Mir-34A Is Involved in Tat-Induced HIV-1 Long Terminal Repeat

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FEBS Letters 586 (2012) 4203–4207

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MiR-34a is involved in Tat-induced HIV-1 long terminal repeat (LTR) transactivation through the SIRT1/NFjB pathway ⇑ Hong-Sheng Zhang , Xin-Yu Chen, Tong-Chao Wu, Wei-Wei Sang, Zheng Ruan

College of Life Science & Bioengineering, Beijing University of Technology, Pingleyuan 100#, District of Chaoyang, Beijing 100124, China

article info abstract

Article history: MicroRNAs (miRNAs) regulate gene expression and may contribute to HIV-1 infection. In this study, Received 11 August 2012 our goal was to investigate the mechanisms by which miR-34a inﬂuenced Tat-induced HIV-1 trans- Revised 1 October 2012 activation through the SIRT1/NFjB pathway. We showed that Tat induced up-regulation of miR-34a Accepted 5 October 2012 expression in TZM-bl cells. MiR-34a signiﬁcantly inhibited SIRT1 expression. Overexpression of Available online 24 October 2012 miR-34a increased Tat-induced LTR transactivation. Forced expression of miR-34a decreased SIRT1 Edited by Ivan Sadowski protein expression and consequently diminished Tat-induced acetylation of p65, while treatment with a miR-34a inhibitor had the opposite effect. These results suggest that regulating SIRT1 by down-regulation of miR-34a levels may be a therapeutic strategy against HIV-1 replication. Keywords: miR-34a Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. SIRT1 Tat NF-jB

1. Introduction tumorigenesis and virus infection [8–10]. Several studies have proposed a regulation paradigm whereby cellular miRNAs target Several key HIV-1 and cellular proteins have been determined mRNAs that encode cellular proteins involved in virus replication. to be necessary for a chronic persistent infection, including the For example, the miR-17/92 cluster, which encodes seven miRNAs, trans-activator Tat [1–3]. The replication rate of integrated HIV-1 among which miR-17-5p and miR-20 may target HATs and HIV-1 is largely controlled at the level of transcription. Though viral tran- Tat cofactor PCAF, was substantially decreased during HIV-1 infec- scription is fully dependent upon host cellular factors, recent ﬁnd- tion [11]. MicroRNA-34a (miR-34a) is mapped in the region of chro- ings indicate a complex interplay between viral proteins and host mosome 1p36.23, and usually it is aberrantly expressed in multiple transcription and signaling machineries (i.e. histone deacetylasess types of diseases [12]. MiR-34a has many potential target genes, (HDACs), histone acetylases (HATs), and other protein-modifying including SIRT1, NMYC, CCND1, CCNE2, CDK4, CDK6, and MET [13]. factors) [4–6]. Dynamic modiﬁcations of chromatin structure and In the study, we showed that upregulated miR-34a promoted nucleosome remodeling have been implicated in HIV-1 transcrip- Tat-induced HIV-1 transactivation. We further demonstrated that tion [4]. HATs can acetylate Tat at lysines 28, 50 and/or 51, all of miR-34a directly targeted 30-UTR of SIRT1 gene. Importantly, SIRT1 which impart different functions. For instance, acetylation at lysine re-expression reduced Tat-induced HIV-1 transactivation induced 50 and 51 by CBP/p300 and GCN5 promotes the dissociation of Tat by miR-34a. from the TAR-binding domain, indicating the start of transcriptional elongation [5]. Human sirutin (SIRT1) is a class III histone 2. Materials and methods deacetylase (HDAC), which mediates de-acetylation of Tat. De-acetylation occurs at the Lys residue at position 50. SIRT1 can 2.1. miRNA target predications inhibit nuclear transcription factor-jB (NF-jB). As a master transcription factor, NF-jB is proposed to be important mediators of Two algorithms, PicTar (http://pictar.mdc-berlin.de) and miRan- HIV-related transcription and replication [7]. da (http://www.microrna.org) were used to predict putative bind- There is increasing evidence that post-transcriptional regulation ing sites in the SIRT1 mRNA for members of the miR-34a family. of gene expression, mediated by microRNAs (miRNAs), plays an important role in the control of cell proliferation, apoptosis, 2.2. Cell culture and reagents

⇑ Corresponding author. Fax: +86 10 67392780. TZM-bl is a HeLa-derived cell line expressing surface CD4, E-mail address: [email protected] (H.-S. Zhang). CXCR4, CCR5 and containing a chromatin-integrated HIV-1 LTR.

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.10.023 4204 H.-S. Zhang et al. / FEBS Letters 586 (2012) 4203–4207

TZM-bl cells and HeLa cells were maintained in DMEM supple- 2.7. Luciferase assay mented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomy- cin, 200 lg/ml G418, and 100 lg/ml hygromycin B at 37 °Cin5% Cells were harvested and the extracts were subjected to lucifer-

CO2 and 95% air in humidified atmosphere. The human lympho- ase assay using the Luciferase Assay System (Promega). Luciferase cytic cells, SupT1 T cells were grown in RPMI 1640 medium activity was normalized with protein concentration. Protein con- supplemented with 10% fetal calf serum at 37 °C and 5% CO2. centration was measured with BCA protein assay kit (PIERCE). Nicotinamide (NAM), phorbol 12-myristate 13-acetate (PMA) and Trichostatin A (TSA) were purchased from Sigma (St. Louis, MO). 2.8. Statistical analysis The following polyclonal antibodies: anti-p65, anti-SIRT1, and anti-b-actin were obtained from Santa Cruz Biotechnology (Santa Results are expressed as mean ± S.E. Differences between Cruz, CA). Anti-Ac-p65 (Lys 310) antibody was purchased from groups were analyzed by Student’s t test or one-way analysis of Abcam (Cambridge, MA). The secondary antibodies IR800 anti- variance. The statistically significant threshold was set at P < 0.05. rabbit, IR800 anti-goat and IR800 anti-mouse IgG were obtained from Li-COR Biosciences (Lincoln, Nebraska, USA). The pCI-Tat 3. Results plasmid was kindly provided by Prof. Ping-Kun Zhou (Beijing Insti- tute of Radiation Medicine, Beijing). The SIRT1 and SIRT1 H363Y (a 3.1. miR-34a level is significantly increased in Tat-treated TZM-bl cells deacetylase-defective mutant of SIRT1) expression plasmids were a gift from Dr. Kouzarides (Cancer Research UK Gurdon Institute, In order to assess the role of miR-34a in Tat-induced HIV-1 Cambridge). All other chemicals were of the highest commercial transactivation, we firstly evaluated the expression of miR-34a in grade available. Tat-treated TZM-bl cells and SupT1 T cells co-transfected with Tat plasmid and LTR-luc plasmid by using quantitative real-time 2.3. Cell transfection PCR. Fig. 1A showed that miR-34a levels were significantly increased in Tat-treated TZM-bl cells and in SupT1 T cells co- For all transfections, TZM-bl cells were used at a confluence of transfected with Tat plasmid and LTR-luc plasmid in a dose- 50–60%. Typically, cells were transfected with Tat plasmid per dependent manner. However, there was no effect on miR-34a well in a 6-well dish by using Genejuice transfection reagent as expression with Tat-treated naïve HeLa cells (data not shown). we previously described [14]. Cell extracts were prepared 48 h 100nM Tat plasmid was chosen in the following experiments. after transfection. SupT1 T cells were performed by transfecting with Tat expressing plasmid and HIV-1 LTR: luciferase plasmid [15]. MiRNA mimics and miRNA inhibitors were designed and syn- thesized by GenePharmar (Shanghai, China). MiRNA inhibitors were designed such that all the nucleotides contained a 20-O- methyl modification. Twenty-four hours prior to transfection, cells were plated onto a 6-well plate (Greiner, Germany) at 40–60% confluence. Transfection of TZM-bl cells grown in 6-well plates were performed using 20 nM miR-34a mimics or negative control, 50 nM miR-34a inhibitor or negative control, and 1 ll of Lipofect- amine 2000 (Invitrogen, USA), according to the manufacturer’s instructions [16]. For miR-34a assay and western blot analysis, cells were harvested for RNA and protein respectively after 24– 48 h.

2.4. Real-time polymerase chain reaction (PCR)

Real-time PCR was performed as previously described [17].

2.5. Luciferase reporter assay

The 30 UTR of SIRT1 (1420 bp) containing the SIRT1-miR-34a response element was cloned into pMIR-REPORT Luciferase vector. A mutant 3’UTR of SIRT1 was synthesizedby PCR, whose sequence contained 50-ACACCCACCAAGGACCATTAGTCCGAGA-30 [18]. Cells were plated in 24-well plates and then cotransfected with 20 nM of either miR-34a mimics or microRNA control, 40 ng of either pMIR-REPORT-SIRT1-WT or pMIR-REPORT-SIRT1-MUT using Lipo- fectamine 2000. Cells were collected 48 h after transfection and analyzed using the Dual-Luciferase Reporter Assay System (Prome- ga, Madison, WI). Transfections were performed in triplicate and Fig. 1. miR-34a level was signiﬁcantly increased by Tat in TZM-bl cells and SupT1 T repeated at least twice in separate experiments. cells. TZM-bl cells and SupT1 T cells. were transfected with (A) 20, 50, 100 Nm Tat plasmid; (B) mimics control (pre-miR-control), miR-34a mimics (pre-miR-34a), 2.6. Western blots inhibitor control (anti-miR-control), miR-34a inhibitor (anti-miR-34a) or 100 nM Tat plasmid for 48 h. Total RNA was subjected to real-time RT-PCR to analyze the expression level of miR-34a in each sample. U6 was used as a reference for miRNAs. Western blots were performed as previously described [14]. Relative miR-34a level of each sample was calculated with respect to the normal Protein concentrations were determined by Bio-Rad assay, as control. Each sample was analyzed in triplicate. ⁄⁄P < 0.01(compared to control). ## directed by the manufacturer (Bio-Rad, Hercules, CA). P < 0.01(compared to Tat). H.-S. Zhang et al. / FEBS Letters 586 (2012) 4203–4207 4205

These data indicate that upregulation of miR-34a may be related to Tat-induced HIV-1 transactivation. To study the biological role of miR-34a in Tat-induced HIV-1 transactivation, TZM-bl cells and LTR-luc plasmid-transfected SupT1 T cells that were treated with miR-34a mimics or inhibitors were analyzed. PMA was the positive control. The miR-34a levels were signiﬁcantly increased in TZM-bl cells and LTR-luc plasmid- transfected SupT1 T cells transfected with miR-34a mimics (Fig. 1B), and upregulated miR-34a promoted Tat-induced HIV-1 transactivation (Fig. 2). Furthermore, downregulated miR-34a by its inhibitor (Fig. 1B) inhibited Tat-induced HIV-1 transactivation (Fig. 2). These results suggest that upregulated miR-34a positively regulates Tat-induced HIV-1 transactivation.

3.2. miR-34a directly targets SIRT1 3’-UTR Fig. 3. Tat induced miR-34a expression through down-regulation of SIRT1. TZM-bl cells were co-transfected with miR-34a mimics, plasmid reporter containing wild- type or mutant 30UTR of SIRT1. After 48 h, luciferase assay was performed and In order to identify the target gene of miR-34a in regulating normalized to the internal ﬁreﬂy luciferase activity. ⁄⁄P < 0.01(compared to control). Tat-induced HIV-1 transactivation, we searched for candidate genes using TargetScan5.2 (http://www.targetscan.org/) and miRBase (http://www.mirbase.org/) microRNA databases. The SIRT1 gene is one of the predicted target genes of miR-34a. To determine whether SIRT1 responded to miR-34a through direct 3’-UTR interaction, we cloned the 3’-UTR of SIRT1 into a reporter plasmid downstream of the luciferase gene to generate pMIR- report-SIRT1-WT or pMIR-report-SIRT1-MUT (with a mutant miR-34a response element). Pre-miR-34a decreased luciferase activity of the reporter vector pMIR-report-SIRT1-WT containing the miR-34a response element (Fig. 3). In contrast, miR-34a has a minimal effect on the reporter vector pMIR-report-SIRT1-MUT with a mutated miR-34a response element. Furthermore, miR- 34a does not affect SIRT1 RNA (data not shown). Taken together, these results indicated that SIRT1 was a direct target of miR-34a.

3.3. miR-34a promotes Tat-induced HIV-1 transactivation by repressingSIRT1

SIRT1 is an important determinant of Tat-induced HIV-1 transactivation. Therefore, we speculated that the role of miR-34a in TZM-bl cells was mediated by regulating endogenous SIRT1 expression. The effects of miR-34a mimics or inhibitors on SIRT1 RNA and protein levels were examined by Q-RT-PCR and western blotting. MiR-34a mimics resulted in 50–70% reduction of SIRT1 protein levels in TZM-bl cells (Figs. 4A). MiR-34a inhibitors significantly upregulated SIRT1 protein levels in comparison to negative

Fig. 4. miR-34a promotes Tat-induced HIV-1 transactivation by repressing SIRT1. (A). TZM-bl cells were transfected with mimics control, miR-34a mimics, inhibitor control, miR-34a inhibitor or 100 nM Tat plasmid for 48 h. SIRT1 expression was assessed by western blot. Loading control was obtained using anti-b-actin antibody. Blots were representative of three experiments. (B) TZM-bl cells and SupT1 T cells were transfected with mimics control, miR-34a mimics, 50nM SIRT1, 50 nM SIRT1H363Y (SIRT1HY), 5 mM NAM, 10 lM TSA or 100 nM Tat plasmid for 48 h. Cells were collected at 48 h post-transfection and analyzed for luciferase activity. The Y axis represents percent increase of luciferase activity relative to the Tat alone. ## a P value <0.01 compared to Tat group (n = 3). P < 0.01(compared to Tat + pre-miR-34a group).

controls in TZM-bl cells (Fig. 4A). MiR-34a mimics or inhibitors had no effect on SIRT1 at RNA level (data not shown). The inhibition of SIRT1 by miR-34a was partially recovered after co-transfection with a plasmid-expressing SIRT1. Fig. 4B showed that Tat-induced HIV-1 transactivation was inhibited when SIRT1 was re-expressed in TZM-bl cells and SupT1 T cells transfected Fig. 2. miR-34a affects Tat-induced HIV-1 LTR transactivation in TZM-bl cells and with LTR-luc plasmid treated with miR-34a, while SIRT H363Y mu- SupT1 T cells. TZM-bl cells and SupT1 T cells were transfected with mimics control, tant plasmid had no effect. While SIRT1 inhibitor nicotinamide miR-34a mimics, inhibitor control, miR-34a inhibitor and 100 nM Tat plasmid or potentiated miR-34a-induced HIV-1 transactivation. These data 1 lM PMA for 48 h. Cells were collected at 48 h post-transfection and analyzed for luciferase activity. The Y axis represents percent increase of luciferase activity conﬁrm that miR-34a promotes Tat-induced HIV-1 transactivation, relative to the Tat alone. ## P value <0.01 compared to Tat group (n = 3). at least in part by repressing endogenous SIRT1. 4206 H.-S. Zhang et al. / FEBS Letters 586 (2012) 4203–4207

Tat miR-34a SIRT1 NF-κB activation HIV-1 transactivation

Fig. 6. The diagram that summarize the interplay of Tat – miR34a – SIRT1 – NF – kB pathway in the process of Tat-induced LTR transactivation. (?: activated; : inhibited).

cause Tat is required for productive transcription from the HIV-1 long terminal repeat (LTR) promoter, suppression of PCAF expression by miR-17-5p and miR-20a results in reduced viral transcription and replication [24]. MiR-27b acts as a novel regulator of Cyclin T1 protein levels which are involved in HIV-1 replication, while miR-29b, miR-223, and miR-150 may indirectly regulate Cy- clin T1 [22]. HIV-1 Tat protein increases the levels of miR-34a and promotes neuronal dysfunction [25]. In the present study, Tat increased the expression of miR-34a, which was consistent with above findings. Tat-induced miR-34a expression promoted HIV-1 transactivation, which was confirmed by transfected with miR- 34a mimics or miR-34a inhibitors. The class III histone deacetylase sirtuin 1 (SIRT1) is increasingly recognized as a critical regulator of stress responses, replicative Fig. 5. Effect of miR-34a on Tat-induced NF-kB p65 acetylation in TZM-bl cells. senescence, inflammation, metabolism, and aging. SIRT1 expres- TZM-bl cells were transfected with (A) mimics control, miR-34a mimics, inhibitor sion is regulated transcriptionally and post-transcriptionally, and control, miR-34a inhibitor; (B) 5 mM NAM, 10 lM TSA or 100 nM Tat plasmid for 48 h. Ac-p65 (lysine 310) expression was assessed by western blot. Loading control its enzymatic activity is controlled by nicotinamide adenine dinu- + was obtained using anti-p65 antibody. Blots were representative of three cleotide (NAD ) levels and interacting proteins [26–28]. Tat also experiments. interacts with SIRT1. Binding of Tat to SIRT1 has two effects. First, SIRT1 deacetylates and inactivates Tat [29]. Second, Tat inhibits the SIRT1-mediated deacetylation of the p65 subunit of NF-jB, thus 3.4. Effect of miR-34a on Tat-induced NF- kB p65 acetylation in TZM-bl promoting the transcriptional activity of NF-jB [30]. Regulation cells of HIV-1 transcription is a complex, multistage process requiring cooperative action of both viral and cellular proteins [31]. The Tat directly interacts with the deacetylase domain of SIRT1 and LTR is regulated by viral proteins as well as host factors, including blocks the ability of SIRT1 to deacetylate lysine 310 in the p65 sub- NF-jB that becomes activated in virus-infected cells. The enhancer unit of NF-kB and inhibited LTR transactivation [9]. As shown in region of HIV-1 LTR-promoter contains two functional active NF- Fig. 5A, transfected with Tat plasmid cause an increase in acety- jB binding sites. NF-jB is a ubiquitous transcription factor critical lated lysine 310 (K310) in the p65 subunit of NF-kB in TZM-bl cells, in inflammation and immunity [32]. Previously, various reagents whereas enforced expression of miR-34a by transfection with miR- targeting the NF-jB signaling pathway have been examined for 34a mimics potentiated Tat-mediated increase in acetylated p65 their inhibitory effects on HIV-1 [33]. NF-jB signaling is indispens- (K310) in TZM-bl cells. However, silencing of miR-34a by transfec- able for T cell activation and HIV-1 preferentially replicates in pro- + tion of a miR-34a inhibitor suppressed Tat-induced increase in liferating CD4 T cells, partly by taking advantage of the activated acetylated p65 (K310) in TZM-bl cells. Treatment with SIRT1 inhib- host transcription machinery [34]. Data from previous studies itor nicotinamide potentiated Tat-mediated increase in acetylated showed that miR-34a directly inhibits SIRT1, a critical regulator p65 (K310); while treated with class I and II HDAC inhibitors TSA of stress responses, replicative senescence, inflammation, metabo- had no effect on Tat-mediated increase in acetylated p65 (K310) lism, and aging. We found that miR-34a inhibits Tat-induced NF- in TZM-bl cells (Fig. 5B). jB activation (as monitored by acetylation of p65). These results suggest that up-regulation of miR-34a may influence Tat-induced 4. Discussion HIV-1 transactivation by activation of NF-jB signaling pathway through down-regulation of SIRT1 expression. In sum, Tat induced Recent studies showed that miRNAs are involved in several up-regulation of miR-34 expression; miR-34a inhibited SIRT1 important biological events including HIV-1 infection. MiRNAs expression and thus led to an increase in acetylation of NF-jB are known to act as regulators in HIV-1 infection and replication p65 with NF-jB activation; NF-jB activation promoted HIV-1 [19–21]. In the study, we firstly verified that the expression level transactivation (Fig. 6). of miR-34a was significantly increased in Tat-treated TZM-bl cells In conclusion, the results of our study suggest that Tat caused and SupT1 T cells co-transfected with Tat plasmid and LTR-luc HIV-1 LTR transactivation as well as NF-jB activation via miR- plasmid. MiR-34a overexpression promoted Tat-induced HIV-1 34a down-regulating SIRT1 expression. The present research could transactivation, whereas its depletion decreased Tat-induced imply that elucidating the mechanism of the signaling pathway HIV-1 transactivation. We further demonstrated that miR-34a di- make it a candidate for treatment of AIDS and its associated rectly targeted 30-UTR of SIRT1 gene, and inhibited its expression. complications. Importantly, SIRT1 re-expression reduced Tat-induced HIV-1 transactivation induced by miR-34a. These data confirm that Acknowledgments miR-34a promoted Tat-induced HIV-1 transactivation, at least in part by repressing endogenous SIRT1. Additionally, we found that This study was supported in part by National Natural Sciences Tat-induced miR-34a expression affected p65 acetylation, which Foundation of China (No. 30800580); Beijing Natural Science Foun- was important for HIV-1 transactivation. dation (No. 5112004 & No. 5093025); Beijing Nova Program (No. Cellular miRNAs can influence HIV replication in two ways: di- 2007B014); National Basic Research Program of China (No. rect targeting of HIV-1 RNA sequences or indirect targeting of RNAs 2009CB930200). We would like to thank Dr. Ping-Kun Zhou for encoding cellular factors involved in HIV replication [22–23]. Be- his kind gift of the plasmid. H.-S. Zhang et al. / FEBS Letters 586 (2012) 4203–4207 4207

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