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. 2014 Dec 9:9:41.
doi: 10.1186/1750-9378-9-41. eCollection 2014.

HIV-1 Tat induces DNMT over-expression through microRNA dysregulation in HIV-related non Hodgkin lymphomas

Anna Luzzi  1 , Federica Morettini  1 , Sara Gazaneo  1 , Lucia Mundo  1 , Anna Onnis  1 , Susanna Mannucci  1 , Emily A Rogena  2 , Cristiana Bellan  1 , Lorenzo Leoncini  1 , Giulia De Falco  3
Affiliations

"V体育ios版" HIV-1 Tat induces DNMT over-expression through microRNA dysregulation in HIV-related non Hodgkin lymphomas

Anna Luzzi et al. Infect Agent Cancer. .

Abstract

Background: A close association between HIV infection and the development of cancer exists VSports手机版. Although the advent of highly active antiretroviral therapy has changed the epidemiology of AIDS-associated malignancies, a better understanding on how HIV can induce malignant transformation will help the development of novel therapeutic agents. .

Methods: HIV has been reported to induce the expression of DNMT1 in vitro, but still no information is available about the mechanisms regulating DNMT expression in HIV-related B-cell lymphomas. In this paper, we investigated the expression of DNMT family members (DNMT1, DNMT3a/b) in primary cases of aggressive B-cell lymphomas of HIV-positive subjects. V体育安卓版.

Results: Our results confirmed the activation of DNMT1 by HIV in vivo, and reported for the first time a marked up-regulation of DNMT3a and DNMT3b in HIV-positive aggressive B-cell lymphomas. DNMT up-regulation in HIV-positive tumors correlated with down-regulation of specific microRNAs, as the miR29 family, the miR148-152 cluster, known to regulate their expression V体育ios版. Literature reports the activation of DNMTs by the human polyomavirus BKV large T-antigen and adenovirus E1a, through the pRb/E2F pathway. We have previously demonstrated that the HIV Tat protein is able to bind to the pocket proteins and to inactivate their oncosuppressive properties, resulting in uncontrolled cell proliferation. Therefore, we focused on the role of Tat, due to its capability to be released from infected cells and to dysregulate uninfected ones, using an in vitro model in which Tat was ectopically expressed in B-cells. .

Conclusions: Our findings demonstrated that the ectopic expression of Tat was per se sufficient to determine DNMT up-regulation, based on microRNA down-regulation, and that this results in aberrant hypermethylation of target genes and microRNAs. These results point at a direct role for Tat in participating in uninfected B-cell lymphomagenesis, through dysregulation of the epigenetical control of gene expression VSports最新版本. .

Keywords: Aggressive B-cell lymphomas; DNMTs; HIV; Tat; microRNAs V体育平台登录. .

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Figures

Figure 1
Figure 1
Expression of DNMTs in HIV + vs. HIV - tumors. (a) Relative expression of DNMT1, DNMT3a/b in HIV+ vs. HIV- tumors by RT-qPCR. Up-regulation of all these genes is observed in HIV+ samples (p < 0.05). (b-c) IHC using an anti-DNMT1 antibody in HIV-negative cases (b) and HIV-positive cases (c). (d-e) IHC for DNMT3a in HIV-negative (d) and positive (e) cases. Up-regulation of DNMT1 and DNMT3a was detected in HIV-positive tumors. None of the antibodies tested for DNMT3b was useful for IHC.
Figure 2
Figure 2
miRNAs predicted to regulate DNMT1, DNMT3a/b were analyzed by RT-qPCR in HIV + vs. HIV - primary tumors. A strong and significant down-regulation of all the selected miRNAs regulating DNMTs is observed in HIV-positive tumors (p < 0.05). The graph is representative of three different RT-qPCR experiments. Error bars represent standard deviation between duplicates.
Figure 3
Figure 3
Immunoistochemical staining for p24 and Tat in HIV + vs. HIV - tumors. (a) IHC using an anti-p24 antibody showed marked positivity of macrophages in a B-cell lymphoma of a HIV-positive subject. (b-c) IHC using an anti-Tat antibody in HIV-negative (b) and HIV-positive cases (c). Marked positivity to Tat of uninfected B-cells is visible in HIV-positive tumors, thus indicating that a soluble form of the protein is released from infected cells and enters neighbouring uninfected ones.
Figure 4
Figure 4
DNMT expression in Tat-positive vs. Tat-negative cells. (a) Relative expression of Tat 24 h after transfection with a Tat-coding vector. (b-c) Ectopic expression of Tat was obtained either upon exposure to the recombinant protein or following transfection of a Tat-coding vector. DNMT1, DNMT3a/b expression was then analysed in Tat-positive (transfected or treated with soluble Tat) vs. Tat-negative cells (empty vector-transfected or untreated cells) at the mRNA level by RT-qPCR (b); up-regulation of all DNMTs is obtained following Tat ectopic expression, both upon exposure to recombinant Tat and following Tat-transfection (p < 0.05). (c) Western blotting analysis for DNMT1 and DNMT3a in Tat-positive vs. Tat-negative cells. Up-regulation of both DNMTs is observed in Tat-positive cells, thus confirming results obtained in HIV-positive primary tumors. None of the tested antibodies for DNMT3b was suitable for WB analysis. Quantification by densitometric analysis is reported.
Figure 5
Figure 5
Expression of DNMTs and DNMT-regulating miRNAs in Tat-positive vs. Tat-negative cells. DNMT1 and DNMT3a/b-regulating microRNA expression was checked by RT-qPCR in Tat-positive vs. Tat-negative cells following transient transfections and exposure to recombinant protein (a). Down-regulation of all miRNAs is observed in Tat-positive cells. The graph is representative of three different RT-qPCR experiments. Error bars represent standard deviation between duplicates. (b-d) Modulation of DNMT-regulating miRNAs was achieved by transient transfections of either mimics or antagomirs of the endogenous miRNAs at different concentration, and the effect on the expression of their respective miRNAs was monitored by RT-qPCR. DNMT1-regulating miRNAs (hsa-miR130a and hsa-miR152) are reported in (b-c), whereas DNMT3a/b regulating miRNAs (hsa-miR29) are shown in (d). (e-f) Relative expression of DNMT1 (e), DNMT3a/b (f) was then evaluated following the ectopic modulation of these miRNAs. Up-regulation of the specific miRNAs results in the down-regulation of DNMTs, whereas increased expression of DNMTs is observed following miRNA inhibition. The graph is representative of three different RT-qPCR experiments (p < 0.05). Error bars represent standard deviation between duplicates. (g-h): WB for DNMT1 and DNMT3a of cells transfected with hsa-miR152 (for DNMT1) and hsa-miR29 (for DNMT3a) mimics and inhibitors. NC: Mimic negative control; M: Either hsa-miR152 or hsa-miR29 mimic; NCI: Inhibitor negative control; I: Either hsa-miR152 or hsa-miR29 inhibitor. None of the tested antibodies for DNMT3b was suitable for WB analysis. Densitometric analysis results are reported.
Figure 6
Figure 6
Analysis of cell cycle regulators in Tat-positive vs. Tat-negative cells. Cell proliferation in Tat-transfected vs. empty vector-transfected cells (a). Tat-transfected cells show a higher proliferation rate, which is in line with the increased expression of cyclin A (b), an S-phase specific marker. (c-d) Relative expression by RT-qPCR of INK4/p16, TP53 and RB1 in Tat-transfected cells (c) and in HIV-positive vs. HIV-negative primary tumors (d). Down-regulation of INK4/p16 and TP53 is observed both in vitro and in vivo. The graph is representative of three different RT-qPCR experiments (p < 0.05). Error bars represent standard deviation between duplicates.
Figure 7
Figure 7
Methylation analysis of DNMT-dependent miRNAs and cell cycle regulatory genes in Tat-positive vs. Tat-negative cells and primary tumors. Methylation analysis of INK4/p16 (a) and TP53 (b) in HIV-positive and -negative primary tumors. M indicates the product using the methyled primers, U indicates the product using the unmethyled primers. Amplification of INK4/p16 resulted in a band lower than 200 bp, whereas the specific product for TP53 was about 100 bp. The specific product for INK4/p16 was obtained only using unmethyled primers in HIV-negative samples, whereas only methyled primers resulted in a INK4/p16 product in HIV-positive tumors, which is indicative of INK4/p16 methylation in HIV-positive tumors. No differences were observed for TP53 between HIV-negative and HIV-positive tumors, as the amplified product was obtained only using unmethyled primers, indicating that no methylation is detectable for TP53 in neither of the two tumor types. Arrows indicate the presence of specific products for both genes. (c) IHC for p16 in HIV-positive tumors; (d) Sequence analysis of hsa-miR-148a in HIV-positive and HIV-negative primary tumors. Arrows indicate the methyled C that are not converted to U in HIV-positive tumors.
Figure 8
Figure 8
Modulation of DNMT-regulating miRNAs affects the expression of INK4/p16 and consequent cell growth control. Treatment with 5-Aza in (a) empty vector-transfected and (b) Tat-transfected cells. Treatment with 5-Aza induces the expression of miRNAs containing CpG islands in Tat-transfected cells, indicating that down-regulation of these miRNAs depends on hypermethylation. (c) Treatment with 5-Aza was also able to restore the expression of INK4/p16 in Tat-transfected cells, thus suggesting that silencing of INK4/p16 is due to hypermethylation. (d) Ectopic modulation of DNMT-regulating miRNAs was achieved using synthetic mimic or inhibitors. Relative expression of INK4/p16 was then checked by RT-qPCR. A decreased expression of the gene was observed following inhibition of miRNAs, which leads to up-regulation of DNMTs and consequent hypermethylation of target genes. Conversely, increased expression of endogenous miRNAs, which leads to downregulation of DNMTs, results in over-expression of INK4/p16 (p < 0.05). NC: Mimic negative control; M: Either hsa-miR152 or hsa-miR29 mimic; NCI: Inhibitor negative control; I: Either hsa-miR152 or hsa-miR29 inhibitor. (e-f) Cell proliferation analysis in cells transfected with miRNA mimics/inhibitors for DNMT1 (e) and DNMT3a/b (f). Inhibition of the endogenous miRNAs enhances cell growth.
Figure 9
Figure 9
A model for Tat-mediated lymphomagenesis. DNMT aberrant activity may be induced by both IL-6 and RB-protein inactivation. As Tat is able to induce both IL-6 expression and RBL2-gene product inactivation, we hypothesize that Tat may induce DNMT up-regulation through these mechanisms, which results in the aberrant methylation of genes and miRNAs.
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