Identification of human miRNAs associated with serum HBsAg.

<p>(a) The circulating HBsAg-miRNA signature: average RQs were obtained from the comparative DDC_T analysis. Values are reported in a bar plot as a logarithmic scale base 10 along with SD. (b) Differentially detected miRNAs between HBsAg positive immunoprecipitates (HBs-IP+) samples (left-most, n = 11; right-most, n = 4) and the group of control HBsAg negative immunoprecipiates (ctrl-IPs) (n = 4) were selected by Mann-Whitney test on –DCt values (left-most, p<0.1; right-most, p<0.05), and an unsupervised hierarchical cluster analysis was finally performed. Venn diagrams indicate the comparison among the pool of HBsAg-associated miRNAs obtained from the comparative DDC_T analysis of panel a (blu circle) and the HBsAg-associated miRNAs obtained from the Mann-Whitney tests (red circle).</p

HBs-immunoprecipitation led to a significant change of detection of some human miRNAs in examined sera.

<p>Heatmap of differentially detected miRNAs in whole HBsAg sera (S+) and HBsAg positive flowthroughs after immunoprecipitation (HBs-F+) was obtained by Mann-Whitney test (p<0.05) followed by hierarchical clustering (-DC_T are represented). Venn diagram: the 157 differentially abundant serum miRNAs between whole HBsAg positive sera (S+) and HBsAg positive flowthroughs (HBs-F+) were compared to miRNAs of clusters 1-to-5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone-0031952-g003" target="_blank">Figure 3b</a> (right panel).</p

Demographic and Virologic Characteristics of Individuals and Sera.

<p>Abbreviations: VP = viral particles, virion; SVP = subviral HBsAg particles; IU = International Units; ng = nanograms; ALT = serum alanine amino trasferase.</p><p>Quantitative values for VP and SVP were obtained as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone.0031952-Gerlich1" target="_blank">[14]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone.0031952-Desire1" target="_blank">[15]</a>: 1 IU of HBsAg corresponds to 1,1E+06 IU HBV DNA and1 ng of HBsAg corresponds to 2,08E+08 SVP or 5,0E+07 VP.</p><p>HBV infection and disease phases were characterized as previuosly reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone.0031952-Brunetto1" target="_blank">[8]</a>: IC = Inactive HBsAg Carriers with serum HBV-DNA persistently below 2000 IU and without liver disease; AC1 = Active HBsAg Carriers with serum HBV-DNA fluctuating below 20.000 IU with normal liver histology; AC2 = Active HBsAg carriers with serum HBV-DNA fluctuating above 20.000 IU with chronic active hepatitis at histology, patients with chronic hepatitis B (CHB).</p

Differences in Western Blotting and human miRNA analyses between immunoprecipitated HBsAg particles and control immunoprecipitations.

<p>Left-most: Western blotting analysis of protein lysates from HBsAg positive immunoprecipitates (HBs-IP+) and control HBsAg negative immunoprecipitates (ctrl-IPs) for detection of Ago2 protein. IP+ lanes contain pooled protein lysates from samples # 1–13 and single lysates from samples # 1 and 9 respectively. Ctrl-IPs lanes contain protein lysates from immunoprecipitates obtained from HBsAg positive sera with mouse monoclonal anti-human c-myc antibody (unrelated-IP+) and HBsAg negative sera using anti-HBs monoclonal antibody (HBs-IP−). HEK is the protein lysate from HEK cells used as positive control for Ago2 protein. The figure is representative of 3 independent experiments. Right-most: HCL is unsupervised hierarchical cluster analysis of detected miRNAs (DC_T values) in both HBs-immunoprecipitated fractions from HBsAg positive immunoprecipitates (HBs-IP+) and control HBsAg negative immunoprecipitates (ctrl-IPs). GDM is supervised gene distance matrix correlating DCt values of HBs-IP+ vs ctrl-IP samples.</p

Canonical pathways enriched by predicted targets of HBsAg-associated miRNAs.

<p>Target genes of HBsAg-associated miRNAs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone-0031952-g003" target="_blank">Figure 3a</a>) were predicted (TargetScan) and ranked. Functional analysis (IPA) was then applied on predicted target genes, and canonical pathways were finally ranked according to their significance in a Fisher exact test (p-value≤0.05, horizontal threshold line in the plot). Left axis: –log(p-values) as histogram boxes; the asterisk (*) marks the three pathways that resulted as significantly enriched by the analyzed gene-list; right axis: for each pathway, black dots indicate the ratio of pathway-defining genes, which is calculated as the number of involved target genes over the total number of genes in that pathway reference dataset.</p

Comparison of miRNA profile of SVR vs NR and REL at different time points by Student’s t test analysis: a total of 21 miRNA (out of 66 tested) showed significant differential expression after Bonferroni correction (cut-off<0.000758).

<p>Comparison of miRNA profile of SVR vs NR and REL at different time points by Student’s t test analysis: a total of 21 miRNA (out of 66 tested) showed significant differential expression after Bonferroni correction (cut-off<0.000758).</p

Baseline and treatment features of study (A) and validation (B) cohorts.

<p>n.d.  =  not detectable, SVR = Sustained virologic response, SC = HBeAg to anti-HBe seroconversion, Rel = relapse, NR = no response, OTR = on treatment response.</p><p>Baseline and treatment features of study (A) and validation (B) cohorts.</p

MiR-B-Index in HBV carriers: diagnostic performance to identify inactive carriers (AUROCs), index kinetics in Peg-IFN treated patients by outcome and distribution of the index values by treatment outcome and phase of HBV infection.

<p><b>A)</b> Receiver operating characteristic curve for MiR-B-Index in IC vs patients with chronic hepatitis (HBeAg positive or negative CHB, HBV/HDV chronic hepatitis, ALD1): 0.9520 (95% CI 0.903 to 1.000, p<0.001); <b>B)</b> ROC for MiR-B-Index in IC vs HBeAg negative CHB (ALD2): 0.954 (95% CI 0.902 to 1.000, p<0.001; <b>C)</b> Kinetics of MiR-B-Index in 14 patients treated with Peg-IFN: MiR-B-Index values progressively increased in SVR, whereas they showed minor fluctuations in REL/NR (p<0.001); <b>D)</b> Whisker plot of the MiR-B-Index (y-axis) values in BL CHB patients treated with Peg-IFN (NR, REL and SVR), 24 week post-T-FU of REL/NR and SVR and IC, separately (CHB-BL vs IC p<0.001; SVR-BL vs SVR PT-FU p<0.001; REL/NR BL vs REL/NR PT-FU p = 0.462; SVR PT-FU vs IC p = 0.792).</p

Differentially expressed miRNAs comparing Baseline (BL) and Week 12 (Wk 12) sera in 14 Peg-IFN HBeAg negative CHB patients.

<p>Of the 8 miRNAs presenting p<0.05 only miR-30e-3p passes the Bonferroni correction (cut-off <0.000758) for multiple testing (<b>ΔΔ</b>Cq 1.7 up-regulation, p = 0.000354).</p><p>Differentially expressed miRNAs comparing Baseline (BL) and Week 12 (Wk 12) sera in 14 Peg-IFN HBeAg negative CHB patients.</p

Dynamic variation of miRNA profiles at Baseline (BL), during (week 12, 24 and End of Treatment, EOT) and after therapy (week 24 post-treatment follow-up, PT-FU) according to treatment response (NR, REL, SVR) in 14 Peg-IFN treated patients.

<p>Statistical analysis by one way ANOVA.</p><p>Dynamic variation of miRNA profiles at Baseline (BL), during (week 12, 24 and End of Treatment, EOT) and after therapy (week 24 post-treatment follow-up, PT-FU) according to treatment response (NR, REL, SVR) in 14 Peg-IFN treated patients.</p