Comparative transcriptome analysis of miRNA in hydronephrosis male children caused by ureteropelvic junction obstruction with or without renal functional injury

Patients and samples

From August 2016 to June 2017, 12 patients that are younger than 6 months with congenital unilateral hydronephrosis were selected as the research subjects. To confirm the diagnosis of hydronephrosis, all patients underwent urinary ultrasound and magnetic resonance urography (MRU), as well as Diuretic radionuclides (DRS) preoperatively. We divided the patients into two groups depending on their preoperative differential renal function (DRF), patients with normal DRF level (>45%) were classified as normal DRF group while those with declined DRF level (<30%) were considered as abnormal DRF group. All of them received open pyeloplasty, the ureteropelvic junctional obstruction were confirmed by operation and pathological postoperatively. The kidney parenchyma tissues were collected during surgery, according to the study of Song et al. (2019), and the tissues were frozen in liquid nitrogen and stored at −80 °C for further study. The study was approved by the Ethics Committee of Shengjing Hospital, China Medical University, Shenyang, China (No. 2012PS81K), and the Ethics Committee waived the need for consent.

Experimental procedure of unilateral ureteral obstruction

The Unilateral Ureteral Obstruction (UUO) is a common prepared animal model in studying obstructive nephropathy. Two litters of 24 newborn Sprague Dawley (SD) rats (weight, 13–18 g, specific pathogen free) were obtained from Liaoning Changsheng Biotechnology Co. Ltd. (Benxi, China). Pups were kept with their dams in a laboratory animal facility with a constant temperature of 23 ± 2 °C and relative humidity of 40 ± 10% under a regular 12 h light/dark cycle. All animal care and experimental protocols were approved by the Committee on the Ethics of Animal Experiments of Shengjing Hospital, China Medical University, Shenyang, China (No. 2018PS228K).

One litter of pups were randomly divided into UUO-2d sham group and UUO-2d group (n = 6, per group), the other litter of pups were randomly divided into UUO-5d sham group, and UUO-5d group (n = 6, per group). Animal surgical procedures were performed using sterile technique under isoflurane anaesthesia. The pups in the UUO groups underwent ligation of the left ureter within 48 h after birth. Briefly, the left ureter was visualized by a flank incision, ligated with 8–0 silk below the renal pelvis and cut between the two ligatures. The sham group underwent the same surgery except for left ureter ligation. Postoperatively, the pups were returned to their dams.

The criteria were established for euthanizing animals prior to the planned end of the experiment. The pups were sacrificed with an overdose of pentobarbital sodium at 150 mg/kg intraperitoneally (i.p.) to reduce the pain or suffering; At days 2ed and 5th after surgery, the kidneys were harvested, frozen in liquid nitrogen, and stored at −80 °C until further processing.

Cell culture and transform growth factor beta 1 (TGF-β1) treatment

HK-2, a human kidney epithelial cell line, was purchased from the Shanghai Institute for Biological Sciences (SIBS), Chinese Academy of Sciences (Shanghai, China) and cultured for less than six months from the time of resuscitation. HK-2 cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) /F-12 1:1 (Hyclone Laboratories, Inc., Logan, UT, USA) supplemented 10% fetal bovine serum supplemented with 1% penicillin/streptomycin at 37 °C, 5% CO₂ in a humidified incubator. At ∼50% confluency, cells were treated with recombinant human TGF-β1 (10 ng/ml) (Proteintech Group, Inc.) for 24 h, 48 h, 72 h, 96 h, 120 h and 144 h or the vehicle control in DMEM/F12 1:1 for the indicated time period.

MiRNA sequence

Briefly, the kidney specimens were collected from hydronephrosis patients, and the RNA sequencing libraries were constructed from the extracted and amplified RNA using the standard Illumina library preparation protocols. miRNA-seq was performed on the Illumina HiSeq 2500 platform using PE150 protocol by Genesky Biotechnologies, Inc. (Shanghai, China). Data were collected as previously described as Liu et al. (2017) reported. The tool of ASprofle was performed for extraction and comparison of Alternative splicing (AS) events (Florea, Song & Salzberg, 2013).

Prediction of target genes

The target genes of the differentially expressed miRNAs were screened by the starBase v2.0 (http://starbase.sysu.edu.cn) (Li et al., 2014), and gene searched in >2 databases simultaneously, such as TargetScan (http://www.targetscan.org), miRTarBase (http://mirtarbase.mbc.nctu.edu.tw) or miRmap databases (http://mirmap.ezlab.org) were predicted to be target genes.

Functional and pathway analysis

For functional enrichment pathway analysis, gene ontology (GO) which was subdivided into three groups, including biological processes, cellular components, and molecular functions, was applied to analyze the function of the predicted target genes. Similarly, the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to find out the significant pathway of the predicted target genes. The GO and KEGG analysis were performed using the OmicShare tools, a free online platform for data analysis (http://www.omicshare.com/tools).

RNA extraction and validation of potential miRNAs using real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from kidney tissues of patients or rats and cultured cells by RNAiso Plus (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. Total RNA (1 µg) was reverse-transcribed to cDNA by using the PrimeScript RT Reagent Kit (TaKaRa, Shiga, Japan) following the manufacturer’ s protocol. Then qRT-PCR was performed with the SYBR Premix Ex Taq (TaKaRa, Shiga, Japan) and primers (Ribobio, Guangzhou, China) in a final volume of 20 µl. All PCR reactions were done in triplicates and repeated at least three times in a 7500 Real-time PCR machine (Applied Biosystems, Waltham, MA, USA). The relative miRNA levels were normalized and quantified by U6 expression levels for each sample. Gene expression analysis were performed using the 2^−ΔΔ Cycle thresholds (ΔΔCt) method. All primers were designed and synthesized by RiboBio Co., Ltd. (Guangzhou, China).

Statistical analysis

SPSS version 13.0 (SPSS Inc. Chicago, IL, USA) and GraphPad Prism version 5.0 (GraphPad, San Diego, CA, USA) were used to analyze the data, which are expressed as the mean ± standard deviation (SD). Differential expression of miRNAs in the kidney tissues of patients with UUO in abnormal renal function group compared to those in normal renal function group was detected using two-sided Student’s t-test. P < 0.05 was considered to be statistically significant.

Clinical information

All the patients with congenital hydronephrosis were Society of Fetal Urology (SFU) grade 4. Among the six patients who underwent miRNA-seq, age distributions were 6–14 weeks in normal renal function group (median age, 10 weeks) and 7–18 weeks in abnormal renal function group (median age, 10 weeks). Meanwhile, the other six patients who underwent RT-qPCR validation assays, age distributions were 12–20 weeks in normal renal function group (median age, 13 weeks) and 6–22 weeks in abnormal renal function group (median age, 12 weeks). All the patients are male.

Differentially expressed renal miRNAs in hydronephrosis children with or without renal dysfunction

After comparative analysis of the miRNA-seq data, 22 miRNAs were differentially expressed in kidney tissues of abnormal renal function group compared with normal renal function group (fold change >1.5; P < 0.01; Table 1). Among them, five miRNAs were up regulation and 17 miRNAs were down regulation in hydronephrosis children with renal dysfunction, Hierarchical clustered heatmap and Volcano plot showed the expression changes of miRNA between the two groups (Fig. 1).

Construction of target prediction network of miRNA-mRNA

To identify the function of the differentially expressed miRNAs, the prediction of miRNA target genes was performed by the starBase v2.0 (http://starbase.sysu.edu.cn) (Li et al., 2014). Subsequently, select genes that overlapped at least two of the three major online miRNA target prediction algorithms: TargetScan, miRTarBase and miRmap databases. We performed the 22 differentially expressed miRNAs (Table 1) to predict the targeting genes using TargetScan, miRTarBase and miRmap databases, 1,499 genes (Supplement 1) were obtained with overlapping at least two of the three major online miRNA target prediction websides. All of these predicted 1,499 mRNAs were used to perform the GO and KEGG pathway analysis. As shown in Fig. 2A, the most significant top 5 GO terms in biological process were respectively cellular process, biological regulation, metabolic process, regulation of biological process and response to stimulus. In molecular function, the top 5 GO terms were binding, catalytic activity, transcription regular activity, molecular function regulator, and transporter activity. In cellular component, cell, cell part, organelle, organelle part and membrane were the top five GO terms. A total of 64 distinct pathways with enrichment test P < 0.05 were identified according to the KEGG pathway database. In addition, KEGG pathway analysis revealed that the identified miRNAs regulated the signaling pathways of cancer, Cushing Syndrome, Longevity regulation, regulation of pluripotency of stem cells, axon guidance, etc (Fig. 2B).

Validation of the microarray data through RT-qPCR in neonatal rat kidney tissues with UUO

To validate the microarray data, twelve candidate human and rat homologous miRNAs were selected for miRNA expression validation using RT-qPCR, including four up-regulated miRNAs (miR-21-5p, miR-224-5p, miR-142-5p, miR-212-5p) and eight down-regulated miRNAs (miR-378a-5p, miR-138-5p, miR-874-3p and miR-378a-3p miR-187-3p, miR-204-3p, miR-873-3p, miR-338-3p) (Table 2). In neonatal rat kidney tissues with 2-day UUO, we found that miR-187-3p, miR-873-3p, and miR-21-5p in twelve selected miRNAs were all up-regulated with statistical significance. Only the trend of change in miR-21-5p was consistent with that observed by miRNA-Seq data (Fig. 3A). Furthermore, in neonatal rat kidney tissues with 5-day UUO, we found that the change trends of one (miR-187-3p) from the down-regulated miRNAs and two from the up-regulated ones (miR-21-5p, miR-142-5p) revealed the same expression tendency as miRNA-Seq data and were statistically significant (Fig. 3B). Therefore, miR-21-5p, miR-187-3p, miR-142-5p were selected for further validation in kidney tissues of patients with congenital hydronephrosis.

Validation of the microarray data through RT-qPCR in kidney tissues of patients with congenital hydronephrosis

We further analyzed the miRNA expression levels of miR-187-3p, miR-21-5p and miR-142-5p in kidney tissues of patients with congenital hydronephrosis using RT-qPCR. The expression levels of miR-187-3p was significantly down-regulated in patients with abnormal DRF compared to normal DRF ones (fold change = 0.277, p = 0.044). Meanwhile, the expression level of miR-21-5p was up-regulated but it was not statistically significant (Fig. 3C). Moreover, there is no significant difference in miR-142-5p expression between abnormal DRF group and normal DRF group (Fig. 3C).

Validation of the microarray data through RT-qPCR in HK-2 cells induced by TGF-β1 in vitro

RT-qPCR analysis revealed that miR-187-3p expression was significantly up-regulated in HK-2 cells after induced by TGF- β1 in vitro, and the expression level of miR-187-3p decreased with time of induction (Fig. 3D). As shown in Fig. 3D, the expression levels of miR-187-3p in HK-2 cells induced by TGF-β1 in vitro were obviously higher than those of control group with ∼10.68-fold, ∼6.49-fold, ∼4.20-fold at 24, 48, 72 h, respectively. However, at 96 and 120 h under TGF-β1 treatment, the expression level of miR-187-3p showed no significant difference between the two groups (p >0.05). Induced for 144 h, there was a marked decrease in expression of miR-187-3p (fold change = 0.001; p = 0.027).