Subjects and study design
All AKI samples were obtained from the patients in NICU of the First Affiliated Hospital of Nanjing Medical University from October 2020 to November 2021. Blood samples of controls were drawn from mild to moderate indirect hyperbilirubinemia at Obstetrics at the third day after birth. Firstly, to perform the circRNA profile, 2mL peripheral venous blood were drawn from five infants with asphyxia induced AKI and five controls. The time point of blood drawing is when the AKI diagnosis was established. Subsequently, for qPCR validation, peripheral blood samples were collected from 20 asphyxial AKI newborns and 16 controls. Samples were mixed with TRIzol and then frozen in liquid nitrogen until they were tested for circRNA sequencing or quantification. The clinical characteristics of AKI infants and controls are showed in Table1.
The indicators of acute perinatal asphyxia were as follows: a) history of fetal distress (bradycardia, late decelerations, absence of heart rate variability); b) the presence of sentinel hypoxia event immediately before or during delivery; c) metabolic acidosis in the arterial umbilical cord (base deficit ≧ -16 mmol/l), and PH≦7.0; d) clinical indications of hypoxic-ischemic encephalopathy and multiple organ involvement .
The definition of AKI was based on the neonatal modified KDIGO criteria , as persistently increased level of serum creatinine (>1.5mg/dl) for at least 24hs or decreased level of urine output (<1.0ml/kg·h).
High-throughput RNA sequencing
Five samples from AKI group and five samples from control group were pooled into two independent tubes for high throughput sequencing. Total RNA was isolated from the above two tubes and purified by Trizol reagent (Invitrogen, Carlsbad, CA, US) following the manufacturer's instruction. The RNA amount and purity of samples was quantified through NanoDrop ND-1000 (NanoDrop, Wilmington, DE, US). The RNA integrity was assessed by Agilent 2100 with RIN number >7.0. 5 ug of total RNA was used to deplete ribosomal RNA according to the instruction of the Ribo-Zero™ rRNA Removal Kit (Illumina, SanDiego, US). Then left RNAs were treated with Rnase R (EpicentreInc, Madison, WI, US) to remove linear RNAs and to enrich circRNAs. After removing linear RNAs and ribosomal, the enriched circRNAs were fragmented into small pieces by divalent cations. Then, the cleaved RNA fragments were reverse-transcribed to create the cDNA, which were further used to synthesize U-labeled second-stranded DNAs with E.coli DNA polymerase I, dUTP and RNase H. An A-base was added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each one contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single-ordual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and final extension at 72℃ for 5min. The average insert size for the final cDNA library was 300bp (±50bp). Finally, we performed the paired-end sequencing on an Illumina Hiseq 4000 (LCBio, China) following the vendor's recommended protocol.
Firstly, cutadapt was used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases. Then sequence quality was verified using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). We used Bowtie2 and Tophat2 to map reads to the genome of species. Remaining reads (unmapped reads) were still mapped to genome using tophat-fusion. CIRCExplorer2 and CIR was used to denovo assemble the mapped reads to circular RNAs at first. Then, back splicing reads were identified in unmapped reads by tophat-fusion. All samples were generated unique circular RNAs. CircRNA expressions from different samples or groups were calculated by scripts in house. Only the comparisons with ‘P value’ less than 0.05 were considered as differential expression by R package. This part was implemented by the company named Obio Technology, Shanghai, China.
Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/) and Gene Ontology (GO; http://www.geneontology.org/) were used to analyze the parent genes to predict circRNA function. GO functional analysis is divided into three parts: molecular function, biological processes, and cellular components. GO analysis was used to annotate gene function based on the GO database to obtain all functions of the gene parameters.
RT-qPCR validation for candidate circRNAs
The extraction and quantification of total RNA were processed as previously described. The RNA quantity control was detected using a NanoDrop2000 Spectrophotometer (Thermo Fisher Scientific, Inc. US). Then, 1µg of total RNA was isolated as previous report and converted into cDNA by the HiScript® II Q Select RT SuperMix for qPCR (R232‑01; Vazyme; China) according to the manufacturer's protocol [4µl 5X HiScript II Select qRTSuperMix, 1µl Random hexamers (50ng/µl) and 1µg RNA]. The RT reaction was conducted at 37˚C for 15min and 85˚C for 2min. Next, the RT-PCR reaction was performed using AceQ® qPCR (Q131‑01; Vazyme; China). For RT-PCR, 1µl cDNA was added to 9µl master mix, including 5µl SYBR®Green Master Mix (Low Rox Premixed; Q131-01; Vazyme; China), 0.2µl reverse and forward primers, and 3.6µl diethypyrocarbonate water. Then, the PCR was performed under the reaction conditions included an initial step at 95˚C for 5min, and 40 cycles at 90˚C for 15sec and at 60˚C for 15sec, 72˚C for 1min and final extension at 72˚C for 10min. The primer sequences are presented in Table2. PCR was performed in each plate using GAPDH as an endogenous control. All primer sequences were designed and synthesized by Guangzhou RiboBio Co. Ltd. The expression level of circRNAs were calculated through the 2-∆∆Ct method.
All data are presented as Mean±SEM. All experiments were repeated at least three times. The data were analyzed by SPSS 17.0 (SPSS, Inc. US) and GraphPad Prism 8.0 (GraphPad Software, Inc. US) statistical packages. For quantitative data, Student’s t-test was applied to analyze statistical significance between two groups. For qualitative data, Fisher's exact test was used to compare between two groups. P < 0.05 was considered as a statistically significant difference.