miRNAs (hsa-miR-3148,hsa-miR-3174,hsa-miR-3613-3p,hsa-miR-3916,hsa-miR-495,hsa-miR-548a-5p, hsa-miR-548b-5p, hsa-miR-548c-5p, hsa-miR-548d-5p, hsa-miR-548h-5p, hsa-miR-548i-5p, hsa-miR-548j-5p, hsa-miR-548w-5p, hsa-miR-548y-5p) (Figure 6). Such miRNA binding sites were selected on the basis of minimum free energy (MFE≤7) and hybridization score (≥140) (Table S3) as per standards normally used for formation of miRNA:mRNA hybrid. Our resequenced data revealed the occurrence of a SNP (T4228C) (Figure S1) in the NCR2 of E variant intact isolates only, which could lead to loss of 9 miRNA binding sites in the corresponding transcripts (hsa-miR-548a-5p, hsa-miR-548b-5p, hsa-miR-548c-5p, hsa-miR-548d-5p, hsa-miR-548h-5p, hsa-miR-548i-5p, hsa-miR-548j-5p, hsa-miR-548w-5p, hsa-miR-548y-5p) (Figure 6), all of which belonged to the hsa-miR-548 family of miRNAs. Interestingly, proportion of E2 intact CaCx cases (54/70, 77%) harboring SNPs in the miRNA binding sites within the NCR2 was significantly higher (p = 0.007) compared to that of non-malignant samples (12/25, 48%). Within E2 intact CaCx cases, it was also observed that none of AA the variants (0/13, 0%) harbored a SNP in the miRNA binding sites in the NCR2. Thus, no loss of miRNA binding sites in the NCR2 was observed in AA variants.

Bottom Line: L2 (mRNA and protein) expression was evident only among cases with episomal viral genomes and L2 mRNA expression correlated significantly with E2 gene copy numbers suggesting expression from all episomal genomes.Among such cases, Asian American (AA) isolates portrayed all of the humanized codons (100%; 4-6/sample) recorded within L2, which was significantly higher (p = 2.02E-7) compared to the European (E) isolates (22.8%; none or 1-2/sample).This resulted in loss of 9 out of 14 miRNA binding sites (hsa-miR-548 family), despite the significant overexpression of miR548a-5p and miR548d-5p among such cases (28.64 and 36.25 folds, respectively), in comparison to HPV negative control samples.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal, India.

ABSTRACT
We tested the hypothesis that (i) synonymous variations within the coding regions, and (ii) variations within the non-coding regions of HPV, influence cervical cancer (CaCx) pathogenesis under the impact of intact HPV16 genomes. Whole genome sequence analysis of HPV16 isolates within 70 CaCx cases and 25 non-malignant samples revealed that synonymous variations were significantly higher within the E6 (p = 0.014), E5 (p = 0.001) and L2 (p = 0.0002) genes of HPV16 isolates within cases, compared to isolates within non-malignant samples. All of the 25 (100%) humanized codons identified within L2 ORF of the samples analyzed, were harbored by CaCx cases, while 8 out of 25 (32%) were harbored by HPV16 positive non-malignant samples (p = 3.87105E-07). L2 (mRNA and protein) expression was evident only among cases with episomal viral genomes and L2 mRNA expression correlated significantly with E2 gene copy numbers suggesting expression from all episomal genomes. Among such cases, Asian American (AA) isolates portrayed all of the humanized codons (100%; 4-6/sample) recorded within L2, which was significantly higher (p = 2.02E-7) compared to the European (E) isolates (22.8%; none or 1-2/sample). Additionally, majority of E variant isolates within cases (54/57; 94.7%) portrayed a variation (T4228C) within the short non-coding region (NCR2) between E5 and L2 genes, which portrays a weak promoter activity specific for L2 mRNA expression. This resulted in loss of 9 out of 14 miRNA binding sites (hsa-miR-548 family), despite the significant overexpression of miR548a-5p and miR548d-5p among such cases (28.64 and 36.25 folds, respectively), in comparison to HPV negative control samples. The findings exemplify the biological relevance of sequence variations in HPV16 genomes and highlight that episomal HPV16 in CaCx cases employ multiple mechanisms to sustain L2 expression, thereby justifying the potential role of L2 in such cancers, as opposed to those harboring viral integration.

Show MeSH
Related in: MedlinePlus