The rats were killed after cervical dislocation, and the whole rats were soaked in 75% ethanol for disinfection. The chests were opened, and the aortas were dissected in sterile PBS. After excision of the fat and fibrous tissue of the vascular adventitia, the aortas were rinsed with PBS to remove the blood and coagulation in the vascular cavity. The aortas were then dissected into 1 mm³ arterial implants with a very sharp scalpel and planted in a culture dish. The intimal surfaces of the arterial grafts were contacted with the bottom of the culture dish, and the planting density was approximately 1 piece/cm². The culture dish was placed in the incubator for 2 h. Culture medium containing 25% fetal bovine serum was added from the top of the tissue to slightly cover the intimal surface of arterial grafts. The plate was transferred into an incubator for 24 h, and after 24 h, the culture medium was changed. On the 3rd day, the endothelial cells were fully grown, and fibroblasts were just about to grow. Then, the arterial grafts were gently removed, and the culture medium was changed every 2 days.

The vascular endothelial cells were inoculated into the culture plate, 2 ml of medium containing 100 μg/ml ox-LDL was added to the culture plate for 12 h, and the cell samples were collected after 12 h of culture.

The lysed sample was placed at room temperature for 10 min to completely separate the nucleoprotein from the nucleic acid. Chloroform (0.2 ml) was added to the lysed sample, and the mixture was violently shaken for 15 s and placed at room temperature for 3 min. Then, the mixture was centrifuged at 12,000 rpm, 4°C, for 10 min. The upper aqueous phase was collected and mixed with an equal volume of isopropanol. The mixture was placed at room temperature for 20 min and then centrifuged at 12,000 rpm, 4°C, for 10 min. The supernatant was discarded, and 1 ml of 75% ethanol was added to wash the precipitate. RNA was purified after centrifugation at 12,000 rpm and 4°C for 3 min. Purified RNA was eluted in 30–50 μl of RNase-free ddH2O.

A Qubit RNA detection kit was used to accurately quantify total RNA to determine the amount of total RNA added to the library construction. The 3′ end of RNA was connected to the connector with T4 RNA ligase 2. The 5′ end of RNA was connected to the connector with T4 RNA ligase 1. The connecting product was reverse-transcribed to obtain the cDNA strand of the connecting product. In this step, the small RNA library was reverse-transcribed into DNA. The reverse transcription product was amplified by PCR to obtain the final library product. The NextSeq 550 (Illumina) system was used for sequencing analysis. Raw reads were obtained after sequencing and then filtered, spliced and decontaminated to obtain clean reads, which were compared with the reference genome for subsequent information analysis. The data were removed with the original program. Clean data were obtained by removing low-quality sequences using the trimmatic program. The FastQC program was used to count the data volume of clean data. Fragments larger than 15 nucleotides were retained for subsequent analysis. The Bowtie program was used to compare clean data to the non-coding RNA database and genome. The edgeR program was used to analyze the differences in non-coding RNA expression. The databases used in this study were list as follow: PiRNABANK (piRNA), miRbase (miRNA) and Ensembl database (snoRNA and snRNA). As for repeat RNA were analyzed by RepeatMasker. The differentially expressed small RNAs are listed in Supplementary Table 1.

Total RNA was extracted from vascular endothelial cells by TRIzol. The EZ-press microRNA Reverse Transcription Kit was used for miRNA and piRNA reverse transcription. PrimeScript RT Master Mix was used for snoRNA and snRNA reverse transcription. SYBR Premix Ex Taq was used to detect the expression of sncRNA in VECs. U6 was used as the internal control. The relative expression of sncRNA was calculated by the 2^–ΔΔCt method. The primers used are listed in Supplementary Table 2.

For piRNA and miRNA overexpression and knockdown, piRNA and miRNA mimics were used for overexpression, and piRNA and miRNA inhibitors were used for knockdown. The piRNA and miRNA mimics and inhibitors were designed and synthesized by General Biosystems (Anhui) Co., Ltd. A total of 50 μl of Opti MEM and 0.2 nmol of RNA were gently mixed to obtain RNA diluent. A total of 50 μl of Opti MEM and 2.0 μl of Lip2000 were gently mixed to obtain Lip2000 diluent, and the Lip2000 diluent was then incubated at room temperature for 5 min. RNA diluent and Lip2000 diluent were mixed and incubated for 20 min at room temperature to form the RNA-Lip2000 complex. RNA-Lip2000 complex was added to the incubated cells, and the culture plate was then gently shaken. Cells were incubated in a 37°C CO₂ incubator for 4–6 h, and then the medium was changed and cultured for 18–48 h.

For snRNA and snoRNA overexpression, the target sequences of snRNA and snoRNA were cloned into the pLVX vector to construct lentivirus, which was used to transfect endothelial cells.

Rat aortic endothelial cells were treated with ox-LDL for 12 h and then subjected to the indicated treatment. After that, cell apoptosis was measured using an Annexin V-FITC/propidium iodide kit (BD Biosciences) according to the manufacturer’s protocol. Apoptotic cells were observed using a flow cytometer. CellQuest software was used to analyze the apoptotic rate.

All data are presented as the mean ± S.D. of three independent experiments. T-tests and ANOVA were used to compare two groups by Excel 2017; p < 0.05 was considered a significant difference, and P < 0.01 was considered highly statistically significant.

PiRNA is involved in transposon silencing, spermatogenesis, genome rearrangement, epigenetic regulation, protein regulation and reproductive stem cell maintenance (28). However, the regulatory effect of piRNAs on endothelial cell injury is unknown. Small RNA sequencing showed that 42 piRNAs were upregulated and 38 piRNAs were downregulated in ox-LDL-treated endothelial cells compared to NC cells (Figures 1A–C). We used PCA to show the relationship between piRNA expression and ox-LDL treatment. The obtained results showed that piRNA expression distinguished the ox-LDL group and NC group (Figure 1D). Next, we used qPCR to verify the small RNA sequencing results of piRNA. The qPCR results showed that compared with the NC group, the expression of 12 piRNAs in the ox-LDL group increased, and the expression of piRNA DQ602402 increased most significantly (Figure 1E). On the other hand, the expression of piRNA DQ606544 and DQ749693 was significantly decreased in the ox-LDL group compared to the NC group (Figure 1F). The expression of piRNA DQ614630 in endothelial cells was increased or knocked down by piRNA DQ614630 mimics or inhibitors (Supplementary Figure 1A). piRNA DQ614630 overexpression promoted the apoptosis of endothelial cells induced by ox-LDL, and piRNA DQ614630 knockdown inhibited the apoptosis of endothelial cells induced by ox-LDL (Figures 1G, H).

Research on snoRNA has developed rapidly, and recent studies have shown that snoRNA is also involved in genetic diseases, human variation, hematopoiesis, metabolism and cancer (19). Small RNA sequencing showed that 80 snoRNAs were upregulated and 68 snoRNAs were downregulated in ox-LDL-treated endothelial cells compared to NC cells (Figures 2A–C). The results of PCA indicated that snoRNA expression can distinguish the ox-LDL group and NC group (Figure 2D). qPCR analysis showed that compared with the NC group, the expression of 20 snoRNAs in the ox-LDL group increased, and the expression of snoRNA ENSRNOT00000079223.1 increased most significantly (Figure 2E). On the other hand, qPCR showed that the expression of ENSRNOT00000079032.1 was decreased in the ox-LDL group compared to the NC group (Figure 2F). The expression of snoRNA ENSRNOT00000079032.1 in endothelial cells was increased by transfection with lentivirus and detected by qPCR (Supplementary Figure 1B). snoRNA ENSRNOT00000079032.1 overexpression inhibited the apoptosis of endothelial cells induced by ox-LDL (Figures 2G, H).

SnRNA includes a large group of non-coding RNAs with large differences, and these non-coding RNAs are mainly involved in the selective splicing of mRNA precursors and the processing of ribosomal RNA (29). We analyzed the expression changes of snoRNA in rat endothelial cells treated with ox-LDL. Small RNA sequencing showed that 20 snRNAs were upregulated and 26 snRNAs were downregulated in ox-LDL-treated endothelial cells (Figures 3A–C). PCA indicated that snRNA expression can distinguish the ox-LDL group and NC group (Figure 3D). We used qPCR to verify the small RNA sequencing results of snRNA. The qPCR results showed that compared with the NC group, the expression of 25 snRNAs in the ox-LDL group increased, and the expression of snRNA ENSRNOT00000081005.1 increased most significantly (Figure 3E). The expression of snRNA ENSRNOT00000081005.1 in endothelial cells was increased by transfection with lentivirus and detected by qPCR (Supplementary Figure 1C). snRNA ENSRNOT00000081005.1 overexpression inhibited the apoptosis of endothelial cells induced by ox-LDL (Figures 3F, G).

MiRNAs are involved in the maintenance of endothelial cell function (30, 31). Small RNA sequencing showed that 106 miRNAs were upregulated and 91 miRNAs were downregulated in ox-LDL-treated endothelial cells compared to NC cells (Figures 4A–C). We used PCA to show the relationship between miRNA expression and ox-LDL treatment. The obtained results showed that miRNA expression could distinguish the ox-LDL and NC groups (Figure 4D). qPCR analysis showed that compared with the NC group, the expression of 21 miRNAs in the ox-LDL group increased, and the expression of miRNA rno-novel-136-mature increased most significantly (Figure 4E). The expression of miRNA rno-novel-136-mature in endothelial cells was increased or knocked down by miRNA rno-novel-136-mature mimics or inhibitors (Supplementary Figure 1D). miRNA rno-novel-136-mature overexpression promoted the apoptosis of endothelial cells induced by ox-LDL, and miRNA rno-novel-136-mature knockdown inhibited the apoptosis of endothelial cells induced by ox-LDL (Figures 4F, G).

All RNAs have a tendency to spontaneously fold into secondary or tertiary structures. Although RNA has only four types of nucleotides, they can form a variety of structures, thus affecting cell function. Aberrantly expanded, repeated RNA sequences can exhibit gain-of-function abnormalities and become pathogenic, giving rise to many diseases (32). The obtained results showed that 4 repeat RNAs were upregulated and 6 repeat RNAs were downregulated in ox-LDL-treated endothelial cells compared to NC cells (Figures 5A–C). PCA showed that repeat RNA expression could distinguish the ox-LDL group and NC group (Figure 5D).