Exosome-delivered and Y RNA-derived small RNA suppresses influenza virus replication
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Multiple interplays between viral and host factors are involved in influenza virus replication and pathogenesis. Several small RNAs have recently emerged as important regulators of host response to viral infections. The aim of this study was to characterize the functional role of hsa-miR-1975, a Y5 RNA-derived small RNA, in defending influenza virus and delineate the mechanisms.
We performed high throughput sequencing of small RNAs in influenza virus-infected cells to identify up- or down- regulated small RNA species. The expression of the most abundant RNA species (hsa-miR-1975) was validated by stem-loop reverse transcription-polymerase chain reaction (RT-PCR). Antiviral effects of hsa-miR-1975 were confirmed by Western Blot, RT-PCR and plaque assay. In vitro perturbation of hsa-miR-1975 combined with exosomes isolation was used to elucidate the role and mechanism of hsa-miR-1975 in the context of antiviral immunity.
Small RNA sequencing revealed that hsa-miR-1975 was the most up-regulated small RNA in influenza virus-infected cells. The amount of intracellular hsa-miR-1975 increased in the late stage of the influenza virus replication cycle. The increased hsa-miR-1975 was at least partially derived from degradation of Y5RNA as a result of cellular apoptosis. Unexpectedly, hsa-miR-1975 mimics inhibited influenza virus replication while hsa-miR-1975 sponges enhanced the virus replication. Moreover, hsa-miR-1975 was secreted in exosomes and taken up by the neighboring cells to induce interferon expression.
Our findings unravel a critical role of Y-class small RNA in host’s defense against influenza virus infection and reveal its antiviral mechanism through exosome delivery. This may provide a new candidate for targeting influenza virus.
KeywordsInfluenza virus Y RNA Hsa-miR-1975 Interferon Exosome
Influenza virus continually threatens public health and antiviral drug resistance has become a major concern for clinical management . Accordingly, identification of host factors involved in viral replication may aid in understanding the interplay between virus and host, as well as in finding new targets for the development of antiviral compounds. Current evidence indicates that host microRNAs (miRNA, typically 21–25 nucleotides long) can play significant roles in the host-virus interaction. Some small RNAs facilitate influenza A virus (IAV) replication. For example, miR-141 suppresses transforming growth factor (TGF)-β2 and miR-9 targets monocyte chemoattractant protein 1-induced protein 1 (MCPIP1) to benefit IAV life cycle [2, 3]. On the other hand, several small RNAs participate in the host defensive response to IAV [4, 5, 6, 7, 8]. Possible mechanisms of how these small RNAs regulate cellular antiviral response were proposed. For instance, miR-483-3p targets RNF5 and CD81, which are regulators of RIG-I pathways . Their downstream signaling augments the expression of IFN-β upon influenza virus infection .
Y RNAs, typically 83–112-nucleotides long, belong to another class of small non-coding RNAs and are involved in a range of cellular processes, including DNA replication, RNA stability and cellular stress responses . Recent studies indicate that Y RNAs can be degraded into small fragments, called Y RNA-derived small RNAs (YsRNAs). The nature and functions of YsRNAs are currently unknown, and, thus, they have been removed from miRBase, which is the primary database for miRNAs . It is speculated that YsRNAs are further processed into microRNA-like small RNAs but its definite signal pathway had not been clearly elucidated .
Extracellular vesicles, including microvesicles and exosomes, contain mRNA, miRNA and other small noncoding RNA species [13, 14, 15, 16]. Of the different extracellular vesicles, exosomes have been the most studied in the context of infection. RNA present within exosomes is biologically active, implying that the RNA can modulate the protein profile and cellular state of the recipient cell . Functions and applications of exosomally transferred RNA include promoting or inhibiting tumor progression, predicting drug response in cancer treatment, mediating cross-talk between the feto-placental unit and the mother during pregnancy and modulating inflammatory response [9, 18, 19, 20, 21, 22, 23, 24]. However, cellular origin and the pathological status of cell will determine the content encapsulated within exosomes. This phenomenon pointed to the fact that packaging of RNA into exosomes is an intricately regulated event [21, 25, 26].
By sequencing small RNAs from cells with and without IAV infection, we identified hsa-miR-1975, one of the Y5 RNA-derived small RNAs, as the most up-regulated small RNA after influenza virus infection. Furthermore, we demonstrated that a synthetic hsa-miR-1975 mimic inhibited influenza virus replication through the production of interferon. Finally, we revealed the mechanism by which hsa-miR-1975 exerted its antiviral effect.
Materials and methods
Cell cultures, reagents, and viruses
Human lung adenocarcinoma epithelial A549 cells were obtained from the Bioresource Collection and Research Centre (BCRC, Taiwan). Madin-Darby canine kidney (MDCK) cells were obtained from ATCC. We cultured A549 cells in F-12 K medium (Invitrogen) by adding 10% FBS (Thermo Scientific), 100 U/mL penicillin G and 100 μg/mL streptomycin. We cultured MDCK cells in DMEM (Invitrogen) by adding 10% FBS (Thermo Scientific), 100 U/mL penicillin G, and 100 μg/mL streptomycin. We maintained cells in a humidified incubator at 37 °C with 5% CO2. The A/WSN/33 (H1N1) (WSN) strain of influenza A virus (IAV) was mainly used in the studies. NC99 (H1N1) and W10 (H3N2) were also used to see whether the expression of small non-coding RNA presented after infection with different viral strains.
Small RNA deep sequencing
A549 cells were infected with A/WSN/33 virus (MOI = 5) for 25 h and harvested for RNA extraction. RNA from uninfected A549 cells was used as a control. The total RNAs were subjected to gel electrophoresis and the RNA bands corresponding to size fractions of 18–40 nucleotide were isolated and extracted for small RNA sequencing. Small-RNA library construction and deep sequencing were done by Welgene Biotech (Taipei, Taiwan). Quality control of RNA revealed the following: the ratios of A230/260 were 2.67 and 1.84, respectively, for WSN infection and without WSN infection. The ratio of A260/280 was 1.96 for both. The ratio of 28S/18S was also 2 for both.
Samples were prepared by using an Illumina sample-preparation kit. In brief, total RNA was ligated with 3′ and 5′ adaptors and reverse-transcribed into cDNA. Polymerase chain reaction amplification was performed to amplify cDNA. cDNA constructs were fractionated by size and purified by using polyacrylamide gel electrophoresis. The libraries were sequenced on an Illumina instrument. Illumina software was used to analyze the sequencing data. Here, 12,057,156 and 11,067,109 reads were obtained for virus-infected and uninfected samples, respectively.
We isolated cellular RNA by using a High Pure RNA Isolation Kit (Roche Diagnostics) according to the manufacturer’s protocol. We created cDNA by using the SuperScript III First-Strand Synthesis System (Invitrogen). The cDNA of YsRNA was prepared by using stem-loop reverse transcription. The standard TaqMan method using the Universal Probe Library System (Roche Diagnostics) was employed for real-time PCR analysis. GAPDH was used as a control for the normalization of cellular RNA and intracellular viral RNA. U24 RNA was used as a control for normalization of human small RNA.
Primers for quantitative RT-PCR
The primers for reverse transcription are oligo (dT) 20 and IAV-specific RT primer (uni-12; 5′-AGCAAAAGCAGG-3′). The primers and probes are the following: for IAV_NP segment: sense 5′-GATGGAGACTGATGGAGAACG-3’and antisense 5′-TCATTTTTCCGACAGATGCTC-3′ with Universal Probe 59; GAPDH: sense 5′-AGCCACATCGCTCAGACAC-3′ and antisense 5′-GCCCAATACGACCAAATCC-3′ with Universal Probe 60; hsa-miR-1975: RT stem-loop primer: 5′-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACAGCTAG-3′; qPCR primers: Forward 5′-CCCCCACAACCGCGC-3′ and Reverse 5′-GTGCAGGGTCCGAGGT-3′ with Universal Probe 21; hsa-Y5: RT stem-loop primer: 5′-TTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACAAAACAG; qPCR primers: Forward 5′-GTCCGAGTGTTGT- GGGTTATTG-3′ and Reverse 5′-GTGCAG- GGTCCGAGGT-3′ with Universal Probe 21; U24: RT stem-loop primer: GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACTGCATCA; qPCR primers: Forward 5′-TTGCTATCTGAGAGATGGTGATGAC-3′ and Reverse 5′-GTGCAGGGTCCGAGGT-3′ with Universal Probe 21; interferon B: Forward 5′-CTTTGCTATTTTCAGACAAGATTCA-3′ and Reverse 5′-GCCAGGAGGTTCTC AACAAT-3′ with Universal Probe 20.
Quantification of miR-1975 and Y5
Serial dilution of hsa-miR-1975 mimic (purchased from MDBio, Taiwan) and human Y5 mimic (purchased from Dharmacon) were prepared. 5 different amounts of hsa-miR-1975 mimic and human Y5 mimic were used for RT-PCR. The amount of RNA mimics and Ct values were used to construct standard equations. We applied the equations to calculate the amount of hsa-miR-1975 and Y5 in cells.
Western blotting analysis
The antibodies for NP, GAPDH, CD63, CD81, TSG101 and HSP70 were purchased from Genetex (no. GTX629633, GTX100118, GTX17441, GTX31831, GTX10255 and GTX11573). The antibodies for HA and calnexin were purchased from Millipore (no. 04–902 and AB2301). Cell lysates were prepared using M-PER mammalian protein extraction reagent (Thermo Scientific) with additional protease inhibitors, subjected to electrophoresis on a SDS-PAGE, and transferred onto a Hybond-P membrane. The membrane was probed with the indicated primary and appropriate secondary antibodies, detected using an enhanced chemiluminescence detection kit, and then imaged by Image-Quant LAS4000.
Y5 mimic, miR-1975 mimic and miR-1975 scramble
Y5 mimic and miR-1975 mimic were purchased from Dharmacon. Scrambled miR-1975 RNA was purchased from MDBio, Taiwan. hsa-miR-1975 mimic, which sequence is identical to hsa-miR-1975, and Scrambled miR-1975 sequences are as follows:
Y5 mimic (5′-AGUUGGUCCGAGUGUUGUGGGUUAUUGUUAAGUUGAUU-UAACAUUGUCUC CCCCCACAACCGCGCUUGACUAGCU-3′) miR-1975 mimic (5′- CCCCCACAACCGCGCUUGACUAGCU-3′) and miR-1975 scramble (5′ –AUAGGCUCCGACGCUCCACACCCUC-3′).
A549 or Vero cells were transfected with miR-1975 mimic, control SiRNA or miR-1975 scramble by using a commercial liposome reagent, DMRIE-C (Invitrogen), according to the protocol provided by the manufacturer. After transfection for 48 h, cells were collected for further analysis or infected with WSN virus.
We infected MDCK cells with serial 10-fold dilutions of IAV for 1 h. We washed them twice with PBS, and then overlaid them with 0.5% agarose-containing MEM-alpha medium. We fixed the cells with 10% formaldehyde and stained cells with 0.1% crystal violet solution 2 days later for counting colony-forming units (CFUs).
Construction of hsa-miR-1975 sponge
Plasmid carrying hsa-miR-1975 sponge was constructed and sequenced at National RNAi Core Facility, Academia Sinica (Taipei, Taiwan). In short, 11 repeats of sponge sequence 5′-AGCTAGTCAAGCGAAATGTGGGGG-3′ were inserted into 3′ of GFP cDNA. Each sponge sequence is separated by spacer sequence CTAC. The sponge sequence is mostly complementary to hsa-miR-1975 sequence.
Exosomes isolation, purification, characterization and RNases treatment
We added ExoQuick-TC (System Biosciences) to the clarified cell culture medium at 1:5 ratio (by volume) to precipitate exosomes. The tube containing the mixture was inverted several times and then incubated overnight at 4 °C. The next day, the sample was centrifuged twice at 1500 g for 30 and 5 min, respectively, in order to remove the supernatant. The pellet was resuspended in 100 μL of PBS for Western Blot of exosome marker, quantitative RT-PCR and treating recipient cells. Western Blot of CD63, CD81, TSG101, HSP70, and calnexin of cellular and exosomal specimens were performed to characterize exosomes. We also diluted exosome pellets in 500 μL PBS. Size of exosomes were determined by using the dynamic light scattering technique (Zetasizer Nano ZS, Malvern Instruments, UK).
To confirm RNA transcripts are contained within the exosomes, RNase was applied when preparation of the exosomes to remove outside contaminants of RNA. Exosomes pellets were diluted in 50 μL PBS and treated with Ambion RNase cocktail 2.5 μL at 37 °C for 15 min.
Polyinosinic:polycytidylic acid potassium salt (PolyI:C) was suspended at 1 μg/ml in nuclease-free water and transfected into cells. At 5 h post-transfection, the cultured media was replaced with fresh media. Cells were harvested for RNA extraction at 24 h post transfection.
Exosomes isolation from donor cells, quantification of exosomes and recipient cells treatment
2 × 106 A549 cells were grown on 3 plates (10-cm dish) and incubated overnight. Two plates of cells were transfected with control SiRNA and one plate of cells was transfected with miR-1975 inhibitor on the second day. We treated 2 plates of cells, one was transfected with control SiRNA and the other was transfected with miR-1975 inhibitor, with Poly(I:C) on third day. We collected cell supernatant on fourth day. We added Exo-Quick to the cell supernatant and incubated at 4 °C overnight. Exosome pellets were diluted in 600 μL PBS and stored in − 80 °C. Protein concentrations of exosomes were measured by Bradford protein assay. We treated 2 × 105 recipient A549 cells with 100 μL of the exosomes and infected cells with WSN 24 h after exosomes treatment.
Cell survival assay
Cell survival was assessed by the MTS assay. The A549 cells were seeded onto 96-well plates and then transfected with mock, control SiRNA, hsa-miR-1975 mimic, hsa-miR-1975 inhibitor at concentrtion of 200 nM for 48 h. For virus infection group, A549 cells were infected with WSN at MOI = 1 for 24 h.
Quantitative variables were compared by one-Way ANOVA or Student’s t-test as appropriate. For all analyses, a P value below 0.05 was considered statistically significant for two-tailed tests. The SPSS (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.
Identification of human small RNA species involved in influenza a virus infection
To investigate the expression profile of hsa-miR-1975 upon IAV infection, we measured the expression levels of hsa-miR-1975 by reverse transcription-quantitative polymerase chain reaction (qPCR) at several time points. The relative amounts of hsa-miR-1975 exhibited substantial increase beginning at 10 h p.i. (hours post infection) (Fig. 1b). The abundance of hsa-miR-1975 increased up to 5 folds at 25 h p.i. (Fig. 1b). To elucidate whether the increase of hsa-miR-1975 is specific to WSN strain, A549 cells were infected with different strains of influenza viruses, including A/WSN/33 (H1N1), influenza A/New Caledonia/20/1999 (NC99) (H1N1) and A/Wisconsin/67/2005 (W10) (H3N2), at a multiplicity of infection (MOI) of 0.01 for 40 h. The expression of hsa-miR-1975 increased significantly after A549 cells were infected with each of the three virus strains (Fig. 1c), suggesting that the increase of hsa-miR-1975 is a general phenomenon associated with the infection by most of the influenza virus strains.
Y5RNA is processed to hsa-miR-1975 during cell apoptosis
Human Y RNAs are rapidly and specifically cleaved into small RNAs of 24 to 34 nucleotides in a caspase-dependent manner, and this occurs under the duress of a range of apoptotic stimuli [29, 30]. Since influenza virus infection induces apoptosis , we examined whether the enhanced expression of hsa-miR-1975 is related to apoptosis upon influenza virus infection. For this purpose, a pan-caspase inhibitor, carbobenzoxy-valyl -alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK), was used to inhibit both intrinsic and extrinsic apoptosis pathways. We provided evidence showing cell apoptosis occurred at 24 h post infection at MOI = 0.1 (Additional file 2: Figure S2). Adding VAD partially mitigated levels of apoptosis.
hsa-miR-1975 mimic inhibits influenza virus replication
The increased expression of hsa-miR-1975 during IAV infection suggests that it may play some roles in IAV life cycle. To address this possible role, we transfected a synthetic hsa-miR-1975 mimic into cells before IAV infection and collected cell lysates at different time points after influenza virus infection. We showed the levels of endogenous and exogenous amount of miR-1975 (Additional file 5: Figure S5). The levels of miR-1975 increased in cells after transfected with miR-1975 mimic is about 300 times of the levels of miR-1975 increased that sorely elevated by virus infection.
hsa-miR-1975 exerts antiviral effect through stimulating interferon production
hsa-miR-1975 is packaged into exosomes after IAV infection
We next examined whether IAV infection affects the levels of hsa-miR-1975 in exosomes. Exosomes pellets were treated with RNase before RNA isolation to make sure that hsa-miR-1975 is contained within exosomes rather than contaminants on the outside of exosomes. We chose another small RNA, U24, as a control for analysis of exosomal hsa-miR-1975 because U24 has been shown to be packaged into exosomes . Furthermore, we showed that U24 in the exosomes was reduced to 40% from the culture medium of GW4869-treated cells as compared to that of DMSO-treated cells (Fig. 5d). The levels of U24 reduction were similar to miR-1975 reduction in exosomes from GW4869-treated cells (Fig. 5c and d). This indicates that the exosome package efficiencies of U24 and miR-1975 are similar. We showed that the relative levels of hsa-miR-1975 were increased in exosomes from WSN-infected A549 cells compared to that from the uninfected cells (Fig. 5e). The abundance of hsa-miR-1975 dropped after adding GW4869 (Fig. 5e). Taken together, these results suggest that hsa-miR-1975 can be packaged into exosomes.
Exosomal hsa-miR-1975 exerts antiviral effect in recipient cells
98.5% of the human genome contains non-coding sequences , and small non-coding RNAs (sncRNAs) make up a significant portion of these transcriptomes. Many of these sncRNAs regulate gene expression and possibly mediate intercellular physiological signals [14, 36, 42, 43]. Human Y RNAs range in size from 83 to 112 nucleotides and fold into stem-loop secondary structures [44, 45]. hsa-miR-1975, located at position 51–75 of Y5 RNA, consisting of 3′ end of stem domain and internal loop domain of human Y5 RNA. In contrast to traditional miRNA biogenesis, YsRNAs neither associate with Argonaute proteins nor are dependent on Dicer [11, 46, 47], implying the difficulty of identifying the potential mRNA targets of hsa-miR-1975 by using immunoprecipitation with AGO2 antibody.
In this study, we explored the biogenesis of hsa-miR-1975. We found that the expression levels of hsa-miR-1975 were increased at late stages of influenza viral life cycle (Fig. 1b), in accordance with the onset of late apoptotic events. Chakrabortty et al. demonstrated that a 23 or 31 nucleotide processed fragments of Y5 from cancer cells induced cell death in primary cells through triggering differential expression of genes associated with the FAS/TGF-β-Smad2/3 apoptotic pathway . Nineteen of 25 nucleotides of hsa-miR-1975 sequence are complementary to the 23- or 31-nucleotide processed fragments of Y5 described by Chakrabortty et al.
The fold increased for miR-1975 at 25 h p.i. was about 20 fold based on the RNA sequence. It increased for 5 fold when we used stem-loop RT-PCR to verify the significance. The different methods used by the two experiments account for the differences in fold change. We further validated the increase of miR-1975 after influenza virus replication is not specific to definite species. In Fig. 1c we infected cell for 40 h with 0.01 MOI of virus because the titer of NC99 and W10 influenza viruses are lower and it will not cause significant increase of miR-1975 within 24 h. When we infected WSN with high MOI for more than 25 h, there is significant cell death and few cells could be collected for analysis. Therefore, we infected A549 cells with lower MOI and longer time.
To clarify the relationship between hsa-miR-1975 and cell viability, we transfected hsa-miR-1975 mimic or inhibitor into cells and examined the cell viability of transfected cells. We found that neither hsa-miR-1975 mimic nor its inhibitor influenced cell viability regardless of influenza virus infection (Additional file 11: Figure S11). Rutjes et al. demonstrated that Y RNA is cleaved and subsequently truncated to YsRNA (29). This process could be inhibited by anti-apoptosis protein Bcl-2 and the caspase inhibitor, implying that nucleolytic activity of Y RNA is activated downstream of caspase cascade . We corroborated that the biogenesis of hsa-miR-1975 is a consequential event upon influenza virus-induced apoptosis by demonstrating that hsa-miR-1975 was decreased in the presence of a pan-caspase inhibitor (Fig. 2a). Nevertheless, the causal relationship between Y RNA and hsa-miR-1975 needs additional confirmation.
Thus far, the role of Y RNA in viral infection has not been addressed. Initially, we thought that since hsa-miR-1975 expression increased during viral replication, it is most likely a positive factor involved in viral replication. However, to our surprise, its presence inhibited viral replication instead. We demonstrated that the viral inhibition effect could not only be found at 24 h but also be found at early stage of infection. We used MOI 1 and infected A 549 cells for 6 h because the difference is not significant with MOI 0.1 at 6 h. Thus, miR-1975 is most likely a cellular factor in the host’s antiviral defense. To further investigate this issue, we overexpressed hsa-miR-1975 mimic in A549 cells. We observed an upregulation of IFNB and diminished abundance of NP in hsa-miR-1975-transfected cells as compared to control SiRNA or mock-transfected cells (Fig. 4b). Lack of antiviral effect of hsa-miR-1975 mimic in interferon-deficient Vero cells indicated that interferon plays a pivotal role in the context of antiviral immunity related to hsa-miR-1975 (Fig. 4d). Goldgraben et al. postulated that differing lengths, rather than sequence, of RNA was responsible for differential interferon responses . We have addressed this issue by comparing the antiviral effect and interferons-stimulating effect of hsa-miR-1975 mimic and a scrambled miR-1975 RNA which is also a 25-mer RNA with the same GC ratio. The results indicated that the sequence of hsa-miR-1975 play vital role in stimulating interferon and inhibiting IAV replication (Additional file 6: Figure S6a, 6b and Additional file 12: Figure S12).
YsRNA molecules have been detected in sera of healthy people . It is intriguing to notice that YsRNA existed in sera wherein the concentrations of RNases are estimated at several hundred nanograms/ml [49, 50]. There are several explanations to this sequence stability. First, Ro60 and La proteins are bound to YsRNA, thereby preventing YsRNA from RNase digestion [29, 31]. Another mechanism is the natural encapsulation of these molecules in vesicles . Y5-derived small RNA had been demonstrated to be packaged into exosomes . We demonstrated that hsa-miR-1975 exerted its function also through packaging into exosomes. These results were expected on the basis of hsa-miR-1975 containing specific sequences which are prone to be secreted in exosomes .
By examining the expression of hsa-miR-1975 and influenza virus NP in recipient cell after receiving exosomes derived from control SiRNA or hsa-miR-1975 inhibitor treated donor cells, we underscored its biologic relevance in cell-to-cell communication. Viral replication in recipient cell was inhibited drastically after receiving exosomes derived from Poly(I:C)-treated donor cells (Fig. 6d). We postulated that exosomes derived from Poly(I:C)-treated donor cells contain proteins or small RNAs that would drive signal transduction to exert antiviral effect. Importantly, by inhibiting hsa-miR-1975 in donor cells, we demonstrated a downregulation of hsa-miR-1975 and enhancement of influenza virus replication in recipient cells (Fig. 6c and d). Together, these results suggest a functional role of exosomal hsa-miR-1975 in defense responses against viral infections.
We would like to thank National RNAi Core Facility, Academia Sinica, for providing hsa-miR-1975 sponge. We also thank Prof. Hui-Chen Chen for helpful discussions and suggestions.
Authors do not have any financial or other disclosures.
YML, CHT, MMCL and WCS conceived the project and designed the experiments; YML, CHT, WYY, MYH, YCC, CYH and WCS conducted the experiments; YML, CHT, MMCL, and WCS contributed to the data analysis, plotting and interpretation; YML wrote the manuscript; MMCL and WCS contributed to the writing and editing of the manuscript. All authors read and approved the final manuscript.
The research described here is supported by Ministry of Science and Technology [MOST 105–2628-B-039-006-MY3 to Wen-Chi Su and 106–2314-B-371-009-MY2 to Yuag-Meng Liu], China Medical University Hospital [DMR-107-118 to Wen-Chi Su] and Changhua Christian Hospital [105-CCH-IRP-003 and 106-CCH-IRP-099 to Yuag-Meng Liu].
Ethics approval and consent to participate
This study was approved by the IRB committee of Changhua Christian Hospital (IRB no. 151213) (Changhua, Taiwan). The study does not violate the rights of other persons or institutions.
Consent for publication
All authors report no potential conflicts of interest.
- 36.Lotvall J, Hill AF, Hochberg F, Buzas EI, Di Vizio D, Gardiner C, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles. 2014;3:26913.CrossRefGoogle Scholar
- 47.Langenberger D, Çakir MV, Hoffmann S, Stadler PF. Dicer-processed small RNAs: rules and exceptions. J Exp Zool part B. Mol Dev Evol. 2013;320(1):35–46.Google Scholar
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