SV40 microRNA miR-S1-3p Downregulates the Expression of T Antigens to Control Viral DNA Replication, and TNFα and IL-17F Expression

Misa Tokorodani; Hirona Ichikawa; Katsutoshi Yuasa; Tetsuyuki Takahashi; Takao Hijikata

doi:10.1248/bpb.b20-00415

Abstract

SV40-encoded microRNA (miRNA), miR-S1, downregulates the large and small T antigens (LTag and STag), which promote viral replication and cellular transformation, thereby presumably impairing LTag and STag functions essential for the viral life cycle. To explore the functional significance of miR-S1-mediated downregulation of LTag and STag as well as the functional roles of miR-S1, we evaluated viral DNA replication and proinflammatory cytokine induction in cells transfected with simian virus 40 (SV40) genome plasmid and its mutated form lacking miR-S1 expression. The SV40 genome encodes two mature miR-S1s, miR-S1-3p and miR-S1-5p, of which miR-S1-3p is the predominantly expressed form. MiR-S1-3p exerted strong repressive effects on a reporter containing full-length sequence complementarity, but only marginal effect on one harboring a sequence complementary to its seed sequence. Consistently, miR-S1-3p downregulated LTag and STag transcripts with complete sequence complementarity through miR-S1-3p-Ago2-mediated mRNA decay. Transfection of SV40 plasmid induced higher DNA replication and lower LTag and STag transcripts in most of the examined cells compared to that miR-S1-deficient SV40 plasmid. However, miR-S1 itself did not affect DNA replication without the downregulation of LTag transcripts. Both LTag and STag induced the expression of tumor necrosis factor α (TNFα) and interleukin (IL)-17F, which was slightly reduced by miR-S1 due to miR-S1-mediated downregulation of LTag and STag. Forced miR-S1 expression did not affect TNFα expression, but increased IL-17F expression. Overall, our findings suggest that miR-S1-3p is a latent modifier of LTag and STag functions, ensuring efficient viral replication and attenuating cytokine expression detrimental to the viral life cycle.

INTRODUCTION

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of many genes by binding to target mRNAs with imperfect complementarity and induce translational repression and/or mRNA decay. They are involved in diverse biological processes, such as development, tumorigenesis, immune function, and cell death.^1–3) In addition to metazoans and plant genomes, they are also encoded by viruses, such as retroviruses, herpesviruses, and polyomaviruses. Growing evidence suggests that viral miRNAs contribute to the regulation of viral replication, host immune evasion, and cellular transformation.^4–12)

The polyomavirus simian virus 40 (SV40) contains a circular double-stranded DNA genome which encodes a regulatory region (bidirectional promoter and replication origin), early viral genome region which encodes the regulatory proteins large and small T antigens (LTag and STag, respectively), and late viral genome region, expressed from complementary strand of the genome, which encodes miRNA and viral capsid protein, VP1-3.^13,14) LTag proteins play pivotal roles as initiators of viral DNA replication.¹⁵⁾ The LTag proteins assemble into hexamers and bind to the replication origin within the regulatory region to unwind the duplex DNA.^16–18) In addition to its role as a helicase during initiation of replication, LTag directly associates with cellular replication protein A (RPA) at the origin to load it on the unwinding DNA template¹⁹⁾ and also recruits other cellular proteins required for the replication, such as DNA polymerase alpha-primase and topoisomerase I, to the viral origin.^18,20,21) Despite these pivotal roles in DNA replication, LTag is downregulated by the SV40 miRNA, miR-S1, which did not affect the yield of infectious viruses.¹³⁾ The miRNA-mediated downregulation of LTag and STag is commonly found among other polyomaviruses, where viral miRNAs are able to efficiently limit DNA replication.^22–25) This suggests that polyomavirus miRNAs, including the SV40 miR-S1, dampen or are dispensable for viral replication, thereby making not only a functional role of the miRNA but also the functional significance of miRNA-mediated LTag downregulation confounding.

SV40 LTag induces the expression of tumor necrosis factor α (TNFα) and interferon β (IFNβ), which inhibit viral replication through the direct induction of an intracellular antiviral state.^26,27) LTag (LT339) and STag from another polyomavirus, Merkel cell polyomavirus (MCV), also strikingly increase the expression of interleukin genes encoding interieukin-1β (IL-1β), IL-6, and IL-8 and chemokine genes encoding CXCL1, CXCL6, and CCL7.²⁸⁾ These cytokines and chemokines are involved in host defense against viral infection by enhancing the response and recruitment of neutrophils and macrophages.²⁹⁾ To reduce these cytokines and chemokines that are unfavorable to the viral life cycle, the polyomavirus may utilize miRNA, which downregulates their inducers, or both LTag and STag. However, whether polyomavirus miRNA can attenuate cytokine induction indirectly by downregulating LTag and STag or directly by inducing translational repression or decay of the cytokine mRNA has not yet been determined.

To elucidate the functional roles of miR-S1 in DNA replication and induction of proinflammatory cytokines and chemokines, we evaluated DNA replication of SV40 genome plasmid with or without miR-S1 expression in various cell types, and also the induction of TNFα, IL17F, and IL8 genes in HEK293 cells transfected with SV40 plasmids with or without miR-S1. Furthermore, we assessed the direct impact of miR-S1 on DNA replication and cytokine expression in HEK293 cells transfected with miR-S1 expression vectors. Our results suggest that miR-S1 prevents LTag overexpression, which negatively affects DNA replication, thereby ensuring efficient DNA replication in most of the cell types examined. Moreover, miR-S1 downregulated LTag and STag to reduce LTag- and STag-mediated induction of TNFα and IL-17F genes. However, miR-S1 did not repress DNA replication or cytokine induction, per se.

MATERIALS AND METHODS

Cell Culture

Human embryonic kidney (HEK293), adenocarcinomic human alveolar basal epithelial (A549), human rhabdomyosarcoma (RD), and human fetal lung fibroblast (WI-38) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. Normal human dermal fibroblasts (HDFs) and keratinocytes (HDKs) were obtained from PromoCell (Heidelberg, Germany) and cultured according to the manufacturer’s instructions.

Plasmid Construction

The plasmids used were SVori-8-16, pMK16, SVori-8-16-dl.miR-S1, wSV40, and wSV40-dl.miR-S1, LTag, STag, and miR-S1 expression plasmids, in addition to a battery of luciferase reporter plasmids for the evaluation of the repressive effects of miR-S1. SVori-8-16 was obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank.³⁰⁾ The pMK16 plasmid was prepared by the excision of approximately 4-kb DNA fragment obtained from BamHI-digestion of SVori-8-16. The SVori-8-16-dl.miR-S1 plasmid was prepared by PCR using the KOD-Plus-Mutagenesis Kit (Promega, WI, U.S.A.) with SVori-8-16 as a template and the following primers: Fwd 5′-gtgaatctatccctgtctcgtgtcctgaatcttccatgttcttctccccaccatc-3′ and Rev 5′-agtcccagggatcgttccaggcacctcagtcctcacagtctgttcatgatc-3′. The wSV40 and wSV40-dl.miR-S plasmids were prepared by inserting the four missing nucleotides into SVori-8-16 and SVori-8-16-dl.miR-S1 plasmids, respectively, using the mutagenesis kit and the following primers: Fwd 5′-ggcctcggcctctgcataaataaaaaaaattagtcagcc-3′ and Rev 5′-gcctcggcctctgagctattccagaagtagtgaggagg-3′. For the construction of LTag expression plasmid, LTag cDNA was amplified by PCR using the following primers: Fwd 5′-ataagaatgcggccgcatggataaagttttaaacagagagg-3′ and Rev 5′-cggaattcttatgtttcaggttcagggggag-3′, and the DNA template of the mutated SVori-8-16 plasmid with deletion of intron 4572–4917 using the mutagenesis kit and the following primers: Fwd 5′-attccaacctatggaactgatgaatg-3′ and Rev 5′-ctcagttgcatcccagaagcctc-3′. STag cDNA was amplified by PCR using the DNA template of SVori-8-16 and the following primers: Fwd 5′-ataagaatgcggccgcatggataaagttttaaacagagagg-3′ and Rev 5′-cggaattcttagagctttaaatctctgtaggtag-3′. These amplified LTag and STag cDNAs were subcloned into pQCXIP plasmids (Clontech, CA, U.S.A.) at the NotI and EcoRI sites. For the construction of the miR-S1 expression plasmid, the pre-miR-S1 DNA fragment was amplified by PCR using the DNA template of SVori-8-16 and the following primers: Fwd 5′-gcgctcgaggatccgaggactgaggggcctgaaatg-3′ and Rev 5′-gcgaattcgaagactcagggcatgaaacag-3′, and subcloned into a pZac plasmid at the XhoI and EcoRI sites. This subcloned plasmid harbored a DNA fragment composed of a CMV promoter and its downstream pre-miR-S1 sequence with a BglII site at the 5′-end and an EcoRI site at the-3′-end. This DNA fragment was further subcloned into pSIREN-RetroQ plasmids (Clontech) digested with BglII and EcoRI. A battery of luciferase reporter plasmids was prepared by PCR-based amplification of the pRL-CMV plasmid using the mutagenesis kit and a pair of primers corresponding to each construct. All constructed plasmids were verified by sequencing (Genetic analyzer 3500, Applied Biosystems, MA, U.S.A.).

Transfection and Cytokine Induction with or without TNFα Treatment

Cells cultured in 12-well plates were transfected using TransIT^®-293 (Mirus Bio) or PEI Max (Polyscience) for HEK293 cells and ViaFect (Promega) for the other cells. Prior to treatment with TNFα, wSV40, and wSV40-dl.miR-S1, or pMK16 plasmid (1200 ng) were cotransfected with an empty pQCXIP plasmid (300 ng) encoding a puromycin-resistance marker into HEK293 cells for 8 h. Subsequently, the transfected cells were selected with culture medium containing 1 µg/mL puromycin for 64 h and then treated with either DMEM alone or DMEM containing 50 ng/mL TNFα for 6 h. In another series of experiments involving treatment with TNFα, miR-S1, LTag, or STag expression plasmids (1500 ng) containing a puromycin-resistance marker were transfected into HEK293 cells. The cells were selected with puromycin and treated with either DMEM or DMEM containing 50 ng/mL TNFα. HEK293 cells were co-transfected with increasing concentrations of LTag or STag plasmid (0, 150, 300, 600, and 1500 ng) and correspondingly decreasing concentration of pQCXIP plasmid (1500, 1350, 1200, 600, and 0 ng, respectively) and treated as described above.

Luciferase Reporter Assay

HEK293 cells were transfected with the internal control pGL3 (Promega), a pRL-miR-S1 reporter plasmid, and SVori-8-16, SVori-8-16-dl.miR-S1, or pre-miR-S1 plasmid at the ratio of 1 : 5 : 25. At 48 h post-transfection, the cells were harvested and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was normalized to the transfection efficiency by calculating the ratio of pRL-miR-S1 reporter activity to pGL3 activity. To exclude any indirect effect of miR-S1 on luciferase activity by affecting the pRL promoter, pRL-miR-S1/pGL3 activity was further normalized to pRL-control/pGL3 activity.

RNA Isolation and Real-Time (RT)-Quantitative (q)PCR

Total RNA was isolated from the transfected cells using Isogen reagent (Nippon Gene) or NucleoSpin^® TriPrep (Macherey–Nagel) according to the manufacturer’s protocol. Mature miRNA expression was quantified using RT-qPCR using specific TaqMan probes (Applied Biosystems) for miR-S1-3p (Cat. #4440886), miR-S1-5p (Cat. #4440886), and U6 snRNA (Cat. #4427975) on a 7500 Fast Real-Time PCR System (Applied Biosystems). For evaluation of gene expression, cDNA was synthesized from 1 µg of total RNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems/Thermo Fisher Scientific) and adapter oligo dT primer 5-ctgatctagaggtaccggatccttttttttttttttt-3′ for LSTag transcripts including LTag and STag or a random hexamer for endogenous cell genes and total LSTag transcripts expressed from both SV40 and LTag expression plasmids. The adapter oligo dT primer enabled us to detect only the LSTag transcripts and to exclude the detection of plasmid DNA encoding LTag, which was an inevitable contaminant due to transfection. RT-qPCR analysis was performed by using SYBR^® Premix Ex Taq II (TaKaRa Bio) and gene-specific primers as follows: LSTag Fwd 5′-cctcagtcctcacagtctgttc-3′ and Rev (adaptor primer) 5′-ctgatctagaggtaccggatcc-3′; β-actin Fwd 5′-tccctggagaagagctacga-3′ and Rev 5′-agcactgtgttggcgtacag-3′; IL8 Fwd 5′-agacagcagagcacacaagc-3′ and Rev 5′-aggaaggctgccaagagag-3′; TNFα Fwd 5′-cagcctcttctccttcctgat-3′ and Rev 5′-gccagagggctgattagaga-3′; IL17F Fwd 5′-gaagcttgacattggcatca-3′ and Rev 5′-gtgtaattccagggggaggt-3′. For quantification of total LSTag expressed from both wSV40 and LTag expression plasmids (shown in Figs. 5B and C), total RNA isolated with NucleoSpin^® TriPrep was used for RT-qPCR analyses with LSTag specific primers (Fwd 5-aggctatcaacccgcttttt-3′ and Rev 5-aaagaacagcccagccacta-3′) because these RNAs were treated on the column with deoxyribonuclease (DNase) to eliminate contamination with plasmid DNA by transfection.

Immunoprecipitation

HEK293 cells were transfected with SVori-8-16 or SVori-8-16-dl.miR-S1 plasmids and the cells were harvested 48 h post-transfection. The harvested cells were lysed with lysis buffer (25 mM Tris–HCl pH 7.4, 150 mM KCl, 0.5% NP-40, 20% glycerol, 1 mM NaF, 0.5 mM DTT, and 2 mM ethylenediaminetetraacetic acid (EDTA)) supplemented with proteinase inhibitors (cOmplete™ ULTRA tablets; Roche). The lysates were precleared with protein G-conjugated magnetic beads (Invitrogen) and then incubated with monoclonal anti-human Ago2 antibody (Wako) and protein G-conjugated magnetic beads. Concentrated magnetic beads harboring the immunoprecipitates were washed with the wash buffer (50 mM Tris–HCl pH 7.4, 300 mM KCl, 0.1% NP-40, and 1 mM MgCl₂) and subjected to RNA isolation for RT-qPCR analysis of miR-S1-3p and LSTag.

DNA Replication Assay

Various cell types were transfected with SVori-8-16, wSV40, or wSV40-dl.miR-S1 plasmids for 4–8 h and harvested at 48 h post-transfection. Total DNA was isolated from the harvested cells using NucleoSpin^® TriPrep. Equal amounts of isolated DNA were treated with 18 U of DpnI or buffer alone at 37 °C for 2.5 h. The treated and untreated DNA specimens were used as templates for RT-PCR analysis with the following primers Fwd 5′-ggcagcctatgattggaatg-3′ and Rev 5′-atacccacgccgaaacaag-3′. Since these primers spanned the amplicon which included two DpnI sites, DpnI treatment was used to exclude detection of the introduced plasmid DNA that remained in the transfected cells. DNA replication, which is represented as the amount of plasmid DNA remaining following DpnI treatment per amount of plasmid DNA introduced into cells, was evaluated. Accordingly, the formula of DNA replication with Ct (cycle of threshold) values can be represented as follows: 2^-^Dpn^{I-treatment(Ct)}/2^{-untreatment(Ct)}−2^-^Dpn^{I-treatment(Ct)}.

Immunoblot Analysis

Lysates were prepared from the transfected cells in sodium dodecyl sulfate (SDS) sample buffer. Protein concentration was determined using the Bradford assay (Bio-Rad). For immunoblotting, equal amounts of lysates were subjected to SDS polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 4% nonfat milk in Tris-buffered saline (TBS) and incubated with an anti-LTag antibody (Abcam). After washing with TBS containing 0.1% Tween 20, the membrane was incubated with secondary HRP-conjugated antibodies (Pierce/Thermo Fisher Scientific), washed with TBS containing 0.1% Tween 20, and then subjected to chemiluminescent detection.

Statistical Analysis

Data from three independent experiments are represented as the mean with error bar displaying standard deviation (S.D.). Significant difference between the two groups was determined using unpaired two-tailed Student’s t-test. Statistically significant differences are indicated as ** p < 0.01 or * p < 0.05.

RESULTS

SV40 Genome Plasmids with a Deletion of the Viral Origin Express Functionally Active miR-S1 in HEK293 Cells

SVori-8-16 plasmid harboring SV40 genomic DNA was used in transfection experiments to determine the temporal expression pattern of SV40 miRNAs. The SVori-8-16 plasmid consists of BamHI-digested SV40 genomic DNA, with a deletion of four nucleotides in the viral origin, in the BamHI-digested pMK16 plasmid backbone (Figs. 1A, B). Therefore, it lacks the regions that encode the capsid proteins VP1-3 and viral DNA replication.³⁰⁾ HEK293 cells were transfected with SVori-8-16 plasmid and harvested at various time points, and the expression of miR-S1-3p and miR-S1-5p (derived from pre-miR-S1, see Figs. 1A, 2A) was evaluated using TaqMan probe-based qPCR assays. While the expression of miR-S1-3p gradually increased by approximately 100-fold at 96 h post-transfection relative to that at 12 h post-transfection, the expression of miR-S1-5p remained at a steady state low level, suggesting its destabilization (Fig. 1C). Concomitantly, the expression of the T antigen transcript (LSTag), which includes both LTag and STag, was also evaluated using RT-qPCR as separate quantification of LTag and STag mRNAs was not possible due to sequence identity. LSTag expression reached a maximum level at approximately 72 h post-transfection (Fig. 1D).

Fig. 1. SVori-8-16 Transfection Induces Expression of Functionally Active miR-S1

(A) Map of SVori-8-16 plasmid lacking four nucleotides (AGGC) in the viral origin (ori). EP, early promoter; LP, late promoter; VP1-3, capsid proteins; Tet^R, tetracycline resistance gene; Kan^R, kanamycin resistance gene. Dotted line represents 3′ UTR of STag mRNA. The green arrows indicate the pair of primers used for the evaluation of DNA replication using RT-qPCR. (B) Schematic representation of the origin of wSV40 and SV ori-8-16. Four repeats of the sequence 5′-GAGGC-3′ are underlined. (C) Temporal expression profile of the SV40 miRNAs, miR-S1-3p and miR-S1-5p. HEK293 cells were transfected with SVori-8-16 plasmid (Fig. 1A) and harvested at various time points. Total RNA was isolated, reverse-transcribed, and real-time (RT) PCR analyses were performed using TaqMan miRNA assays. The miRNA expression was normalized to the expression of the small nuclear RNA, U6. (D) Temporal expression profile of LSTag transcript. Total RNA isolated from SVori-8-16 transfected cells was reverse-transcribed into cDNA using the adaptor-oligo dT15 primer. SYBR RT-qPCR analyses were performed using adapter and gene-specific primers. β-Actin was used as a reference gene. (E) Evaluation of the repressive effects of pre-miR-S1 expressed by SVori-8-16 transfection. The luciferase reporter plasmids harboring nucleotide sequence complementary to pre-miR-S1 were cotransfected with either the SVori-8-16 plasmid or pMK16 and pGL3 plasmids into HEK293 cells. Renilla luciferase activity was normalized to firefly luciferase activity. The obtained data were further normalized to exclude the effects of miR-S1 on the pRL promoter (see Materials and Methods). (C–E) Data are presented as the mean ± standard deviation (S.D., n = 3). ** p < 0.01. (Color figure can be accessed in the online version.)

To assess whether the miR-S1 expressed from the SVori-8-16 plasmid was functionally effective, luciferase reporter assay was carried out using a reporter plasmid harboring oligonucleotide complementary to the pre-miR-S1 sequence downstream of the luciferase gene. This reporter plasmid was co-transfected with SVori-8-16 or control pMK16 plasmids into HEK293 cells, followed by the measurement of luciferase activity. SVori-8-16 transfection caused approximately 50% decrease in the luciferase signal compared to that of control pMK16 transfection (Fig. 1E). Collectively, these results indicate that SVori-8-16 plasmids can express a functionally effective miR-S1, possibly miR-S1-3p.

Identification of Target Sequences Responsible for the Repressive Effects of miR-S1

To compare the repressive effects of miR-S1-3p and miR-S1-5p, and to identify the target sequences responsible for their repressive effects, we performed reporter assays in HEK293 cells in which pre-miR-S1 expression plasmids were cotransfected with reporter plasmids harboring complementarities to various parts of the pre-miR-S1 sequence downstream of the luciferase gene, as shown in Fig. 2A. Upon pre-miR-S1 expression, the pre-miR-S1 reporter containing a perfect complementarity to the pre-miR-S1 sequence exhibited markedly reduced luciferase activity (approx. 15% of the control activity). The miR-S1-3p reporter containing nucleotide sequence complementary to miR-S1-3p also displayed markedly decreased activity (approx. 20% of control activity), whereas the miR-S1-5p reporter with miR-S1-5p-complimentary sequence also exhibited a statistically significant reduction, but to a much lesser extent than the miR-S1-3p reporter (approx. 90% of the control activity) (Fig. 2B). These results, along with the temporal expression patterns (Fig. 1C), indicate that miR-S1-3p is the mature miRNA most represented by the dominant repressive effects. Next, we examined the sequence responsible for miR-S1-3p -mediated repressive effects. Upon pre-miR-S1 expression, the levels of the reporter harboring a 10-nucleotide (nt) sequence complementary to positions 1−10 nt at the 5′-end of miR-S1-3p, containing its full seed sequence, exhibited a slight but statistically significant reduction (Fig. 2B). However, activity of the reporter containing a 10-nt sequence complementary to the 3′-end or the middle part of mature miR-S1-3p (positions 11−20 or 6−15 at the 5′-end) was not reduced.

Fig. 2. Identification of miR-S1 Target Sequences Responsible for Its Repressive Effects

(A) Schematic representation of luciferase reporter constructs containing nucleotide sequence complementary to various parts of the pre-miR-S1 nucleotide sequence. (B) Pre-miR-S1 expression plasmid was cotransfected with control or luciferase reporter plasmids into HEK293 cells. pRL plasmid was used as control, and its normalized luciferase activity was set as 1 for relative luciferase activity. Data are shown as mean ± S.D. (n = 4). ** p < 0.01. (Color figure can be accessed in the online version.)

SV40 miR-S1-3p Downregulates LSTag Expression through miR-S1-Ago2-Mediated mRNA Decay

SV40 miR-S1 downregulates the expression of the LSTag transcripts, as both miR-S1-3p and miR-S1-5p are perfectly complementary to LSTag mRNAs, thereby directing the cleavage of LSTag mRNAs through the RNA-mediated interference complex (RISC).^13,31) To verify this RISC-mediated downregulation of LSTag mRNA, we performed Ago2 immunoprecipitation (IP) experiments wherein, as a control plasmid, we constructed and used SVori-8-16 plasmid defective in pre-miR-S1 expression. This SVori-8-16-dl.miR-S1 plasmid contains mutated sequences at the pre-miR-S1 site on the late strand, as illustrated in Fig. 3A, but intact amino acid-coding potential of LTag to eliminate pre-miR-S1 expression.

Fig. 3. SVori-8-16-dl.miR-S1 Transfection Does Not Induce Expression of miRNAs That Downregulate LSTag Expression

(A) Schematic representation of the mutated nucleotides (blue) within the miR-S1-5p and miR-S1-3p nucleotide sequences (red) in the SVori-8-16-dl.miR-S1 (lower) and SVori-8-16 plasmids (upper). The nucleotide sequence of miR-S1 is depicted in reverse orientation to the sequence illustrated in Fig. 2A. (B) Schematic diagram showing the secondary structure of pre-miR-S1 and mutated pre-miR-S1. Highlighted sequences are the miRNAs corresponding to 5p-arm (blue) or 3p-arm (red). (C) Reporter plasmids containing nucleotide sequence complementary to either pre-miR-S1 of SVori-8-16 or mutated pre-miR-S1 of SVori-8-16-dl.miR-S1 sequences were cotransfected with pMK16, SVori-8-16, or SVori-8-16-dl.miR-S1 plasmids into HEK293 cells. The reporter activity observed in pMK16 transfection was normalized and set as 1 for relative luciferase activity. Data are shown as mean ± S.D. (n = 3). (D) SVori-8-16 or SVori-8-16-dl.miR-S1 plasmids were transfected into HEK293 cells, and the cell lysates were analyzed for LTag expression using immunoblotting. β-actin was used as a loading control. (E) Temporal expression profiles of LSTag transcripts in SVori-8-16- and SVori-8-16-dl.miR-S1-transfected HEK293 cells. The relative expression of LSTag transcripts was measured using RT-qPCR. Data are shown as mean ± S.D. (n = 4). (F) miR-S1-3p and LSTag transcripts were significantly higher in Ago2-immunoprecipitates of SVori-8-16-transfected HEK293 cells, compared to those of SVori-8-16-dl.miR-S1-transfected cells. Cell lysates from SVori-8-16- and SVori-8-16-dl.miR-S1-transfected HEK293 were immunoprecipitated with anti-Aog2 antibody or normal IgG, followed by RNA isolation and RT-qPCR analysis. The results are presented as LSTag mRNA precipitated by the anti-Ago2 antibody relative to that precipitated by normal IgG. Data are shown as mean ± S.D. (n = 3). (C and F) ** p < 0.01. (Color figure can be accessed in the online version.)

Prior to the IP experiments, we verified whether the SVori-8-16-dl.miR-S1 plasmid expresses a functionally effective mutated-pre-miR-S1 to downregulate mutated LSTag through base-pairing of complementary sequences. First, SVori-8-16-dl.miR-S1, SVori-8-16, or pMK16 plasmids were cotransfected with the reporter plasmid carrying the sequence complementary to that of pre-miR-S1 and evaluated for their repressive effects on the reporter activity. SVori-8-16 transfection reduced the reporter activity, but SVori-8-16-dl.miR-S1 transfection did not (Fig. 3B). However, this lack of reduction in luciferase activity by SVori-8-16-dl.miR-S1 transfection may be due to imperfect complementarity between the sequences of the reporter gene and mutated pre-miR-S1, which may eventually be expressed. To assess this possibility, a reporter plasmid carrying a nucleotide sequence complementary to the mutated pre-miR-S1 sequence was cotransfected with SVori-8-16-dl.miR-S1 or SVori-8-16 and used to evaluate luciferase activity. Neither SVori-8-16-dl.miR-S1 nor SVori-8-16 reduced the reporter activity (Fig. 3C). Moreover, Mfold secondary structure analysis of the mutated pre-miR-S1 predicted a distorted secondary hairpin structure with internal bulges (Fig. 3B, also see Supplementary Fig. 1 for details). Collectively, these results indicate that the SVori-8-16-dl.miR-S1 plasmid abrogated miRNA expression and lost the ability to downregulate LSTag expression. Consistently, SVori-8-16-dl.miR-S1 transfection remarkably increased the expression level of LTag protein and LSTag transcripts, compared to that of SVori-8-16 transfection (Figs. 3D, E).

The reduced LSTag expression observed in SVori-8-16 transfection resulted from Ago2-miR-S1-mediated LSTag mRNA decay. To verify this, we performed IP experiments wherein whole cell lysates obtained from HEK293 cells transfected with SVori-8-16 or SVori-8-16-dl.miR-S1 were immunoprecipitated with either an anti-Ago2 antibody or normal immunoglobulin G (IgG). Total RNA was isolated from Ago2-IP or IgG-IP complexes and subjected to RT-qPCR analysis to evaluate the relative amounts of LSTag mRNA and miR-S1-3p. Anti-Ago2 antibody immunoprecipitated more LSTag mRNA along with miR-S1-3p in SVori-8-16-transfected cells than in SVori-8-16-dl.miR-S1 (Fig. 3F), indicating that the reduction of LSTag mRNA occurred through miR-S1-Ago2-mediated mRNA decay.

SV40 Plasmid with an Intact Viral Origin Exhibits Higher Level of Viral DNA Replication than the SVori-8-16 Plasmid

To explore the functional roles of miR-S1 in viral DNA replication, we prepared an SV40 plasmid with intact viral origin by inserting the four missing nucleotides of AGG C into the SVori-8-16 plasmid, as shown in Figs. 1A and B. First, this wSV40 plasmid was compared with the SVori-8-16 plasmid for its DNA replication ability. Each plasmid was transfected into HEK293 cells, from which total DNA (including plasmid DNA) was isolated and treated with or without DpnI, followed by RT-qPCR analysis of DNA replication. This analysis revealed significantly increased DNA replication in the wSV40 plasmid-transfected cells compared to that of the SVori-8-16 plasmid, which remained replication-competent albeit with 1/3 replication rate relative to wSV40 plasmid (Figs. 4A, B).

Fig. 4. wSV40 Transfection Increases SV40 DNA Replication but Decreases LSTag and miR-S1-3p Expression

(A) Schematic diagram showing the principle of the viral DNA replication assay. wSV40 and wSVori-8-16 plasmids, extracted from Esherichia coli (dam⁺ strain), were transfected into cells. Cells were harvested at 48 h post-transfection and subjected to DNA extraction. Replicated DNA (red) lacking methylation at DpnI site (white arrowhead) and template plasmid DNA that retains methylation at DpnI site (black arrowhead) were treated with or without methyl residue-sensitive DpnI. The amount of plasmid DNA in the untreated and treated specimens was quantified using RT-qPCR with the primers (green arrow). DNA replication was calculated by dividing the amount of plasmid DNA remaining after DpnI treatment by the amount of plasmid DNA introduced into the cells. (B) DNA replication of wSV40 and SVori-8-16 in HEK293 cells. (C) LSTag expression in wSV40- and wSVori-8-16-transfected HEK293 cells was evaluated using RT-qPCR (left) and immunoblotting (right). (D) miR-S1-3p expression was quantified in wSV40- and wSVori-8-16-transfected HEK293 cells using TaqMan miRNA assays. (B– D) Data are shown as mean ± S.D. (n = 4). * p < 0.05 and ** p < 0.01. (Color figure can be accessed in the online version.)

We next compared the expression levels of LSTag and miR-S1-3p between wSV40- and SVori-8-16-transfected cells. RT-qPCR and immunoblot analyses showed that the expression levels of LSTag transcripts and LTag proteins were significantly lower in wSV40-transfected cells compared to the SVori-8-16-transfected cells (Fig. 4C). Similarly, miR-S1-3p expression was also lower in wSV40-transfected cells (Fig. 4D), suggesting that the viral origin site, especially the central palindrome containing the four repeats of 5′-GAG GC-3′ sequence, may be involved in the control of miR-S1 expression.

Exogenous LTag Overexpression Reduces DNA Replication of wSV40 Plasmids in HEK293 Cells

SV40 LTag promotes DNA replication of the viral genome.¹⁸⁾ Thus, it is reasonable to expect that transfection of wSV40-dl.miR-S1 plasmid defective in miR-S1 expression (see Fig. 3A) would increase LTag expression and induce higher replication than that of wSV40. As expected, HEK293 cells transfected with wSV40-dl.miR-S1 showed increased LSTag expression and induced higher replication than wSV40 transfection (Fig. 5A). Next, to test whether additional exogenous LTag expression further increase DNA replication, increasing concentrations of LTag expression plasmid were cotransfected with the wSV40 plasmid. The results showed that DNA replication increased with increasing expression levels of LTag until a threshold was reached, and then decreased when LTag was overexpressed beyond the threshold (Fig. 5B). These results suggest the importance of an optimal amount of LTag for efficient DNA replication.

Fig. 5. Expression Level of LTag in HEK293 Cells Affects DNA Replication

(A) DNA replication and LSTag and miR-S1-3p expression were determined in wSV40- and wSV40-dl.miR-S1-transfected HEK293 cells using RT-qPCR. Data are shown as mean ± S.D. (n = 5). (B) Increasing concentration of LTag expression plasmid (0, 150, 450, and 1300 ng) in combination with pQCXIP plasmid (1300, 1150, 850, and 0 ng, respectively) was cotransfected with wSV40 plasmid (300 ng), and DNA replication and total LSTag expression were evaluated using RT-qPCR. Data are shown as mean ± S.D. (n = 3). (C) Either miR-S1 expression plasmid or control plasmid was cotransfected with wSV40-dl.miR-S1 plasmid, and DNA replication and LSTag and miR-S1-3p expression levels were determined using RT-qPCR. Data are shown as mean ± S.D. (n = 4). (D) Increasing concentrations of miR-S1 expression plasmid (0, 150, 450, and 1300 ng) in combination with control plasmid (1300, 1150, 850, and 0 ng,) were cotransfected with wSV40 plasmids (300 ng), and DNA replication and LSTag and miR-S1-3p expression levels were evaluated using RT-qPCR. Data are shown as mean ± S.D. (n = 3). (A–E) * p < 0.05 and ** p < 0.01.

MiR-S1 expression, per se, did not affect DNA replication without LTag downregulation. This was verified by evaluating LSTag transcript expression and viral plasmid DNA replication in HEK293 cells cotransfected with wSV40-dl.miR-S1 plasmid and either a control or pre-miR-S1 expression plasmid. This experiment was performed using wSV40-dl.miR-S1 plasmid. This plasmid enabled the assessment of the effects of miR-S1 on DNA replication which was not through its LTag downregulation because miR-S1 could not downregulate LSTag transcripts expressed from the plasmid (see Fig. 3C). The results exhibited similar DNA replication and expression levels of LSTag transcripts, compared to co-transfection of the control plasmids (Fig. 5C).

Next, increasing concentrations of pre-miR-S1 expression plasmids were cotransfected with wSV40 plasmids to determine DNA replication and LSTag expression levels at each concentration. Although miR-S1-3p expression increased in a concentration-dependent manner, DNA replication and LSTag transcript levels were reduced to the same extent regardless of the amount of miR-S1 plasmid (Fig. 5D). These findings indicated that excess miR-S1 expression neither depleted LSTag expression nor abrogated DNA replication. These results are consistent with those of recent studies showing that despite high levels of viral miRNA, RacPyV tumors still expressed early gene transcripts.^32,33)

wSV40 Transfection Induced Higher DNA Replication than wSV40-dl.miR-S1 Transfection in Various Cell Types

To assess the importance of an optimal amount of LTag for efficient DNA replication, we performed transfection experiments with wSV40 and wSV40-dl.miR-S1 plasmids in various types of cells, including RD, A549, WI-38, HDFs, and HDK cells because the amount of LSTag transcripts expressed from the plasmids depends on cellular transcription factors, which are differentially expressed in these distinct cell types. As expected, the expression of LSTag transcript varied among the examined cells (Fig. 6A). RD cells and HDFs showed a much higher expression level of LSTag transcripts and LTag proteins than HEK293 cells (Figs. 6A, B). However, these differences in LSTag expression levels may be ascribed to the differences in transfection efficiencies between the cell types. Therefore, we determined the transfection efficiency of each cell type by transfecting them with a GFP plasmid. As shown in Fig. 6C, HEK293 cells were the most efficiently transfected cells among the examined cells, and approximately 41% of the cells were positive for GFP. Data on both the transfection efficiency and relative LSTag expression in various cell types enabled us to estimate LSTag expression within a single cell and compare it among the cell types. These estimations and comparisons revealed that all the examined cells exhibited higher expression levels of LSTag than HEK293 cells. Based on the above, we evaluated DNA replication in each type of cell transfected with wSV40 or wSV40-dl.miR-S1 plasmids. Contrary to the observation in HEK293 cells, transfection with wSV40 induced higher DNA replication than that with wSV40-dl.miR-S1, which induced higher LSTag expression in all the examined cell types (Figs. 6A, D). Collectively, these data suggest that miR-S1 prevents LTag overexpression in all the examined cell types, except HEK293 cells.

Fig. 6. Transfection with wSV40 Induced More DNA Replication than Transfection with wSV40-dl.miR-S1 in Various Types of Cells

(A) Relative expression of LSTag transcript in various types of cells transfected with wSV40 or wSV40-dl.miR-S1 plasmids was determined using RT-qPCR. Bar plots show relative expression levels relative to wSV40-transfected HEK293 cells arbitrarily set to 1. β-Actin was used as an internal control. Data are shown as mean ± S.D. (n = 4). (B) HEK293 and RD cells, transfected with wSV40 or wSV40-dl.miR-S1 plasmids, were subjected to immunoblot analysis for LTag protein expression. (C) Table showing transfection efficiency and relative expression of LSTag transcripts in various types of cells. Transfection efficiency of the cells was determined by transfecting with a GFP plasmid. Relative LSTag expression in each cell line transfected with wSV40 plasmid is presented relative to LSTag expression in the transfected HEK293 cells, which was arbitrary set to 1, because the expression level of β-actin varied among the cell and was unsuitable as an internal control. Based on the transfection efficiencies and relative LSTag expression, LSTag expression within a single wSV40-transfected cell was estimated for each cell line. (D) DNA replication was determined using RT-qPCR in each cell type transfected with wSV40 or wSV40 dl.miR-S1 plasmids. Data are shown as mean ± S.D. (n = 4). * p < 0.05 and ** p < 0.01.

MiR-S1 Reduces LTag- and STag-Mediated Induction of TNFα and IL-17F Genes by Downregulating LSTag

The expression levels of IL-8, TNFα, and IL-17F transcripts were evaluated in HEK293 cells transfected with either wSV40 or wSV40-dl.miR-S1 plasmid. RT-qPCR analysis showed that transfection with both wSV40 and wSV40-dl.miR-S1 induced an increase in TNFα and IL-17F transcripts but not IL-8, compared to mock transfections (Fig. 7A). Comparison of the expression levels of TNFα and IL-17F transcripts between wSV40 and wSV40-dl.miR-S1 transfectants revealed a subtle but statistically significant reduction in TNFα and IL-17F transcripts in wSV40-transfected cells that express miR-S1. These results prompted us to test whether miR-S1 itself suppresses cytokine expression. MiR-S1 transfection did not affect TNFα transcript expression and conversely increased IL-17F transcript expression (Fig. 7B). Therefore, we next assessed the effect of LTag and STag expression on the induction of the cytokine genes. Forced expression of LTag or STag significantly increased the expression of TNFα and IL-17F transcripts but not that of IL-8, compared to the control (Fig. 7C), clearly indicating LTag- and STag-mediated induction of TNFα and IL-17F transcripts. One explanation for the reduced expression of TNFα and IL-17F observed in wSV40 transfectants may be the result of miR-S1-mediated downregulation of LTag or STag level. This was verified through RT-qPCR analysis of cytokine expression in HEK293 cells transfected with increasing concentrations of STag or LTag plasmids. As expected, the expression levels of TNFα and IL-17F transcripts increased with increasing concentrations of LTag or STag plasmids (Figs. 7D, E). However, IL-8 expression level was only slightly increased at the maximum concentration of STag plasmid, but not with the LTag plasmid (Figs. 7D, E).

Fig. 7. Effect of Forced Expression of wSV40, wSV40 dl.miR-S1, miR-S1, LTag, and STag on Induction of IL-8, TNFα, and IL-17F Genes in HEK293 Cells (A) wSV40 or wSV40-dl.miR-S1 Plasmids Were Cotransfected with Puromycin-Resistant Plasmids into HEK293 Cells, Followed by Selection with Puromycin

The stably transfected and mock-transfected cells without puromycin selection were harvested and subjected to RNA isolation and RT-qPCR for analysis of IL-8, TNFα, and IL-17F expression. (B) Pre-miR-S1 expression plasmid or control plasmids containing the puromycin-resistance gene were transfected into HEK293 cells, which were then selected with puromycin, harvested, and subjected to RNA isolation for the analysis of IL-8, TNFα, and IL-17F expression using RT-qPCR. (C) IL-8, TNFα, and IL-17F expression was evaluated using RT-qPCR in HEK293 cells transfected with LTag and STag expression plasmids harboring puromycin-resistant gene and selected with puromycin. TNFα and IL-17F expression levels were significantly increased in the LTag- and STag-transfected cells compared to that in the mock- and control-transfected cells. (D, E) Increasing concentrations of STag (D) or LTag expression plasmids (E) were transfected, and their effect on IL-8, TNFα, and IL-17F expression was evaluated using RT-qPCR. (A–E) Data are shown as mean ± S.D. (n = 4). * p < 0.05 and ** p < 0.01.

Treatment with TNFα Differentially Affects LTag- and STag-Mediated Cytokine Induction

An inflammatory milieu, as might occur in vivo during a viral infection, can induce cytokine production and immune response in SV40-infected cells in response to which viruses may evolve a strategy to counteract the cytokine production through miR-S1 expression. To investigate this possibility, HEK293 cells transfected with wSV40 or wSV40-dl.miR-S plasmids were treated with TNFα and the expression of IL-8, TNFα, and IL-17F transcripts was analyzed using RT-qPCR. Following treatment with TNFα, both wSV40 and wSV40-dl.miR-S1 transfected cells showed increased expression of IL-8 and IL-17F and decreased expression of TNFα compared to the mock transfectants (Fig. 8A). However, the presumptive effect of miR-S1 on cytokine induction was noted only for IL-17F expression, which exhibited a subtle but statistically significant difference between wSV40- and wSV40-dl.miR-S1-transfected cells. Nevertheless, lower induction of IL-17F in wSV40-transfected cells may also be attributed to lower LSTag expression (downregulated by miR-S1), since apart from the forced miR-S1 expression the increased expression of LTag and STag also contribute to increased IL-17F expression following TNFα treatment (Figs. 8B, D, E).

Fig. 8. Effect of TNFα Treatment on IL-8, TNFα, and IL-17F Expression in HEK293 Cells Transfected with wSV40, wSV40-dl.miR-S1, miR-S1, LTag, or STag Expression Plasmids

(A) wSV40 or wSV40-dl.miR-S1 plasmids were cotransfected with puromycin-resistant plasmid into HEK293 cells, followed by selection with puromycin. The stably transfected and mock-transfected cells were treated with 50 ng/mL TNFα for 6 h, harvested, subjected to RNA isolation and RT-qPCR analysis for IL-8, TNFα, and IL-17F expression. (B) Pre-miR-S1 expression plasmid or control plasmid were transfected into HEK293 cells, which were then selected with puromycin, treated with TNFα, and subjected to RNA isolation and RT-qPCR for analysis of IL-8, TNFα, and IL-17F expression. (C) IL-8, TNFα, and IL-17F expression was evaluated by RT-qPCR in HEK293 cells transfected with LTag or STag expression plasmids. The cells were selected with puromycin and treated with TNFα. IL-17F expression markedly increased in the LTag- and STag-transfected cells, whereas TNFα expression decreased in the STag-transfected cells compared to that in both mock- and control-transfected cells. (D, E) Increasing concentrations of STag (D), or LTag expression plasmids (E) were transfected, and their effect on IL-8, TNFα, and IL-17F expression levels were evaluated using RT-qPCR following treatment with TNFα. (A–E) Data are shown as mean ± S.D. (n = 4). * p < 0.05 and ** p < 0.01.

Treatment with TNFα differentially affected LTag- and STag-mediated cytokine induction in HEK293 cells. Expression of either LTag or STag caused a slight decrease in TNFα expression following treatment with TNFα, but markedly increased TNFα expression in the absence of treatment. However, they induced IL-17F expression irrespective of TNFα treatment (Figs. 8C−E, 7C−E). IL-8 expression was also induced by either LTag or STag expression followed by TNFα treatment (Figs. 8C−E) but not by LTag expression in the absence of the treatment (Figs. 7C−E).

DISCUSSION

The polyomavirus SV40 genome encodes two mature miRNAs, miR-S1-3p and miR-S1-5p, of which miR-S1-3p is the dominantly expressed and temporally increased form. MiR-S1-3p markedly reduced luciferase activity of the reporter containing a nucleotide sequence perfectly complementary to miR-S1-3p. Consistently, it downregulated the expression of LSTag transcripts with a perfect complementary sequence through miR-S1-Ago2-mediated mRNA decay. Transfection of SV40 genome plasmid that expressed miR-S1 reduced LSTag expression and increased DNA replication compared to transfection with a mutated genome plasmid without miR-S1 expression in most of the cell types examined except HEK293. However, forced miR-S1 expression did not affect DNA replication without LTag downregulation. MiR-S1 reduced LTag- and STag-induced TNFα and IL-17F gene expression through LSTag downregulation, although its forced expression alone did not affect TNFα but increased IL-17F expression.

MiR-S1 preferentially targets and downregulates the viral regulatory proteins, LTag and STag, whereas it may not target cellular mRNAs or, if so, may only have a marginal effect on posttranscriptional repression of the mRNAs. MiRNA-mediated LTag and STag downregulation is a common feature among polyomavirus miRNAs, which are encoded on the late strand complementary to LTag and STag open reading frame sequences.^{13,22,34–36)} Canonical miRNAs post-transcriptionally repress mRNAs through base-pairing between the miRNA seed site and seed matching sites within the 3′ UTRs of the target mRNAs.³⁷⁾ However, through perfect complementarity between the seed and seed matching sites of the reporter, miR-S1 exhibited only a marginal reduction in luciferase activity. There is limited information about the cellular targets of polyomavirus miRNAs.⁸⁾ SV40 miR-S1-5p targets the seed site of cellular hsa-miR423 5p molecule,³⁸⁾ while human polyomavirus BKV and JCV 3p miRNAs target a stress-induced ligand, ULBP3, which is recognized by the killer receptor, NKG2D, of natural killer cells.³⁹⁾ Given that LTag and STag are the dominant targets of miR-S1, it is likely that miR-S1 plays a pivotal role in modulating LTag- and STag-involved biological processes, such as DNA replication and cytokine induction.

Among the infected cell types, the expression level of LTag protein differed significantly due to the differential expression or activation of cellular transcription factors. While LTag is expressed at very low levels in some cell types, it is highly expressed in other cell types, as was observed in the present study in HEK293 and RD cells, respectively. In either cell type, miR-S1 may have a functional significance in LTag downregulation. Low level of LTag expression is further downregulated by miR-S1, resulting in insufficient viral replication, which may enable SV40 to remain latent within the cells of the tissue, as reported for JCV miRNAs.³⁴⁾ In this context, it is worth noting that excessive expression of miR-S1 did not deplete LTag expression or abrogate DNA replication (Fig. 5D). On the other hand, miR-S1 prevented excessive expression of LTag to ensure effective replication, as shown in the transfection experiments using SV40 genome plasmids and miR-S1-defective plasmids. A similar reduction in DNA replication associated with increased LTag expression was noted in renal proximal tubule epithelial cells infected with a mutant BKV without miRNAs, compared to that in control cells infected with wild-type BKV.²³⁾ Although the underlying mechanism of reduced DNA replication following LTag overexpression has not yet been explored, it is possible that the cellular proteins essential for viral DNA replication, such as RPA, DNA polymerase alpha-primase, and topoisomerase, may be sequestered by the excess LTag proteins due to their LTag-binding properties.^40–44)

This is the first report to demonstrate that SV40 LTag and STag proteins induce IL17F expression and that miR-S1 downregulates LTag- and STag-induced TNFα and IL17F expression. These LTag- and STag-induced cytokines are deleterious to the virus life cycle, especially to viral replication and persistence within the host cells.²⁷⁾ To attenuate their deleterious effects, SV40 may utilize miR-S1 to downregulate LTag and STag, thereby indirectly inhibiting the cytokine production. IL-17F, which is highly homologous to IL-17A, is considered to be an inflammatory cytokine because it induces the expression of proinflammatory cytokines (IL-1β, IL-9, IL-6) and chemokines (IL-8, CXCL1, CCL2, CCL3, CCL7, CCL20) in many different cell types.^45–49) Besides the induction of these cytokines and chemokines, both IL-17A and IL-17F are involved in host defense against extracellular bacterial and viral pathogens through the recruitment of neutrophils and macrophages, granulopoiesis, and production of anti-microbial molecules, such as β-defensins, lipocalin 2, CRAMP, and mBD-3.^46,50–53)

IL-17-mediated recruitment of neutrophils also induces tissue destruction.⁵⁴⁾ IL-17 signaling mediated tissue injury in a mouse kidney ischemia–reperfusion injury,⁵⁵⁾ whereas overexpression of both IL-17 and TNFα in the knee joints of normal mice synergistically enhanced chondrocyte death and cartilage surface erosions.⁵⁶⁾ The inflammatory and tissue-destructive properties of IL-17 as well as the present finding of LTag- and STag-inducible IL-17F prompts us to suggest that polyomavirus-associated nephropathy (PVAN) may be attributable to LTag- and STag-induced IL-17F and TNFα expression and to further propose a potentially novel therapeutic strategy for treating PVAN with a combination of anti-IL-17 agents, such as anti-IL-17A/F and anti-IL-17R antibodies, and miRNA. PVAN is mostly caused by BKV in renal allografts of immunocompromised patients, in which BKV becomes reactivated, promotes replication, and induces a tubulointerstitial inflammatory response.⁵⁷⁾ To prevent PVAN from being significantly associated with renal transplant dysfunction and allograft loss, the only existing therapeutic strategy is the reduction of immunosuppression, which also increases the risk of acute rejection and potential allograft loss.⁵⁸⁾

Acknowledgments

The authors would like to thank Prof. Muto Y, Faculty of Pharmacy and Research Institute of Pharmaceutical Sciences, Musashino University, for his helpful advice on the Mfold software for analyzing the secondary structures of miRNA. This work was supported in part by a JSPS KAKENHI Grant (Grant No. 25460075) and a Grant from the Musashino University.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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