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

Background 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. Methods 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. Results 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. Conclusions 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. Supplementary Information The online version contains supplementary material available at 10.1186/s11658-022-00346-4.


tsRNA-seq libraries 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 by RNA Simple Total RNA Kit (TIANGEN, Beijing, China). Total RNA samples were pretreated to eliminate the interference of RNA modi cation in the construction of small RNA-seq libraries: 1) 3aminoacyl (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 the commercial kit for tsRNA sequencing library preparation (Illumina, California, USA). The kit includes 3'-adapter and 5'-adapter ligation adaptor ligation, cDNA synthesis, and library PCR ampli cation. PCR ampli ed 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 quanti ed using Agilent 2100 Bioanalyzer (Agilent Technologies, California, USA) and then sequenced using Illumina NextSeq 500 (Illumina). Sequencing was performed by Kangchen Bio-tech (Shanghai, China).

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 pro le 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://gtrnadb.ucsc.edu/). Remaining reads were aligned to the transcriptome including mRNA/rRNA/snRNA/piRNA/snoRNA/miRNA biotypes. Expression pro le and differential expression of tsRNAs were calculated based on standardized TPM (Transcripts Per Million).

Quantitative real-time PCR (qRT-PCR) validation
Total small RNAs (smRNAs) of proliferative and quiescent HASMCs were isolated by MiRcute miRNA Isolation Kit (TIANGEN). Quantity and integrity of total smRNAs were measured by NanoDrop ND-1000 (Thermo Fisher Science, Massachusetts, USA) and 1.2% agarose gel electrophoresis. Total smRNAs were reverse transcribed by miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN). qRT-PCR was performed using SYBR Green analysis of miRcute Plus miRNA qPCR Kit (TIANGEN). Reaction conditions of all samples were: Initial denaturation at 95°C for 10 min, heat denaturation at 95°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 10 s, with 40 cycles. All samples were normalized using U6 as an internal control. 2 −∆∆Ct method was applied to calculate the fold change of expression of tsRNAs; Student's t-test was performed for statistical signi cance. Primers of AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, AS-tDR-000076, and U6 were shown in Supplementary Table 1. Nuclear and cytoplasmic RNA extraction Nuclear and cytoplasmic components of proliferative HASMCs were isolated using the Nuclear/Cytosol Fractionation Kit (BioVision, California, USA) following the manufacturer′s instructions. Extraction, quanti cation, and integrity detection of smRNAs were consistent with the above. Reaction condition of qRT-PCR was consistent with the above. GAPDH and U6 were applied as positive controls for the cytoplasm and nucleus, respectively. Primers were listed in Supplementary Table 1.

GO and pathway analyses
GO and KEGG pathway analyses of the proliferation-related gene sets containing target promoters and proliferation-related target DEmRNA sets were performed to explore the potential of AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076 in HASMC proliferation using the DAVID database [22].

Antisense oligonucleotide transfection
Antisense oligonucleotide (ASO) of AS-tDR-000076 and negative control (NC) were designed and synthesized by GENEWIZ (Beijing, China). According to the procedures of Lipofectamine 3000® Transfection Reagent (Invitrogen, California, USA), Lipofectamine 3000 was used to transfect 5,000 ng ASO into HASMCs cultured in a 25 cm 2 cell culture ask. After 7 h of incubation in a 37°C and 5% CO 2 humidi ed incubator, the transfection medium was replaced with fresh medium and cultured to the appropriate time point. Sequences of NC and ASO were listed in Supplementary Table 2. Western blot analysis Total protein in HASMCs transfected with ASO or NC for 48 h was extracted using RIPA buffer (Solarbio, Beijing, China) and 1 mM PMSF (Solarbio). Protein concentration was detected by the Bradford method (Solarbio). Equal amounts of protein were separated by SDS-PAGE and transferred onto polyvinylidene uoride (PVDF) membranes (Merck, Darmstadt, Germany). At 37°C, 5% skimmed milk was used to block the membrane for 2 h, followed by washing with Tris-buffered saline (TBS), and then incubated overnight at 4°C with the following speci c primary antibodies: MFN2 (dilution at a 1: 500, Abcam), GAPDH (dilution at a 1: 1000, Wanleibio). After washing three times in Tris-buffered saline within Tween 20 (TBST) for 5 min, the membrane was incubated in goat anti-rabbit secondary antibody (dilution at a 1: 20,000, Sino Biological) for 1 h at room temperature. Subsequently, ChemiDoc™ MP Imaging System (BIO-RAD) visualized the protein signals and quanti ed band strength.

Statistical analysis
All data are from at least three independent experiments, expressed in the form of mean ± standard deviation (SD). Student's t-test was performed to compare the differences between the two groups. When p-value < 0.05, the difference was statistically signi cant.

Knockdown of AS-tDR-000067 inhibits the proliferation of HASMCs
In the AS-tDR-000067-promoter interaction network, we found an important apoptosis-related target gene p53 [75] (Fig. 2b). Through RNAhybrid [76], it was found that AS-tDR-000067 contains two binding sites on the p53 promoter (Fig. 7a). ASO was used to speci cally knockdown AS-tDR-000067 in proliferative HASMCs, and the knockdown e ciency of AS-tDR-000067 was as high as 60% (Fig. S1). Western blot con rmed that knockdown of AS-tDR-000067 promoted the expression of p53 (Fig. 7b, c). Subsequently, EdU was executed to test the proliferation rate of HASMCs, and AS-tDR-000067 suppression resulted in a reduction of up to 80% in EdU positive cells (Fig. 7d, e). Therefore, we speculate that AS-tDR-000067 may promote HASMC proliferation by targeting the p53 promoter to inhibit its transcription.
Knockdown of AS-tDR-000076 inhibits the proliferation of HASMCs AS-tDR-000076 was selected for further analysis because GO analysis showed that the target DEmRNAs of AS-tDR-000076 were involved in negative regulation of smooth muscle cell proliferation (ontology: biological process, GO: 0048662) (Fig. 5g). Another important reason is that MFN2, an important proliferation inhibitor [77], is one of the target DEmRNAs of AS-tDR-000076 (Fig. 4d). Through RNAhybrid [76], it was found that the binding site of AS-tDR-000076 was located in CDS (coding sequence) and 3′-UTR (untranslated region) of MFN2 (Fig. 8a). ASO was utilized to speci cally knockdown AS-tDR-000076 in proliferative HASMCs, and the knockdown e ciency of AS-tDR-000076 was as high as 60% (Fig. S2).
Western blot con rmed that knockdown of AS-tDR-000076 promoted the expression of MFN2 (Fig. 8b, c). Subsequently, EdU was executed to test the proliferation rat of HASMCs, and AS-tDR-000076 suppression resulted in a reduction of up to 80% in EdU positive cells (Fig. 8d, e). Therefore, we speculate that AS-tDR-000076 may promote cell proliferation by targeting MFN2. Discussion tsRNAs, as a new type of non-coding RNA derived from tRNA, are receiving more and more attention. tsRNAs are rich in content, evolutionarily conservative, and widely present in all areas of life. These characteristics suggest that they are not the by-product of tRNA production or degradation, and they may be involved in the regulation of the body [23,78]. There are two hot spots in the mechanism research of tsRNAs. One is that it interacts with various proteins [54,[79][80][81]. The other is its miRNA-like function to inhibit the expression of target genes [23,82] 1280 is a tsRNA derived from tRNA Leu , which binds to the 3'-UTR of JAG2 and silences its expression, thereby inhibiting the proliferation, migration, and self-renewal of colorectal cancer cells mediated by the Notch signaling pathway [83]. This provides a new idea for exploring the role of tsRNAs in vascular diseases, that is, tsRNAs can regulate abnormal proliferation-related vascular diseases by inhibiting the expression of target genes.
The purpose of this paper is to screen and identify tsRNAs related to HASMC proliferation. Here, we performed high-throughput RNA sequencing and screened out 1,838 DEtsRNAs. QRT-PCR con rmed that AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076 were up-regulated in proliferative HASMCs, and mainly located in the nucleus. Then, we predicted and screened their target genes respectively to construct tsRNA-promoter, tsRNA-mRNA, and circRNA-tsRNA interaction networks. Bioinformatics analysis showed that target genes of 4 tsRNAs (AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076) are involved in various proliferation-related terms and pathways. In tsRNApromoter interaction networks, we found that AS-tDR-001370 can target cell proliferation-related proteins CCND1 [90], SPRY2 [91], and BMPR2 [92]. The expression of CCND1 is increased in proliferative VSMCs and can promote its proliferation [90]. Thus, AS-tDR-001370 may regulate VSMC proliferation by promoting CCND1 transcription. Besides, AS-tDR-000076 can also target CCND1, indicating that AS-tDR-000076 can coordinate with AS-tDR-001370 to regulate VSMC proliferation. SPRY2 can inhibit the VSMC proliferation and migration, thereby reducing neointimal growth after vascular injury [91]. Loss of function of BMPR2 is often found in patients with pulmonary arterial hypertension (PAH) induced by abnormal proliferation of VSMC, which has the effect of inhibiting the VSMC proliferation [92]. Thus, AS-tDR-001370 may promote the proliferation of VSMC by targeting SPRY2 and BMPR2 promoters and inhibiting their transcription. Cell proliferation-related proteins YAP1 [93], CDK6 [94], ATG4B [95], and p53 [48] are the target genes of AS-tDR-000067. YAP1 promotes VSMC proliferation by interacting with TEA domain transcription factor 1 (TEAD1) on the enhancer of platelet-derived growth factor receptor beta (PDGFRB) [93]. Thus, AS-tDR-000067 may regulate VSMC proliferation by regulating PDGFRB downstream pathway. CDK6, like CCND1, is an important cell cycle regulator and can promote VSMC proliferation [94]. ATG4B can promote the proliferation, invasion, migration [95], and autophagy [96] of cancer cells. Thus, AS-tDR-000067 may regulate VSMC proliferation by promoting CDK6 and ATG4B transcription. p53 is an important tumour suppressor gene, and the transcription factor it encodes is essential for the regulation of the cell cycle and apoptosis [75]. A large number of studies have con rmed that p53 can prevent atherosclerosis [97], hypertension [98], vascular stenosis [99], and other vascular remodelling diseases by inhibiting the proliferation, invasion, and migration of VSMCs and inducing their apoptosis. Our research has con rmed that knocking down AS-tDR-000067 can promote the expression of p53, indicating that AS-tDR-000067 may promote HASMC proliferation by inhibiting the transcription of p53 (Fig. 7). However, this mechanism is not explained in this article, and further experimental veri cation is needed. Cell proliferation-related proteins ITPR1 [100], CALD1 [101], and RTN4 [102] are the target genes of AS-tDR-009512. ITPR1 is also called IP3R1, and its regulated Ca + signal is essential for VSMC proliferation [100]. CALD1 is related to the contractile function of VSMC [101]. RTN4 is also called Nogo, which can inhibit VSMC proliferation and migration [102]. Therefore, AS-tDR-009512 can regulate VSMC proliferation by targeting ITPR1, CALD1, and RTN4 promoters. Cell proliferation-related proteins TGFB1 [103], MAPK9 [104], and SFRP1 [105] are the target genes of AS-tDR-000076. TGFB1 can inhibit VSMC proliferation and promote its apoptosis by regulating long noncoding RNA MEG3 [103]. Bioinformatics analysis showed that MAPK9 was related to the Wnt signaling pathway, an important VSMC proliferation-related pathway [106]. Besides, SFRP1 was an important inhibitor of the Wnt signaling pathway, suggesting that AS-tDR-000076 may regulate VSMC proliferation through it [107].
In tsRNA-mRNA interaction networks, AS-tDR-001370's target mRNA NUMBL is involved in the inhibition of the Notch signaling pathway [67, 108], an important VSMC proliferation-related pathway [109]. It is suggested that AS-tDR-001370 can promote VSMC proliferation by reducing the effect of NUMBL. Also, the target genes PHB2 [58] and CLMN [59] of AS-tDR-001370 can inhibit cell proliferation, suggesting AS-tDR-001370 may also promote proliferation through these two ways. AS-tDR-000067's target mRNA PYCARD is a pro-apoptotic molecule [54]. Thus, AS-tDR-000067 may inhibit the process of apoptosis by targeting PYCARD. As the downstream target genes of AS-tDR-009512, TPM1 [110], TMEFF2 [111], and PTPRJ [112,113] are involved in cell proliferation and metastasis. It has been reported that TPM1 is involved in the inhibition of VSMC proliferation and metastasis [110]. MicroRNA-21 regulates VSMC function of lower extremity arteriosclerosis obliterans by targeting TPM1 [62]. Therefore, AS-tDR-009512 may drive VSMC proliferation by downregulating TPM1. As a target of many molecules, TMEFF2 participates in the inhibition of tumour cell proliferation, migration, and invasion by inhibiting the MAPK signaling pathway [111]. The involvement of the MAPK signaling pathway in VSMC proliferation has been con rmed [114]. Here, we propose that AS-tDR-009512 participates in VSMC proliferation through the MAPK signaling pathway. Similarly, PTPRJ (also known as DEP-1 or CD148) can inhibit the proliferation and migration of multiple cells through the ERK [112] or PI3K signaling pathways [113] and is closely related to cytoskeletal rearrangements [59]. TMEFF2 and PTPRJ also serve as target genes of AS-tDR-000076, indicating that AS-tDR-009512 and AS-tDR-000076 may coordinate the proliferation of VSMC through the MAPK and PI3K signaling pathways. MFN2 [77], SF1 [66,115], and OGN [65, 116] are target genes of AS-tDR-000076, which are down-regulated in proliferative HASMCs. It has been reported that MFN2 can inhibit the proliferation and promote the apoptosis of VSMCs by inhibiting the Ras-Raf-ERK1/2 pathway [77] and PI3K-Akt pathway [117], respectively. Therefore, AS-tDR-000076 may promote the VSMC proliferation by inhibiting the MFN2-Ras-Raf-ERK1/2 pathway. Wnt signaling pathway is an important proliferation-related pathway, which plays an important role in the VSMC proliferation [118].
SF1 is a downstream molecule of the Wnt signaling pathway [119] and can inhibit the VSMC proliferation [66,115]. Hence, AS-tDR-000076 may regulate the VSMC proliferation by silencing SF1. Similarly, OGN is also involved in the inhibition of proliferation. It can negatively regulate the epidermal growth factor receptor (EGFR) signaling pathway [116] and vascular endothelial growth factor receptor 2 (VEGF2) signaling pathway [65], but there is no study on its regulation of VSMC proliferation. It has been reported that OGN inhibits the proliferation and migration of cancer cells through the PI3K/Akt/mTOR signaling pathway [120]. Therefore, whether AS-tDR-000076 also regulates the VSMC proliferation through ONG/PI3K/Akt/mTOR signaling pathway is also worthy of further discussion. In addition to the VSMC proliferation, AS-tDR-000076 is also involved in the regulation of cardiomyocyte function related molecules, such as the transcription factor Nkx2.5 that regulates cardiomyocyte contraction [121], EH domain containing 3 (EHD3) that maintains the excitability and physiological function of myocardial cell membranes [122], cardiac conduction related SCN10A (sodium voltage-gated channel alpha subunit 10) [123], their insu cient expression are involved in the occurrence of diseases such as abnormal cardiac conduction, ventricular brillation, and heart failure. Therefore, further research on the function of AS-tDR-000076 is helpful to understand the mechanism of heart disease and may provide a new therapeutic target.
The purpose of this article is to screen the tsRNAs involved in the HASMC proliferation and explore their miRNA-like functions. The tsRNA-promoter interaction networks show that AS-tDR-000067 can regulate more than 800 target genes, of which p53 is involved in VSMC proliferation [48]. EdU uorescent stain and western blot con rmed that AS-tDR-000067 may promote HASMC proliferation by targeting p53 promoter. The tsRNA-mRNA interaction networks show that AS-tDR-000076 can regulate more than 300 target genes, of which MFN2 is involved in VSMC proliferation [77]. Studies have reported that miRNA can promote cell proliferation by down-regulating MFN2, such as MiR-93 [124] and MicroRNA-497 [125]. Therefore, we speculate that AS-tDR-000076 promotes proliferation by inhibiting the expression of MFN2.
EdU uorescent stain and western blot con rmed that AS-tDR-000076 may promote HASMC proliferation by targeting MFN2. However, the speci c regulatory mechanism of AS-tDR-000067 and AS-tDR-000076 on target gene was not covered in this article, and experiments are needed to further explore.

Conclusions
In this article, we conducted a comprehensive analysis of the tsRNA expression pro les of proliferative and quiescent HASMCs. Subsequently, qRT-PCR was performed to con rm the sequencing results. The expression of AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076 was up-regulated in proliferative HASMCs and mainly located in the cytoplasm. We constructed their tsRNA-promoter, tsRNA-mRNA, and circRNA-tsRNA interaction networks. DAVID database was used to analyze the function of target genes, and pointed out that AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076 may positively regulate VSMC proliferation. Finally, it was con rmed that AS-tDR-000067 and AS-tDR-000076 may participate in the proliferation of HASMC through down-regulation of p53 and MFN2, respectively, indicating that they may become new targets for the treatment of vascular diseases. This paper reports the role of tsRNAs in human vascular disease for the rst time, emphasizing the extensive regulation of tsRNAs, and providing new insights into the mechanism of vascular disease.

Availability of data and materials
The original data discussed in this study have been deposited in NCBI Gene Expression Omnibus and are accessible with the GEO Series accession number GSE164540 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE164540).

Competing interests
The authors declare no competing interests.