Patients and study design
This study included 187 participants and their clinical data are listed in Supplementary Table 1. Four SLE patients and three healthy controls (HCs) were used in the primary small RNA profiling study and the validation cohort consisted of 97 SLE patients with SLE, 10 rheumatoid arthritis (RA) patients, 15 ankylosing spondylitis (AS) patients and 20 HCs. An additional 38 SLE patients were enrolled for further IFN-α-related assay. All patients fulfilled the classification criteria for SLE , RA , or AS , respectively, and the enrollment was completed at the Department of Rheumatology, Renji Hospital South Campus, between June 2017 and December of 2018. The SLE disease activity index (SLEDAI)  was assessed and all patients with > 500mg/24h proteinuria were defined as experiencing active lupus nephritis at the time of enrollment. The prednisone dose was divided into low/median/high dose (<20mg/d / ≤ 20mg/d and <50mg/d / ≤50mg/d) group. Any subjects with acute or chronic infections were excluded and the study protocol was reviewed and approved by the institutional ethics committee, and written informed consent was obtained from all participants.
Isolation of CD4+ T cells
The peripheral blood lymphocytes (PBMCs) were isolated from HCs and SLE patients using Ficoll gradient centrifugation within 6 h of collection of the samples. CD4+ T cells were then purified after immunomagnetic separation with the human CD4+ T cell isolation kit (BD Biosciences) according to the manufacturer's instructions. The isolated human primary CD4+ T lymphocytes were lysed in Trizol Reagents (Sigma) or used to perform in vitro transfection. Samples from the validation cohort were used for qRT-PCR.
Cell culture and transfection
CD4+ T cells from HCs were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Hyclone) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL IL-2 at 37 °C in a humidified atmosphere containing 5% CO2 (Thermo Fisher). For transfection, CD4+ T cells were seeded at a density of 0.5 × 106 cells/well and three types of tRF-3009 or three types of negative control (single-stranded RNA, ssRNA) (Guangzhou RiboBio Co., Ltd.) were transfected using RNAimax (Thermo Fisher) according to the manufacturer’s protocols. After 48h of incubation at 37 °C, cells were harvested for small RNA sequencing.
To evaluate the metabolic changes CD4+ T cells were seeded at a density of 1 × 106 cells/well and stimulated with/without 1 × 105 U/mL IFN-α and transfected with or without 20 µM tRF-3009 siRNA or 20 µM tRF-3009 mimic for 24 hours. Metformin (Met) or 2-Deoxy-D-glocose (2-DG) were added at 1mM for 24 hours, and phytohemagglutinin (PHA) was added at 1ug/ml as indicated.
Total RNA was extracted from the primary human CD4+ T cells using the TRIzol Reagent (Invitrogen) and the quality of the RNA samples was evaluated by using a Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific Inc.) and then size-fractionated on a 15% polyacrylamide gel electrophoresis (PAGE) gel to collect the 18–60nt fraction. The 5’ and 3’ RNA adapters were ligated to the RNA pool, and then this was subjected to RT-PCR to generate the sequencing libraries. PCR products were purified and then sequenced using a HiSeq 2000 sequencing system (BGI Tech). tRFs were aligned to the tRFdb (http://genome.bioch.virginia.edu/trfdb) and the transcripts were trimmed and aligned to the human genome (hg19). Differentially expressed tRFs and the transcripts were filtered using the following criteria: |Fold change ≥ 2.0| and P-value < 0.05 and |Fold change ≥ 1.5| and FDR < 0.05, respectively.
qRT-PCR validation of candidate tRFs and other mRNAs
Quantification of tRFs was completed using the same methods as those for analyzing piRNAs with some modifications . Isolated total tRFs RNA or mRNA (1.0μg) was polyadenylated at 37°C for 20 min with poly(A) polymerase (NEB) and reverse transcription was performed using the Superscript II Reverse Transcriptase kit (Takara). RNA transcript levels were measured by qRT-PCR using SYBR Green PCR Master Mix. After adding forward and reverse primers, the mixtures were incubated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s and 58 °C for 10 s. All experiments were performed in biological triplicate and the data were analyzed using the comparative cycle threshold (Ct) method. U6/β-actin was used as an internal control and the relative quantification was calculated using the following equation: Amount of target gene = 2−ΔCt, where ΔCt = Ct target genes − Ct U6/ β-actin. Gene-specific primers are listed in Supplementary Table 2. All samples were performed in triplicate.
Serum IFN-α level in lupus patients
Serum was obtained from SLE patients and serum IFN-α levels were measured using human IFN alpha ELISA kits (Raybiotech, lnc) according to the manufacturer’s instructions .
The functions of the differentially expressed tRF target genes were investigated using Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis. Hierarchical clustering of the differentially expressed genes (DEGs) according to their biological process (BP), cellular component (CC) and molecular function (MF) categories were completed using GO analysis and used to elucidate genetic regulatory networks (http://www.geneontology.org). Pathway analysis using graphical diagrams was performed to explore the DEG pathways using the Pathway database (http://www.genome.jp/kegg/) and a co-expression network was built using the Cytoscape software (version 3.6.1). Significance was determined by P-value and FDR. And the protein-protein interaction network (PPI) of the tRF-3009 interacting proteins was constructed and the module analysis was performed using STRING database.
The target binding sites of the tRFs (targeting both the 3’UTR region and promoter regions of all annotated genes) were predicted using the miRanda database (http://www.microrna.org/microrna/home.do). Significant 3'UTR regions and promoter regions were identified using the following criteria: energy ≤ − 15 and score ≥ 140 and energy ≤ − 20 and score ≥ 150, respectively.
Measurements of metabolic changes
Oxygen consumption rate (OCR) was measured using an XF96 Extracellular Flux Analyzer under mitochondrial stress test conditions (Seahorse). Assay buffer was prepared from non-buffered RPMI medium (Sigma) supplemented with 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate. Baseline OCR values were averaged between technical replicates for the first three successive time intervals. Extracellular lactate production was measured using a Glucose-lactic acid analyzer (SBA-40E). Mitochondrial membrane potential (△Ψm) was assessed using 5,5,6,6’-tetrachloro-1,1,3,3’-tetraethyl benzimidazolyl carbocyanine iodide (JC-1, green +red -cells%, Sigma). JC-1 is an ideal fluorescent probe whose mitochondrial uptake is directly related to the magnitude of the mitochondrial membrane potential . The greater the mitochondrial uptake, the greater concentration of JC-1 aggregate forms which have a red fluorescent emission signal, as opposed to the JC-1 monomer that fluoresces green [25, 26]. The ratio of red to green fluorescence is often applied to measure the ratio of mitochondrial depolarization. Intracellular ATP was measured using the ATP Determination Kit. ROS were measured using DCFH-DA dye (Thermo Fisher), flow cytometry and a Fluorometric Intracellular Ros Kit (Sigma) . Blank medium was used as the negative control, and PHA was used as the positive control. PHA belongs to high molecular glycoprotein family and has the activity of promoting the mitosis of PBMC .
RNA pull down
The healthy donor-derived CD4+ T cells were transfected with 5′-biotinylated synthesized tRF-3009 or a random sequence. CD4+ T cells were collected in an extraction buffer 48 hours post transfection. After separating the lysate by centrifugation at 14 000 rpm for 15 min, the cell extract was incubated with streptavidin-coated microbeads overnight at 4°C, following the manufacturer's protocol (μMACS, Miltenyi Biotec.). The proteins interacting with the tRF-3009 were pulled down using magnetic separator, followed by three washes. The proteins were resolved on PAGE, followed by silver staining. Gels fragments that contained bands of the protein of interest were excised and analyzed by mass spectrometry (LC/MS).
Data are presented as the mean ± SD. Two-tailed Student’s t-test or Mann-Whitney tests were used to evaluate the differences between the pairs of groups. Correlations between clinical information and tRF expression were evaluated using Pearson’s correlation test. All statistical analyses were performed using GraphPad Prism (version 6.0) and SPSS software (version 21.0). A P-value of < 0.05 was considered statistically significant.