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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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Relative quantification of L2 mRNA expression.(A) Amplification plot based on quantitative real time PCR of HPV16 L2 expression. L2 is transcribed in E2 intact/episomal (episomal or concomitant) but in E2 disrupted/integrated cases. (B) Dissociation curve depicting the first-derivative melting curve for the reaction characterizing the expression of L2 (Tm of 80.5°C). (C) Amplification plot based on quantitative real time PCR of ACTB expression. ACTB is expressed by both episomal and integrated HPV16 positive cases. (D) Dissociation curve depicting the first-derivative melting curve for the reaction characterizing the expression of ACTB (Tm of 81.0°C).
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pone-0065647-g001: Relative quantification of L2 mRNA expression.(A) Amplification plot based on quantitative real time PCR of HPV16 L2 expression. L2 is transcribed in E2 intact/episomal (episomal or concomitant) but in E2 disrupted/integrated cases. (B) Dissociation curve depicting the first-derivative melting curve for the reaction characterizing the expression of L2 (Tm of 80.5°C). (C) Amplification plot based on quantitative real time PCR of ACTB expression. ACTB is expressed by both episomal and integrated HPV16 positive cases. (D) Dissociation curve depicting the first-derivative melting curve for the reaction characterizing the expression of ACTB (Tm of 81.0°C).

Mentions: In our earlier study, we confirmed the intactness of the E2 gene by analysing the presence of the viral transcript (E7-E1∧E4) that produces the repressor E2, by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR of E7 and E4 (nested to the E2 gene) genes [31]. Based on such analysis, samples were classified as pure episomal or concomitant (episomal and integrated) with intact E2 genes, and integrated with disrupted E2 genes. The study [31] also revealed that these two types of cancers differed in the expression of E7 and E2 mRNAs. We therefore determined L2 mRNA expression, by quantitative real time PCR on 23 episomal/concomitant HPV16 positive CaCx cases, and compared the data with that of 11 integrated CaCx cases. No L2 expression was recorded among the integrated cases, as opposed to distinct L2 mRNA expression in episomal/concomitant CaCx cases (Figure 1), which was quite similar to that recorded in case of E2 expression in our earlier study [31]. All of the samples analysed, portrayed the expression of ACTB mRNA transcripts as internal control. Further analysis failed to reveal significant (p = 0.224, t-test) differences in L2 mRNA expression between AA [mean (L2 CT/ACTB CT) ± sd = 0.834±0.127] and E [mean (L2 CT/ACTB CT) ± sd = 0.904±0.128] variants (Figure 2). The ratio, L2 CT/ACTB CT, was also found to be significantly correlated with the E2 copy numbers (p = 0.004; R2 = 0.336) within the episomal CaCx cases (Figure 3), justifying the expression of L2 from episomal viral genomes.


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)

Relative quantification of L2 mRNA expression.(A) Amplification plot based on quantitative real time PCR of HPV16 L2 expression. L2 is transcribed in E2 intact/episomal (episomal or concomitant) but in E2 disrupted/integrated cases. (B) Dissociation curve depicting the first-derivative melting curve for the reaction characterizing the expression of L2 (Tm of 80.5°C). (C) Amplification plot based on quantitative real time PCR of ACTB expression. ACTB is expressed by both episomal and integrated HPV16 positive cases. (D) Dissociation curve depicting the first-derivative melting curve for the reaction characterizing the expression of ACTB (Tm of 81.0°C).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065647-g001: Relative quantification of L2 mRNA expression.(A) Amplification plot based on quantitative real time PCR of HPV16 L2 expression. L2 is transcribed in E2 intact/episomal (episomal or concomitant) but in E2 disrupted/integrated cases. (B) Dissociation curve depicting the first-derivative melting curve for the reaction characterizing the expression of L2 (Tm of 80.5°C). (C) Amplification plot based on quantitative real time PCR of ACTB expression. ACTB is expressed by both episomal and integrated HPV16 positive cases. (D) Dissociation curve depicting the first-derivative melting curve for the reaction characterizing the expression of ACTB (Tm of 81.0°C).
Mentions: In our earlier study, we confirmed the intactness of the E2 gene by analysing the presence of the viral transcript (E7-E1∧E4) that produces the repressor E2, by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR of E7 and E4 (nested to the E2 gene) genes [31]. Based on such analysis, samples were classified as pure episomal or concomitant (episomal and integrated) with intact E2 genes, and integrated with disrupted E2 genes. The study [31] also revealed that these two types of cancers differed in the expression of E7 and E2 mRNAs. We therefore determined L2 mRNA expression, by quantitative real time PCR on 23 episomal/concomitant HPV16 positive CaCx cases, and compared the data with that of 11 integrated CaCx cases. No L2 expression was recorded among the integrated cases, as opposed to distinct L2 mRNA expression in episomal/concomitant CaCx cases (Figure 1), which was quite similar to that recorded in case of E2 expression in our earlier study [31]. All of the samples analysed, portrayed the expression of ACTB mRNA transcripts as internal control. Further analysis failed to reveal significant (p = 0.224, t-test) differences in L2 mRNA expression between AA [mean (L2 CT/ACTB CT) ± sd = 0.834±0.127] and E [mean (L2 CT/ACTB CT) ± sd = 0.904±0.128] variants (Figure 2). The ratio, L2 CT/ACTB CT, was also found to be significantly correlated with the E2 copy numbers (p = 0.004; R2 = 0.336) within the episomal CaCx cases (Figure 3), justifying the expression of L2 from episomal viral genomes.

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