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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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Representative Immunoblot analysis of L2 and ACTB protein expression.Upper panel depicts L2 expression. Lanes 1 and 2: HPV16 positive E2 disrupted/integrated CaCx case samples (D1 and D2); Lanes 3, 5, and 7: HPV16 positive E2 intact/episomal (episomal or concomitant) European variants (EV1, EV2, EV3, respectively); Lanes 4, 6, and 8: HPV16 positive E2 intact/episomal (episomal or concomitant) Asian American variants (AAV3, AAV2, AAV1, respectively). Lower panel depicts ACTB expression among all the samples analysed. Sample details are illustrated in Table 5.
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pone-0065647-g004: Representative Immunoblot analysis of L2 and ACTB protein expression.Upper panel depicts L2 expression. Lanes 1 and 2: HPV16 positive E2 disrupted/integrated CaCx case samples (D1 and D2); Lanes 3, 5, and 7: HPV16 positive E2 intact/episomal (episomal or concomitant) European variants (EV1, EV2, EV3, respectively); Lanes 4, 6, and 8: HPV16 positive E2 intact/episomal (episomal or concomitant) Asian American variants (AAV3, AAV2, AAV1, respectively). Lower panel depicts ACTB expression among all the samples analysed. Sample details are illustrated in Table 5.

Mentions: We determined L2 protein expression by immunoblot analysis, on a subset of 12 CaCx cases (integrated or E2 disrupted CaCx cases = 4, Asian American episomal or E2 intact CaCx cases = 3 and European episomal or E2 intact CaCx cases = 5) from the set that was used for L2 mRNA expression analysis. L2 expression was recorded among the episomal CaCx cases, AA and E variant isolates, while such expression could not be identified among the integrated CaCx cases (Figure 4). All the CaCx samples, irrespective of episomal or integrated, portrayed the expression of ACTB protein (endogenous control). The status of humanized codons within the AA and E variant isolates of samples revealing L2 protein expression is depicted in Table 5. L2 protein expression was quantified by densitometric analysis of immunoblot results by IMAGE J software (http://rsb.info.nih.gov/ij/docs/index.html) and no significant difference (p = 0.562, t-test) was recorded between AA [mean (area of L2 protein band/area of ACTB protein band) ±sd = 2.66±1.85] and E variants [mean (area of L2 protein band/area of ACTB protein band) ± sd = 1.95±0.61] as portrayed in Figure 5.


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)

Representative Immunoblot analysis of L2 and ACTB protein expression.Upper panel depicts L2 expression. Lanes 1 and 2: HPV16 positive E2 disrupted/integrated CaCx case samples (D1 and D2); Lanes 3, 5, and 7: HPV16 positive E2 intact/episomal (episomal or concomitant) European variants (EV1, EV2, EV3, respectively); Lanes 4, 6, and 8: HPV16 positive E2 intact/episomal (episomal or concomitant) Asian American variants (AAV3, AAV2, AAV1, respectively). Lower panel depicts ACTB expression among all the samples analysed. Sample details are illustrated in Table 5.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065647-g004: Representative Immunoblot analysis of L2 and ACTB protein expression.Upper panel depicts L2 expression. Lanes 1 and 2: HPV16 positive E2 disrupted/integrated CaCx case samples (D1 and D2); Lanes 3, 5, and 7: HPV16 positive E2 intact/episomal (episomal or concomitant) European variants (EV1, EV2, EV3, respectively); Lanes 4, 6, and 8: HPV16 positive E2 intact/episomal (episomal or concomitant) Asian American variants (AAV3, AAV2, AAV1, respectively). Lower panel depicts ACTB expression among all the samples analysed. Sample details are illustrated in Table 5.
Mentions: We determined L2 protein expression by immunoblot analysis, on a subset of 12 CaCx cases (integrated or E2 disrupted CaCx cases = 4, Asian American episomal or E2 intact CaCx cases = 3 and European episomal or E2 intact CaCx cases = 5) from the set that was used for L2 mRNA expression analysis. L2 expression was recorded among the episomal CaCx cases, AA and E variant isolates, while such expression could not be identified among the integrated CaCx cases (Figure 4). All the CaCx samples, irrespective of episomal or integrated, portrayed the expression of ACTB protein (endogenous control). The status of humanized codons within the AA and E variant isolates of samples revealing L2 protein expression is depicted in Table 5. L2 protein expression was quantified by densitometric analysis of immunoblot results by IMAGE J software (http://rsb.info.nih.gov/ij/docs/index.html) and no significant difference (p = 0.562, t-test) was recorded between AA [mean (area of L2 protein band/area of ACTB protein band) ±sd = 2.66±1.85] and E variants [mean (area of L2 protein band/area of ACTB protein band) ± sd = 1.95±0.61] as portrayed in Figure 5.

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