Total RNA samples were extracted from Wistar WU rat retinal tissues of five developmental stages (P5-P21). In order to monitor the general quality of extracted RNA samples RNA integrity number (RIN) was calculated by Agilent Bioanalyzer Software. High quality and low degradation samples (RIN≥7) were only processed further. To evaluate the reproducibility of the experimental procedures in 316 v2 chip two independent P7 samples were assayed (biological replicates) and P21 samples were sequenced as technical replicates in every sequencing procedure. The miRNA levels between the two independent P7 tissue samples and every P21 samples were characterized by a high Pearson’s correlation coefficient (Pearson r =0.92 and 1.00, respectively), that means high reproducibility of the sequencing (Figure 1A). Some samples were also sequenced in 318 v2 chip (P5, P10, P15 and P21; as biological replicate) also with a high Pearson’s correlation coefficient (Figure 1B). Results were averaged to reduce experimental errors. In 316 v2 chip we obtained in total 143 796 965 bases and in average 12 182 661 reads for each sample after removal of low quality reads and contaminants, with the peak length of each sample at about 24 nt (Additional file 1). RNA reads were annotated based on their sequences (in average 420 178 reads/sample), and their relative abundances were determined by their counts, normalized to the number of reads per kilobase of the exon model per million mapped reads (RPKM) methods. To minimize the false positive signal, only reads that were detected in both 316 and 318 v2 sequencings (biological and technical replicates) were used for further bioinformatics analysis.
Based on the non-coding RNA classification methods of ENSEMBL our data generated by sequencings were classified into five functionally important categories including ribosomal RNAs (rRNAs), as well as small RNAs such as microRNAs, snoRNAs and snRNAs (Table 1).
Table 1: Classification of non-coding RNAs detected by IonTorrent PGM sequencing as time-scale manner.
samples
|
rRNA
|
miRNA
|
snRNA
|
snoRNA
|
P5_P7
|
97
|
643
|
623
|
686
|
P7_P10
|
97
|
630
|
603
|
668
|
P10_P15
|
91
|
610
|
580
|
621
|
P15_P21
|
96
|
627
|
617
|
680
|
It is well known the rRNAs playing important roles in the protein synthesis machinery, the highest rRNA number were at least 97, but the lowest value at the comparison of P10 and P15. The total expression levels for small RNAs that contribute to the biogenesis of rRNAs or to the protein synthesis, mainly characterized by the same tendency during the development period. Slight decrease of the number of non-coding RNAs at P10 suggests an important role of regulation of protein synthesis for the retinal synaptogenesis. Focusing on the known miRNAs in non-coding RNAs from ENSEMBL, we identified approximately 275 miRNAs at least one of the five developmental stages. The expression profile of miRNAs could provide indication of their potential functions during development. We observed that although there was no obvious difference in the total number of miRNAs detected in retina during development process, the expression level of different miRNAs in retina was very dynamic over stages. There are some miRNAs that show constant high abundance during the development process such as; mir-19, mir-101; mir-181, mir-183, mir-124 and let-7. While there are miRNAs that appear as most abundant only in the early stages such as mir-20a, mir-206, mir-133, mir-466, mir-1247 or mir-3582, others are characteristic with high abundance at later stages or increasing with development for example mir-29, mir-96, mir-125, mir-344 or mir-664. The most abundant miRNAs at each developmental stage are collected in Table 2.
Table 2: The top 20 most abundant miRNAs (RPMM) at each developmental stage.
P5
|
P7
|
P10
|
P15
|
P21
|
rno-mir-376c
|
rno-mir-376c
|
rno-mir-106a
|
rno-mir-106a
|
rno-mir-376c
|
rno-mir-106a
|
rno-mir-106a
|
rno-mir-376c
|
rno-mir-376c
|
rno-mir-106a
|
rno-mir-206
|
rno-mir-206
|
rno-mir-206
|
rno-mir-206
|
rno-mir-206
|
rno-mir-19a
|
rno-mir-377
|
rno-mir-377
|
rno-mir-124-1
|
rno-mir-124-1
|
rno-mir-141
|
rno-mir-124-1
|
rno-mir-124-1
|
rno-mir-129-1
|
rno-mir-129-1
|
rno-mir-143
|
rno-mir-19a
|
rno-mir-129-1
|
rno-mir-377
|
rno-mir-377
|
rno-mir-let7c-2
|
rno-mir-143
|
rno-mir-19a
|
rno-mir-19a
|
rno-mir-320a
|
rno-mir-377
|
rno-mir-129-1
|
let7c-2
|
rno-mir-34b
|
rno-mir-19a
|
rno-mir-124-1
|
rno-mir-320a
|
rno-mir-141
|
let7c-2
|
rno-mir-3571
|
rno-mir-152
|
rno-mir-152
|
rno-mir-103a1
|
rno-mir-298
|
rno-mir-100
|
rno-mir-129-1
|
rno-mir-let7c-2
|
rno-mir-34b
|
rno-mir-18a
|
let7c-2
|
rno-mir-320a
|
rno-mir-141
|
rno-mir-18a
|
rno-mir-141
|
rno-mir-3570
|
rno-mir-100
|
rno-mir-344b-2
|
rno-mir-298
|
rno-mir-103a1
|
rno-mir-18a
|
rno-mir-103a1
|
rno-mir-103a1
|
rno-mir-152
|
rno-mir-144
|
rno-mir-144
|
rno-mir-3570
|
rno-mir-100
|
rno-mir-34a
|
rno-mir-320a
|
rno-mir-let7b
|
rno-mir-18a
|
rno-mir-3570
|
rno-mir-29b1
|
rno-mir-222
|
rno-mir-29b1
|
rno-mir-34b
|
rno-mir-18a
|
rno-mir-143
|
rno-mir-34a
|
rno-mir-103a1
|
rno-mir-298
|
rno-mir-222
|
rno-mir-320a
|
rno-mir-29b1
|
rno-mir-222
|
rno-mir-344b-2
|
rno-mir-29b1
|
rno-mir-222
|
rno-mir-152
|
rno-mir-141
|
To further validate the sequencing results by quantitative polymerase chain reaction (qPCR) we choose miR-9 and mir-23miRNAs which based on our previous investigations and literature search seemed to have major importance during postnatal retinal development(Leucht et al. 2008; Arora et al. 2010; Gao 2010; La Torre et al. 2013; Qi 2016; Pöstyéni et al. 2021b, a).The relative expression level of miR-9 exhibited the up-regulated pattern while that of mir-23 exhibited the down-regulated pattern compared to P21. In addition, the expression patterns of miRNAs revealed by qPCR were mostly consistent with sequencing results (Figure 2) with some exceptions. For example, miRNA-9at P5 and P7 or mir-23 at P15, when the sequencing results suggest down-regulation comparing to P21, while qPCR data propose minor up-regulations. However these minor discrepancy exhibited between qPCR and deep-sequencing results, but dataset intend generally high correlation and accuracy of sequencing in detection and quantification of the relative expression levels of miRNAs.
Prediction and Functional Analysis of Differentially Expressed miRNA Target Genes
Principal component analysis (PCA) revealed partial separation of samples based on postnatal day of development as shown in Additional file 2. Differential expression analysis identified 37 miRNAs which were differentially expressed on consecutive time-points with FDR < 0.05 (Additional file 3.pdf). When applying only the cut off for the value of log2 fold change more or less than ±2 there were collected 37 miRNAs listed in Additional file 4.pdf. Interestingly using any of settings only down-regulated miRNAs were reached the cut-off by P5 vs. P7 and P15 vs. P21, while only up-regulated miRNAs were detected by P7 vs. P10. Eight miRNAs changed significantly by more than 2-fold between time-points (Table 3) and the largest difference was observed for rno-mir-598, which increased 4.69-fold on postnatal day of 10 compared to P7.
Table 3: Differentially expressed miRNAs with FDR<0.05.*Values are mean RPMMs.
gene_symbol
|
id
|
Days
|
value_1*
|
value_2*
|
log2 (fold_change)
|
p_value
|
rno-mir-30c2
|
ENSRNOG00000035562
|
P5
|
P7
|
199873.00
|
7386.51
|
-4.76
|
0.0001
|
rno-mir-30c1
|
ENSRNOG00000035567
|
P5
|
P7
|
168793.00
|
8637.79
|
-4.29
|
0.0001
|
rno-mir-143
|
ENSRNOG00000035603
|
P5
|
P7
|
10014.50
|
2004.60
|
-2.32
|
0.0250
|
rno-mir-1b
|
ENSRNOG00000057048
|
P5
|
P7
|
14769.90
|
725.95
|
-4.35
|
0.0329
|
rno-mir-3579
|
ENSRNOG00000035473
|
P7
|
P10
|
1142.48
|
5550.71
|
2.28
|
0.0333
|
rno-mir-218-2
|
ENSRNOG00000035481
|
P7
|
P10
|
2617.80
|
13853.00
|
2.40
|
0.0214
|
rno-mir-3565
|
ENSRNOG00000035486
|
P7
|
P10
|
685.42
|
4756.47
|
2.79
|
0.0145
|
rno-mir-137
|
ENSRNOG00000035491
|
P7
|
P10
|
854.78
|
4819.50
|
2.50
|
0.0179
|
rno-mir-32
|
ENSRNOG00000035512
|
P7
|
P10
|
1922.49
|
28685.70
|
3.90
|
0.0122
|
rno-mir-153
|
ENSRNOG00000035515
|
P7
|
P10
|
541.30
|
4352.94
|
3.01
|
0.0024
|
rno-mir-135a
|
ENSRNOG00000035529
|
P7
|
P10
|
4917.51
|
22359.10
|
2.18
|
0.0204
|
rno-mir-3597-1
|
ENSRNOG00000035551
|
P7
|
P10
|
16311.20
|
86967.90
|
2.41
|
0.0101
|
rno-mir-30c2
|
ENSRNOG00000035562
|
P7
|
P10
|
7386.51
|
336258.00
|
5.51
|
0.0001
|
rno-mir-30c1
|
ENSRNOG00000035567
|
P7
|
P10
|
8637.79
|
295971.00
|
5.10
|
0.0001
|
rno-mir-3597-3
|
ENSRNOG00000035580
|
P7
|
P10
|
11775.60
|
63874.70
|
2.44
|
0.0099
|
rno-mir-143
|
ENSRNOG00000035603
|
P7
|
P10
|
2004.60
|
9290.18
|
2.21
|
0.0335
|
rno-mir-135b
|
ENSRNOG00000035621
|
P7
|
P10
|
2411.21
|
15906.80
|
2.72
|
0.0497
|
rno-mir-96
|
ENSRNOG00000035624
|
P7
|
P10
|
33220.40
|
286882.00
|
3.11
|
0.0017
|
rno-mir-3597-2
|
ENSRNOG00000035628
|
P7
|
P10
|
19556.50
|
99022.70
|
2.34
|
0.0106
|
rno-mir-211
|
ENSRNOG00000035648
|
P7
|
P10
|
13667.30
|
91139.80
|
2.74
|
0.0400
|
rno-mir-190b
|
ENSRNOG00000041245
|
P7
|
P10
|
7222.03
|
50375.40
|
2.80
|
0.0087
|
rno-mir-190
|
ENSRNOG00000047497
|
P7
|
P10
|
1332.48
|
8445.52
|
2.66
|
0.0378
|
rno-mir-141
|
ENSRNOG00000035577
|
P10
|
P15
|
1059.79
|
131.89
|
-3.01
|
0.0314
|
rno-mir-1247
|
ENSRNOG00000048809
|
P10
|
P15
|
6088.90
|
1189.15
|
-2.36
|
0.0483
|
rno-mir-3556a
|
ENSRNOG00000035458
|
P15
|
P21
|
14381.70
|
85066.60
|
2.56
|
0.0026
|
rno-mir-29b1
|
ENSRNOG00000035463
|
P15
|
P21
|
29531.50
|
177063.00
|
2.58
|
0.0016
|
rno-mir-339
|
ENSRNOG00000035479
|
P15
|
P21
|
760.71
|
5231.66
|
2.78
|
0.0066
|
rno-mir-326
|
ENSRNOG00000035506
|
P15
|
P21
|
1078.36
|
6358.60
|
2.56
|
0.0056
|
rno-mir-32
|
ENSRNOG00000035512
|
P15
|
P21
|
7525.46
|
1685.89
|
-2.16
|
0.0257
|
rno-mir-184
|
ENSRNOG00000035522
|
P15
|
P21
|
10837.30
|
78876.80
|
2.86
|
0.0206
|
rno-mir-99a
|
ENSRNOG00000035524
|
P15
|
P21
|
56161.10
|
312037.00
|
2.47
|
0.0328
|
rno-mir-30c2
|
ENSRNOG00000035562
|
P15
|
P21
|
354473.00
|
18818.80
|
-4.24
|
0.0001
|
rno-mir-30c1
|
ENSRNOG00000035567
|
P15
|
P21
|
277205.00
|
16863.80
|
-4.04
|
0.0002
|
rno-mir-130a
|
ENSRNOG00000035570
|
P15
|
P21
|
26959.40
|
123611.00
|
2.20
|
0.0150
|
rno-mir-141
|
ENSRNOG00000035577
|
P15
|
P21
|
131.89
|
855.18
|
2.70
|
0.0474
|
rno-mir-3554
|
ENSRNOG00000035606
|
P15
|
P21
|
2286.65
|
12293.90
|
2.43
|
0.0094
|
rno-mir-29b2
|
ENSRNOG00000035637
|
P15
|
P21
|
30986.10
|
206699.00
|
2.74
|
0.0006
|
rno-mir-6327
|
ENSRNOG00000050007
|
P15
|
P21
|
519.38
|
25211.20
|
5.60
|
0.0174
|
rno-mir-3584-1
|
ENSRNOG00000051517
|
P15
|
P21
|
1230.86
|
9485.82
|
2.95
|
0.0049
|
rno-mir-340-1
|
ENSRNOG00000047385
|
P15
|
P21
|
20327.60
|
4995.53
|
-2.02
|
0.0209
|
To gain insight on the roles of the differentially expressed miRNAs during postnatal-development of the retina, DIANA web-tool was used to predict potential target genes and to apply pathway enrichment analysis. For differentially expressed miRNAs, 850 predicted target genes were annotated with function. It is important to know that each miRNA can have different targets, and different miRNAs can have the same target gene. Between enriched pathways targeted by miRNAs cut off for the value of log2 fold change more or less than ±2 there were as follows: lipid metabolisms (such as unsaturated fatty acid, arachidonic acid or glycerophospholipid), amino acid metabolisms (such as valine, leucine, isoleucine or lysin degradation, tyrosine metabolism) and glycan metabolisms in large numbers. The dataset of P5-P7 transition has shown the crucial role of glutamatergic synapse formation (Additional file 5.pdf). Among significantly down-regulated miRNAs rno-mir-30c1 and 2, rno-mir-205 and rno-mir-503 were detected to target Prkx (ENSRNOG00000003696), Adcy6 (ENSRNOG00000011587), Gnai3 (ENSRNOG00000019465) and Homer2 (ENSRNOG00000019297) genes as shown in Figure 3 highlighted. Furthermore, also the data analysis of P5-P7 transition has revealed the importance the gap junctions (rn04540) KEGG pathway. In addition to Prkx (ENSRNOG00000003696), Adcy6 (ENSRNOG00000011587) and Gnai3 (ENSRNOG00000019465) genes, Gja1 (ENSRNOG00000000805) shows essential role in this pathway. These results suggest roles of miRNAs in regulating cell metabolism, synapse formation (both chemical and electric) as well as proliferation/survival/migration processes involved in retinal development process.