Integrated analysis of tRNA-derived small RNAs in proliferative human aortic smooth muscle cells

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to vascular remodeling diseases. Recently, it has been discovered that tRNA-derived small RNAs (tsRNAs), a new type of noncoding RNAs, are related to the proliferation and migration of VSMCs. tsRNAs regulate target gene expression through miRNA-like functions. This study aims to explore the potential of tsRNAs in human aortic smooth muscle cell (HASMC) proliferation. High-throughput sequencing was performed to analyze the tsRNA expression profile of proliferative and quiescent HASMCs. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate the sequence results and subcellular distribution of AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076. Based on the microRNA-like functions of tsRNAs, we predicted target promoters and mRNAs and constructed tsRNA–promoter and tsRNA–mRNA interaction networks. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to reveal the function of target genes. EdU incorporation assay, Western blot, and dual-luciferase reporter gene assay were utilized to detect the effects of tsRNAs on HASMC proliferation. Compared with quiescent HASMCs, there were 1838 differentially expressed tsRNAs in proliferative HASMCs, including 887 with increased expression (fold change > 2, p < 0.05) and 951 with decreased expression (fold change < ½, p < 0.05). AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076 were increased in proliferative HASMCs and were mainly located in the nucleus. Bioinformatics analysis suggested that the four tsRNAs involved a variety of GO terms and pathways related to VSMC proliferation. AS-tDR-000067 promoted HASMC proliferation by suppressing p53 transcription in a promoter-targeted manner. AS-tDR-000076 accelerated HASMC proliferation by attenuating mitofusin 2 (MFN2) levels in a 3′-untranslated region (UTR)-targeted manner. During HASMC proliferation, the expression levels of many tsRNAs are altered. AS-tDR-000067 and AS-tDR-000076 act as new factors promoting VSMC proliferation.


Background
The death toll of cardiovascular diseases increases year by year, among which ischemic heart disease and cerebrovascular disease account for more than 80% of deaths [1]. Vascular smooth muscle cells (VSMCs), one of the cell types in blood vessels, mainly contract and relax to maintain blood pressure and flow [2]. Abnormal proliferation of VSMCs is a critical process in the pathogenesis of numerous vascular remodeling diseases, such as atherosclerosis [3], hypertension [4], vascular stenosis [5], and diabetic vascular complications [6]. Therefore, exploration of VSMC proliferation can contribute to the early diagnosis and treatment of pathological vascular conditions. With the development of deep sequencing technology, tsRNAs, a new class of small noncoding RNAs derived from tRNAs, have been discovered in various organisms [7]. Interestingly, the roles of tsRNAs in mediating cardiovascular diseases by regulating VSMC proliferation have been gradually revealed recently [8][9][10]. For example, tRF GlnCTG and tRF-Gly-GCC promote VSMC proliferation [8,9], whereas 5′-tiRNA-Cys-GCA inhibits VSMC proliferation, migration, and dedifferentiation by reducing the levels of its target gene signal transducer and activator of transcription 4 (STAT4) [10]. However, the actual repertoire of tsRNAs affecting VSMC proliferation remains unclear.

tsRNA-seq library preparation and sequencing
To screen tsRNAs related to HASMC proliferation, we performed high-throughput RNA sequencing of three sets of proliferative and quiescent HASMCs. Total RNA of proliferative and quiescent HASMCs was extracted using an RNA simple total RNA kit (TIANGEN, Beijing, China). Total RNA samples were pretreated to eliminate the interference of RNA modification in the construction of small RNA-seq libraries: (1) 3-aminoacyl (charged) was deacylated to 3′-OH for 3′ adaptor ligation; (2) 3′-cP (2′,3′-cyclic phosphate) was removed to 3′-OH for 3′ adaptor ligation; (3) 5′-OH (hydroxyl group) was phosphorylated to 5′-P for 5′-adaptor ligation; (4) m1A and m3C were demethylated for effective reverse transcription. Subsequently, tsRNA-seq libraries were constructed using a commercial kit for tsRNA sequencing library preparation (Illumina, CA, USA). The kit includes 3′-adapter and 5′-adapter ligation, complementary DNA (cDNA) synthesis, and library PCR amplification. PCR-amplified fragments with a size of 135-160 bp (corresponding to the size range of 15-40 nt small RNA) were selected as tsRNA-seq libraries. Finally, the prepared tsRNA-seq libraries were quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA) and then sequenced using an Illumina NextSeq 500 (Illumina). Sequencing was performed by AKsomics (Shanghai, China). The tsRNA nomenclature used in this paper is derived from AKsomics, where AS indicates the abbreviation of AKsomics, tDR stands for tRNA-derived small RNA, and the number represents the order in which the tsRNA was found during sequencing. In addition, in our sequencing data, we also present the tRFdb_IDs of tsRNAs documented in the tRFdb database [11].

Sequencing data analysis
Image analysis and base calling were performed by Solexa pipeline v1.8 (Off-Line Base Caller software, v1.8). Valid sequences were preserved by alignment statistical analysis for subsequent tsRNA expression profile analysis and differential expression analysis. Sequencing quality was tested by FastQC software, and NovoAlign software (v2.07.11) was applied to align the trimmed reads (with 5′, 3′-adaptor bases removed) with the mature tRNA and pre-tRNA sequences of GtRNAdbb: Genomic tRNA Database (http:// gtrna db. ucsc. edu/). Remaining reads were aligned to the transcriptome including mRNA/rRNA/snRNA/piRNA/snoRNA/miRNA biotypes. The expression profile and differentially expressed tsRNAs (DEtsRNAs) were calculated based on standardized transcripts per million (TPM). DEtsRNAs can be obtained through GEO series accession number GSE164540 (https:// www. ncbi. nlm. nih. gov/ geo/ query/ acc. cgi? acc= GSE16 4540).

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total small RNAs (smRNAs) of proliferative and quiescent HASMCs were isolated using a MiRcute miRNA Isolation Kit (TIANGEN). The quantity and integrity of total smR-NAs were measured using NanoDrop ND-1000 (Thermo Fisher Science, Massachusetts, USA) and 1% agarose gel electrophoresis. Total smRNAs were reverse-transcribed using a miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN). qRT-PCR was performed using SYBR Green analysis in the miRcute Plus miRNA qPCR Kit (TIANGEN). The reaction conditions for all samples were initial denaturation at 95 °C for 10 min, followed by 40 cycles of heat denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, and extension at 72 °C for 10 s. All samples were normalized using U6 as internal control, and the 2 −∆∆Ct method was applied to calculate the fold change of expression of tsRNAs. The primers are presented in Additional file 1. The experiment was repeated three times for each gene.

Nuclear and cytoplasmic RNA detection
Nuclear and cytoplasmic components of proliferative HASMCs were isolated using a Nuclear/Cytosol Fractionation Kit (BioVision, CA, USA) following the manufacturer's instructions. Extraction, quantification, and integrity detection of smRNAs were consistent with the above. The reaction conditions for qRT-PCR were in keeping with the above. GAPDH and U6 were applied as positive controls for the cytoplasm and nucleus, respectively. The primers are listed in Additional file 1. The experiment was repeated three times.

GO and pathway analyses
For the GO and pathway analyses of target DEmRNAs and HASMC proliferationrelated genes containing target promoters, we explored the potential function of AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076 on HASMC proliferation using the DAVID database [16].

Cell proliferation analysis
After the corresponding treatment of HASMCs, a 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay was performed according to the instructions of the BeyoClick ™ EdU Cell Proliferation Kit and Alexa Fluor 594 (Beyotime, Shanghai, China). EdU-positive cells were observed by fluorescence microscope (Olympus IX71, Tokyo, Japan). The experiment was repeated three times.

Dual-luciferase reporter gene assay
Dual-luciferase reporter assays were performed to evaluate the direct binding of AS-tDR-000067 to the p53 promoter, as well as AS-tDR-000076 to the MFN2 3′-UTR. Using GPtransfect-Mate (GenePharma, Shanghai, China), luciferase reporter gene vector (psi-CHECKTM-2 Vector, Promega, USA) containing the wild-type (WT) p53 promoter was cotransfected into 293A cells with AS-tDR-000067 mimic or AS-tDR-000067 mutant (GenePharma). Similarly, the vector containing the WT MFN2 3′-UTR was cotransfected with AS-tDR-000076 mimic or AS-tDR-000076 mutant (GenePharma). After 24 h, the Dual-Luciferase ® Reporter Assay System (Promega) was performed based on the manufacturer's instructions. For each analysis, the Renilla luciferase signal was normalized to the firefly luciferase signal. Sequences of tsRNA mimics and tsRNA mutant are listed in Additional file 5. The experiment was repeated three times.

Statistical analysis
All data are from at least three independent experiments, expressed as mean ± standard deviation (SD). Student's t-test was performed to compare the differences between two groups. p < 0.05 was considered statistically significant.

Expression profile analysis and qRT-PCR detection of tsRNAs
Compared with quiescent HASMCs, PCNA levels were increased together with decreased α-SMA levels, and cell proliferation rate was significantly augmented in proliferative HASMCs (Additional file 6), suggesting successful induction of two cell phenotypes for screening HASMC proliferation-related tsRNAs. Using RNA sequencing, we screened 3891 tsRNAs to explore their expression profiles in two HASMC phenotypes. The scatter plot in Fig. 1A, volcano plot in Fig. 1B, and hierarchical clustering in Fig. 1C illustrate that, in contrast to quiescent HASMCs, proliferative HASMCs possessed 887 increased DEtsRNAs and 951 decreased DEtsRNAs (fold change > 2 or < ½, p < 0.05). According to the fold change and expression abundance, four DEtsRNAs (AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076) were selected for qRT-PCR detection. Consistent with the sequencing, AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076 were higher in proliferative HASMCs (Fig. 1D). Nuclear/cytoplasmic RNA detection revealed that the four DEtsRNAs were predominantly localized in the nucleus of HASMCs (Fig. 1E).

Construction of tsRNA-promoter interaction networks
The functions of tsRNAs binding to Argonaute (AGO) proteins are similar to miR-NAs [17]. The binding of nuclear miRNAs to promoters leads to activation [18] or silencing [19] of the transcription of target genes. We thus speculated that tsRNAs are similar to miRNAs and play regulatory roles by targeting promoters. Four DEtsRNAs (AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076) were selected to construct tsRNA-promoter interaction networks (Fig. 2).

AS-tDR-000076 silencing restrains HASMC proliferation by augmenting MFN2 levels
In the light of the RNA-seq and qRT-PCR results, AS-tDR-000076 was increased in proliferative HASMCs (Fig. 1D). Moreover, GO analysis showed that target DEmRNAs of AS-tDR-000076 were enriched in negative regulation of smooth muscle cell proliferation (ontology: biological process, GO: 0,048,662) (Additional file 8). These two clues suggest that AS-tDR-000076 might be a regulator of HASMC proliferation. ASO specifically restrained approximately 60% of AS-tDR-000076 expression in HASMCs (Additional file 9). AS-tDR-000076 silencing resulted in a reduction of up to 80% in EdU-positive cells (Fig. 5A, B). Intriguingly, MFN2, a crucial VSMC proliferation inhibitor confirmed by our group [22] and others [23][24][25][26], is a target gene of AS-tDR-000076 (Fig. 3D). AS-tDR-000076 silencing promoted MFN2 protein levels in HASMCs (Fig. 5C). Utilizing RNAhybrid [12], we found a possible binding site between AS-tDR-000076 and MFN2 3′-UTR (Fig. 5D). Dual-luciferase reporter gene assay revealed that AS-tDR-000076 mimic decreased the fluorescence activity of MFN2-WT plasmid (Fig. 5E), manifesting that the 3′-UTR of MFN2 was a target of AS-tDR-000076. These findings elucidate that AS-tDR-000076 promotes HASMC proliferation, at least in part, by restricting MFN2 levels in a 3′-UTR-targeted manner. Discussion tsRNAs, a new type of noncoding RNAs derived from tRNAs, are receiving increasing attention. tsRNAs are abundant, evolutionarily conserved, and widely distributed, suggesting that they are not the by-products of tRNA production or degradation, and may be involved in organism regulation [27][28][29]. There are two hot spots in the mechanism of tsRNAs; one is the interaction with proteins [30,31], and the other is the miRNA-like function that inhibits target gene expression [32]. Through these mechanisms, tsRNAs regulate cell proliferation [33], migration [34], apoptosis [35], and other biological processes [36]. At present, studies on tsRNAs are mainly focused on revealing their roles in cancer [37]. Intriguingly, the mystery that tsRNAs are involved in cardiovascular disease by regulating VSMC proliferation has been gradually resolved [8][9][10]. tRF GlnCTG , which is highly increased in injured rat common carotid arteries, promotes rat VSMC proliferation by inhibiting Fas cell surface death receptor (FAS) levels in a 3′-UTR-targeted manner [8]. tRF-Gly-GCC is increased in patients with atherosclerosis and promotes VSMC proliferation [9]. 5′-tiRNA-Cys-GCA, which is decreased in human and mouse aortic dissection, inhibits VSMC proliferation by reducing STAT4 levels in a 3′-UTR-targeted manner [10]. However, understanding the regulation of VSMC proliferation by tsRNAs is still in its infancy.
Here, we identified 1838 DEtsRNAs between proliferative and quiescent HASMCs using RNA-seq ( Fig. 1A-C). Four DEtsRNAs (AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076) were increased in proliferative HASMCs (Fig. 1D) and mainly located in the nucleus of HASMCs (Fig. 1E). It is worth noting that the regulatory role of tsRNAs in the nucleus has not been well explored, including in VSMC proliferation. Given that miRNAs activate or restrain the transcriptional process in the nucleus by targeting promoters [18,19], we speculated that tsRNAs might have the same function. We predicted target promoters of the four DEtsRNAs and constructed tsRNA-promoter interaction networks (Fig. 2). Four gene sets containing target promoters were enriched in some VSMC proliferation-related GO terms and pathways (Additional file 7). Analysis of tsRNA-promoter interaction networks indicated that AS-tDR-001370 might target the promoters of cyclin D1 (CCND1) and sprouty RTK signaling antagonist 2 (SPRY2) (Fig. 2A). CCND1 has been confirmed to promote VSMC proliferation [38]. SPRY2 reduces neointimal growth after vascular injury by inhibiting VSMC proliferation and migration [39]. Therefore, AS-tDR-001370 might promote HASMC proliferation by activating CCND1 transcription but inhibiting SPRY2 transcription. Reticulon 4 (RTN4), which inhibits VSMC proliferation and migration [40], contained the target promoter of AS-tDR-009512 (Fig. 2C). Hence, AS-tDR-009512 might facilitate HASMC proliferation by suppressing RTN4 transcription. AS-tDR-000076 was likely to target the promoters of CCND1 and transforming growth factor beta 1 (TGFB1) (Fig. 2D). TGFB1 inhibits VSMC proliferation and promotes VSMC apoptosis [41]. Accordingly, AS-tDR-000076 might accelerate HASMC proliferation by motivating CCND1 transcription but restricting TGFB1 transcription. Excitingly, we found that the promoter of p53 might be targeted by AS-tDR-000067 (Fig. 2B). p53 is a crucial tumor suppressor gene, and the transcription factor it encodes is essential for the cell cycle and apoptosis [21]. A large number of studies have confirmed that p53 prevents atherosclerosis [42], hypertension [43], vascular stenosis [44], and other vascular remodeling diseases by inhibiting VSMC proliferation, invasion, and migration and inducing VSMC apoptosis. In this study, we revealed that AS-tDR-000067 suppression inhibited HASMC proliferation (Fig. 4A, B) accompanied by augmented p53 levels (Fig. 4C), and further elucidated that the promoter of p53 is a target of AS-tDR-000067 (Fig. 4D, E). Taken together, we document that AS-tDR-000067 promotes HASMC proliferation, at least in part, via inhibiting transcription of p53 in a promoter-targeted manner. However, the mechanism of AS-tDR-000067 translocation into the nucleus is not explored in this work and requires further verification.
In addition, four target DEmRNA sets were also enriched in some VSMC proliferation-related GO terms and pathways (Additional file 8). Investigation of tsRNA-mRNA interaction networks revealed that AS-tDR-009512 might target tropomyosin 1 (TPM1) and transmembrane protein with EGF-like and two follistatin-like domains 2 (TMEFF2) (Fig. 3C). miR-21 promotes VSMC proliferation and migration by targeting and reducing TPM1 [45,46]. TMEFF2 blocks proliferation of pancreatic cancer cells by inhibiting phosphorylation of the MAPK signaling pathway [47], and the MAPK signaling pathway is highly correlated with VSMC proliferation [48], which hints that TMEFF2 might inhibit VSMC proliferation via the MAPK signaling pathway. Hence, AS-tDR-009512 was likely to promote HASMC proliferation by targeting and suppressing TPM1 and TMEFF2. AS-tDR-000076 was likelihood to target TMEFF2 and splicing factor 1 (SF1) (Fig. 3D). SF1 has been confirmed to inhibit VSMC proliferation [49]. Therefore, AS-tDR-000076 might accelerate HASMC proliferation by silencing SF1 and TMEFF2.