Human Cytomegalovirus Mir-UL70-3p Downregulates the H2O2 Induced Apoptosis in HEK293T Cells by Targeting the Modulator of Apoptosis-1 (MOAP1)

Abhishek Pandeya Babasaheb Bhimrao Ambedkar University Anup Mishra Babasaheb Bhimrao Ambedkar University Raj Kumar Khalko Babasaheb Bhimrao Ambedkar University Sukhveer Singh CSIR-Indian Institute of Toxicology Research Nishant Singh CSIR-Indian Institute of Toxicology Research Sanjay Yadav All India Institute of Medical Sciences Raebareli Sudipta Saha Babasaheb Bhimrao Ambedkar University Sangeeta Saxena Babasaheb Bhimrao Ambedkar University Sunil Babu Gosipatala (  gsbabu@bbau.ac.in ) Babasaheb Bhimrao Ambedkar University https://orcid.org/0000-0002-5377-817X


Introduction
Human Cytomegalovirus (HCMV) is a ubiquitous human pathogen belonging to the Betaherpesvirinae subfamily, which is able to maintain latent infection that persists throughout the life of the host [1]. It shows subclinical symptoms in an immunocompetent host but causes signi cant mortality and morbidity in infants and immunocompromised hosts. It is an enveloped DNA virus (≅ 230Kb) encoding more than 170 proteins and numerous long and small non-coding RNAs, making it the largest among the herpesviruses [2][3][4]. The genome of HCMV is divided into two segments, designated as UL (unique long) and US (unique short), bounded by inverted repeats [5] and contains ~ 150 open reading frames that encode proteins [6]. The gene expressions in this virus occur in temporal order; i.e., the rst set of viral gene products are classi ed as immediate early genes (IE), followed by the expression of early genes (E), and nally, the late gene (L) products. These gene products help the virus in causing latent infections by evading the host effective immune responses. Apoptosis, an innate antiviral immune response, is also inhibited by the viral proteins and miRNAs that helps in establishing and maintaining the latency in the infected host. HCMV encodes anti-apoptotic proteins through both the US [US4, US28] and UL [UL37, UL38 and UL144] regions of its genome [7,8]. The antiapoptotic proteins of HCMV, a viral inhibitor of caspase-8-induced apoptosis (vICA/pUL36), and mitochondria localized inhibitor of apoptosis (vMIA/pUL37X1) interferes in the cellular apoptosis by targeting pro-caspase 8 and Bcl2, respectively [9].

Materials And Methods
Cell Lines and Cell culture: HEK293T cell lines were procured from American Type Cell Culture (ATCC, USA) and grown in Dulbecco's Modi ed Eagle Media (Gibco) supplemented with 10% (v/v) Fetal Bovine Serum (Gibco, Brazil origin) and 1% antibiotic and antimycotic (Gibco). Cells were incubated at 37°C supplemented with 5% CO 2 in a humidi ed atmosphere and were routinely passaged after con uency reached up to 80%. The cells were routinely tested for mycoplasma infections.
Transfection and co-transfection: The miR-UL70-3p and its inhibitor were transfected with Dharmafect1 No T-2001-03 (Dharmacon, USA), whereas all the co-transfection experiments were carried out with Lipofectamine 3000 as per the manufacturer's protocol (L3000008-Invitrogen, USA). Vectors: The 3' UTR of MOAP1 was cloned in the pEZX-MT06 vector of Genecopoeia, USA, (cat No: HmiT016794-MT06 & Cat No: Cmi000001-MT06), which contains re y luciferase as the reporter gene (controlled by the SV40 promoter) and Renilla luciferase as the tracking gene (Controlled by the CMV promoter). 4',6-Diamidino-2-phenylindole (DAPI) staining: Apoptotic cell nuclei were observed by staining with 4′,6-diamidino-2-phenylindole (DAPI) (Himedia Cat No: TC 229) under the uorescent Microscope using the blue lter at emission/ excitation 358nm ⁄ 461nm. (Axiovert A1-Carl Zeiss-Jena-Germany) as per the protocol mentioned in Fig 1. Image analysis: The apoptotic cell nuclei and their condensation were analysed through ImageJ ver. 1.53 (National Institute of Health, Bethesda, MD). In brief, a nuclear morphology measurement, 16-bit photomicrographs of DAPI stained nuclei were converted to the 8-bit image before being the auto threshold to a binary photo using the default method "Make Binary" function in ImageJ. Touching cell nuclei were separated by the "watershed" function, and small fragments of nuclei were discarded on the basis of the area by the "analyzing particle" function. The latter function also provided other morphological parameters, including nuclear area, circumference, and form factor. By comparing the total numbers of cell nuclei obtained with watershed, an estimated percentage of apoptotic cells were computed by human intervention as the number of apoptotic cell nuclei (irregular shape and degraded shape)/ the total number of DAPI stained nuclei (with watershed) * 100, which provided the average percentage of apoptotic cells in each and every experimental setup.
Caspase-Glo 3/7 assay: The apoptotic induction and inhibition were assessed via Caspase-Glo 3/7 assay kit from Promega (Madison, WI, USA Cat No: G8090) using the luminometer (GloMax® Navigator System, Promega). Brie y, the cells were seeded in 96 well white plates followed by overnight incubation.
The HEK293T cells transfected with 30nM of hcmv-miR-UL70-3p & its inhibitor was treated with 0.4 mM H 2 O 2. The culture plate containing the cells were removed from the incubator and equilibrated at room temperature for 30 min. 100μL of Caspase-Glo reagent was added to each well and gently mixed in a plate shaker. The plate was then incubated at room temperature for 2 hrs. Luminescence was read using the Luminometer (GloMax® Navigator System, Promega). Caspase 3 and 7 activity was measured using raw values of luminescence to obtain a relative to control value. The nal caspase activity was calculated by averaging three replicates from two independent experiments.
Flow Cytometry: HEK293T cells were cultured in a 6-well plate followed by transfection, using DharmaFACT1 Then, samples were incubated with the 5µL of Propidium Iodide; (Invitrogen; Cat No: P1304MP respectively) in the dark for 10 min. Then samples were analyzed by diluting the cells with 400µL of 1X binding buffer by Flow Cytometry using FACS Canto II and FACSDiva software (Becton Dickinson Biosciences). The data represents the percentage of apoptotic cells in Q2 and Q4 quadrants, in which Q4 shows cell population having Annexin V + and PIdemonstrating early apoptosis, whereas Q2 shows cell population having Annexin V + and PI + demonstrating late apoptosis.

Scanning Electron Microscopy (SEM):
The SEM images were taken for normal and apoptotic cells from the Scanning Electron Microscope as described by Fischer et al., 2012 [23]. The Apoptotic/miRNA treated / control cells were added with freshly prepared 1ml of 2.5 % glutaraldehyde in PBS and incubated at 4ºC for 2-4 hrs. The culture petri dishes were thoroughly rinsed with PBS, three times for 10 minutes each. After primary xation of Glutaraldehyde, 1 ml of 1% osmium tetroxide in dH 2 O was added and incubated at room temperature for 1 hr. The cells were thoroughly rinsed with PBS, three times for 10 minutes each; then, cells were dehydrated through graded concentration of Acetone (30, 50, 70, 90, 95, and 100 %) for 5-15 minutes each. Then the cells were dried in a desiccator overnight. The samples were individually mounted on double-sided carbon tape, which was attached to a metal stab. These stabs were coated with electrically conducting metal, i.e., platinum, using a Sputter coater (JFC 1600; JEOL, Tokyo, Japan) at 20mA. The images were taken at different magni cations, i.e., 200X, 800X and 4000X, through Scanning Electron Microscope (JOEL-JSM6490LV).
qRT-PCR: HEK293T were harvested and total RNA was extracted using Pure Link RNA mini kit (Invitrogen, USA Cat No: 12183018A) according to the manufacturer's instruction. cDNA was synthesized using 1 μg total RNA using Proto Script® II First Strand cDNA Synthesis Kit (New England Biolabs Inc, USA Cat no: E6560S) as per the manufacturer instructions. qRT-PCR for MOAP1 was carried out using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems Cat No: A25742). All the primers were procured through IDT technologies, USA with following sequences: MOAP1; F-5' CACGAGCACTAGATCACGGCTGCTGGA 3' and R-CTGCCACACAGCAGCTCTGGGAGATGCC 3' [24], GAPDH; F-5' ACATCGCTCAGACACCATG 3' and R-5' TGTAGTTGAGGTCAATGAAGGG 3' [25]. The reactions were performed in an Agilent Aria Mx Real-Time PCR machine. The PCR conditions were as follows: UDG activation at 50ºC for 2 min, Hot start (Activation of SYBR Green) at 95 0 C for 2 min followed by denaturation at 95 0 C for 15 sec, annealing at 60 0 C for 1 min for 40 cycles along with the melt curve analysis as per the machine protocol. To con rm speci c ampli cation of the PCR product, dissociation curves were checked routinely, and uorescence was measured continuously. The relative mRNA expression was normalized to that of GAPDH in the corresponding sample by 2 -ΔΔCT . The measurement was done in triplicates, and the results are presented at the means ±SEM.
Dual-luciferase reporter assays: To assess the ability of hcmv-miR-UL70-3p for binding with the 3' UTR of MOAP1, these assays were performed. Brie y, HEK293T cells were seeded in a 12-well plate (3 × 10 5 cells/well), and after 24 hrs, they were transfected along with 30nM of miR-UL70-3p mimic; its inhibitors; both wild and deleted 3'UTR of MOAP1 cloned in pEZX-MT-06 vector using lipofectamine 3000 (Invitrogen) in triplicate wells. Luciferase activity was measured using the Dual-Luciferase Reporter assay system No E1910 (Promega Corporation, Madison, WI) in GloMax® Navigator Luminometer System, Promega as per manufacturer's protocol at 24 hrs post-transfection. All measurements were done in triplicate wells, and signals were normalized for transfection e ciency against the Renilla control. Data from three independent repetitions were presented as the mean ±SEM for the statistical analysis. shows a signi cant decrease in the Cas3/7 activity, which suggest that the miRNA downregulated the apoptosis. Further, to con rm whether the observed down regulatory effect was due to miR-UL70-3p, the cell group was simultaneously transfected with miR-UL70-3p, and it increased the Cas3/7 activity to RLU 4.77 X10 5 (Fig 2c).

SDS-PAGE and
c) Flow Cytometry: In addition to the above, we further evaluated the antiapoptotic effect of miR-UL70-3p through ow cytometry through Annexin-V and Propidium Iodide (PI). The cells were divided into 4 groups as mentioned above, and the apoptotic cell ratio in H 2 O 2 treated group was 11.21 percent, whereas the cells group transfected with miR-UL70-3p was 3.74 percent (decrement of 7.47%). Once we transfected with the miRNA and its inhibitor, the apoptotic cell ratio became 9.59 percent. It clearly showed that whenever we simultaneously transfected the cells with miR-UL70-3p and its inhibitor, the effect of miR-UL70-3p was be reversed (Fig. 2d & e).

D) SEM analysis:
We further analyzed the morphological differences through the Scanning electron microscope for the cell groups by taking images at 200x, 800x and 4000X magni cation in all the cells. Further, these images were converted to histograms based on mean cell surface coverage area. The results showed that the reduced surface area due to the treatment of H 2 O 2 was reversed when we transfected the cells with the miR-UL70-3p, suggesting that it decreased the process of apoptosis ( Fig. 2f & g).

hcmv-miR-UL70-3p targets MOAP1, thereby regulates the H 2 O 2 induced apoptosis?
Our earlier in-silico studies indicate that the proapoptotic gene, i.e., modulator of apoptosis 1 (MOAP1) was a potential target for hcmv-miR-UL70-3p.  (Fig 3a). In order to con rm, MOAP1 downregulation was due to miR-UL70-3p. We analysed the MOAP1 mRNA expression in the cells simultaneously transfected with miR-UL70-3p and its inhibitor; the results showed that the mRNA expression levels were increased compared to the miR-UL70-p treated cells. These results con rm that the miR-UL70-3p can downregulate the mRNA levels of MOAP1. b). miR-UL70-3p has potential binding sites in the 3'UTR of MOAP1 The above results con rm the downregulation of MOAP1 mRNA by hcmv-miR-UL70-3p; by performing the dual luciferase-based assays, we tried to prove the functionality of the binding site of miR-UL70-3p in the 3' UTR of the MOAP1. Our earlier studies show that the 3'UTR of MOAP1 has a potential binding site at the position of 527 for hcmv-miR-UL70-3p. The entire wild type (WT) and deleted (del) 3'UTR of MOAP1 dual-luciferase vector constructs were commercially procured and transfected in the cells to demonstrate the functionality of this binding site with miR-UL70-3p and measured for the luminescence. The results show that the luminescence was suppressed to 46.68% in the cells transfected with wild 3' UTR, compared to the 3'UTR deleted vector constructs, suggesting that the binding site predicted the 3'UTR region of MOAP1 was functional. Further, the luminescence was restored when we co-transfected with mir-UL70-3p inhibitor (Fig 3 b & c). These results con rm that the binding site at 527-549 of 3'UTR of MOAP1 is functional for hcmv-miR-UL70-3p. simultaneously treated with the mir-UL70-3p and its inhibitor. Further, we compared the downregulation of MOAP1 protein levels by the miR-UL70-3p with the commercially available siRNA for MOAP1. The extent of MOAP1 downregulation was similar for both these molecules suggesting that the miR-UL70-3p downregulates the MOAP1 (Fig. 3d & e).
3. Comparison of MOAP1 mRNA and its protein inhibitions by the hcmv-miR-UL70-3p and siRNA of MOAP1: After con rming the proapoptotic gene MOAP1 as a functional target for miR-UL70-3p, we further compared the extent of inhibition by miR-UL70-3p and the siRNA against MOAP1 on MOAP1 mRNA and its protein levels. The siRNA (27nt) was commercially procured and designed against 3' UTR of MOAP1 at position 38 to 66. The MOAP1 mRNA levels (Fig 4b & C)

Discussion
In the present study, we demonstrated the antiapoptotic role of HCMV encoded miRNA miR-UL70-3p in in vitro studies by using HEK293T cells. Further, we ascribed the antiapoptotic activity of this miRNA ability to bind with the 3'UTR of the proapoptotic mRNA modulator of Apoptosis 1 (MOAP1) and thereby downregulating the MOAP1 protein. MOAP1 is a proapoptotic protein capable of initiating the apoptosis by interacting with Bax and RASSF1A. Its expression in different cells vary and is expressed in the adipose tissues, adrenal, blood, breast, colon, and comparatively at higher levels in the brain and heart. It interacts with apoptosis regulator Bcl-2 associated X protein (BAX) through its Bcl-2 homology 3 (BH3)like motif and translocates the Bax on to the mitochondrial outer membrane leading to the polymerization. The Bax polymerization on the outer mitochondrial membrane results in to the increased permeability, thereby releases cytochrome C to the cytosol, initiating the mitochondrial dependent apoptosis [27][28][29][30]. At normal conditions, MOAP1 stays in an inactive conformation. However, it gets activated when it binds to RASSF1A and forms a death receptor complex after stimulated by TNFα and results in the opening of BH3 domain to allow to interact with Bax (Bcl-2 associated X protein). MOAP1 rapidly increases its concentration in the cytosol due to apoptotic stimuli and plays a pivotal role in initiating the mitochondrial-dependent apoptosis [31]. Targeting this proapoptotic protein by the HCMV miRNA miR-UL70-3p, HCMV downregulates the cellular apoptosis so that it can do productive infection at the early stages of infection. Being a Herpesviridae family of viruses, HCMV encodes 26 mature miRNAs, and they act in concert with the viral proteins in regulating the apoptosis for viral survival. Many research groups across the world actively engaged in deciphering the antiapoptotic role of HCMV miRNAs and understanding the ne tuning of HCMV proteins and miRNAs concordance in regulating apoptosis. The rst HCMV miRNA reported to have antiapoptotic activity was miR-UL148D, and in that study, the authors demonstrated inhibition of IEX-1 induced apoptosis in HEK293 by ectopically expressing the miR-UL148D [17]. By performing in silico studies, we also reported the antiapoptotic activity to the HCMV miRNAs, miR-UL70-3p and miR-UL148D [22], and predicted the proapoptotic gene, MOAP1 as a potential target for the miR-UL70-3p. In continuation, Kim et al. showed that ectopic expression of HCMV miRNAs, i.e., mir-UL36-3p, miR-US25-2p and miR-UL22A-3p downregulated the apoptosis by targeting the different caspases in Human Foreskin broblasts [19].
hcmv-miR-UL70-3p was rst reported by Grey F et al in 2005 through northern blot analysis and further reported to be expressed both in the latent and lytic phases of HCMV infections [32]; however, the authenticity of this miRNA need to be established [33]. miR-UL70-3p reported to regulate the cellular apoptosis [22] and also reported to regulate focal adhesions, gap junctions, MAPK signalling and Erb pathway, thus effecting epithelial cell migration and adhesion [34]. It was reported that hcmv-miR-UL70-3p expression was different in different cells; for example, it was poorly induced in human embryonic lung cells (HELs); moderately in undifferentiated THP-1 cells and strongly in differentiated THP-1 cells (monocyte cell lines) [14]. Recent literature shows that an elevated expression of miR-UL70-3p in Glioblastoma cells suggest the cancer regulatory potential of this miRNA [35]. Further, Zhong et al. proposed that, in addition to human cells, the expression of hcmv-miR-UL70-3p and the other HCMV miRNAs miR-US4 is observed in the diseased human dental pulp. According to their bioinformatics study, the HCMV encoded miRNAs target human in ammation, antiviral mechanisms, and angiogenesis-related genes of the human. [36].
In this study we evaluated the antiapoptotic activity of miR-UL70-3p in HEK293T cells on H 2 O 2 induced apoptosis. The apoptotic inhibition by this miRNA was measured as a percentage of apoptotic nuclei/apoptotic rate, inhibtion of effector Caspases 3/7 activity and membrane ru ing/blebbing in the cells. The uorescence microscopic studies reveal that the apoptotic nuclei were signi cantly downregulated in the cells transfected with the miR-UL70-3p, and the inhibition was reversed when the treated cells were co-transfected along with miR-UL70-3p inhibitor (Fig. 2b). Further, we measured the apoptosis inhibition by the miRNA by measuring the effector Caspase(s) 3/7 levels, and these levels were downregulated in the cells transfected with miR-UL70-3p followed by H 2 O 2 treatment, thereby showing inhibition of apoptosis when compared to the cells without the presence of this miRNA. The ow cytometry results con rm the antiapoptotic effect of miR-UL70-3p as there was a signi cant reduction of apoptotic cells (both early and late apoptotic nuclei) in the cells transfected with miR-UL70-3p. There was no signi cant reduction in early apoptotic cells but there was 7.3 percent reduction of apoptotic cells as observed in the late phase of apoptosis.
Our earlier in silico studies predicted that MOAP1 is the potential target for the mir-UL70-3p [22]; and has a single binding site in the 3'UTR at the position 527 to 549, to con rm this, we performed the dualluciferase assays with both wild and deleted UTR's of MOAP1vectors tarsnfected along with miR-UL70-3p and measured the reduction in raw luminescence unit through luminometer. The results show 46.68 percent reduction in the luminescence observed in the cells transfected with the 3' UTR of MOAP1 compared to the deleted 3' UTR suggesting that the miR-UL70-3p bind to the site 527-549 and the binding ability of the miRNA is functional. Though the predicted target sequence was not completely complementary to the whole sequence of miR-UL70-3p; however, it was completely complementary to the seed region of the miR-UL70-3p (2-8 position of the 5' miRNA). Human miRNAs hsa-miR-1228, miR-25 such as are also reported to target the MOAP1 and modulate the cellular apoptosis [24,37]. These human miRNAs do not share any sequence homology with the hcmv-miR-UL70-3p (supplementary information S1) and the binding site is also not common (supplementary information S1), suggesting neither their sequences nor their binding sites were common.
The MOAP1 protein expression levels were examined in the groups treated with and without miR-UL70-3p transfection, and the results show the MOAP1 protein expression was decreased in the cells transfected with the mir UL 70-3p. This clearly indicates that the miR-UL70-p downregulating the MOAP1 protein levels by targeting the mRNA of MOAP1 in the 3' UTR region. We further compared the inhibitory action of this miRNA with the commercially available siRNA against the MOAP1 UTR region and found that though the extent of protein inhibition was more for the siRNA when compared to miR UL 70-3p.

Supplementary Files
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