3.1 Construction of expression profile of sRNAs for BC by sRNA-sequencing
SRNA sequencing was carried out to construct the differentia expression of non-coding RNAs in BC compared to NAT. Two small libraries were sequenced for two paraffin blocks (NAT and BC tissue). Histopathological properties of this specimen were shown in table 1 (Case1). By comparing non-coding RNA expression profiles of BC and NAT, 1400 sRNAs (p<0.05) were filtered out by a SAM software. The raw data were then standardized and log2-transformed to show on a scatter plot (Fig. 1A). Among 1400 sRNAs, 723 non-coding RNAs were downregulated, but 678 sRNAs were upregulated. Each dot on the scatter plot represents the sRNA. Among 1400 sRNAs, 520 microRNAs were differentially expressed. 185 microRNAs were down-expressed, but 335 were overexpressed. The x-axis denotes the data of the NAT and y-axis denotes the data of the BC. Correlation plot was constructed to show expression levels of non-coding RNAs between BC and NAT (Fig. 1B). Blue color denoted the correlation of sRNAs between the BC and NAT. Heat map was designed to show 29 miRNAs which were markedly downregulated in BC compared to NAT (Fig. 1C). Table 2 shows the information on these 29 miRNAs which are notably downregulated. In this study, has-miR-1275, which underlined with red color in heat map, was focused to identify sequence and expression level.
3.2 Confirmation of miR-1275 expression level by RT-qPCR
The miR-1275 expression level in RNA-sequencing was observed to be significantly downregulated in BC tissue, as compared to NAT. The p.value of this miRNA was 0.614 (Fig. 2A). Then, this miRNA was selected to confirm. RT-qPCR machine was used to confirm the differential expression of miR-1275 in the laboratory. 40 FFPE blocks for 20 cases was used in this experiment, including 20 BC tissues and 20 normal tissues next to tumoral tissues (2cm away from tumoral tissues). The clinical properties of these cases were briefly explained in table 1. The miR-1275 expression level was detected to be markedly decreased in BC cells compared to adjacent normal cells and the P-value of this was 0.001** (Fig.2B). The mature sequence of which in the BC and NAT was made up of 17 nucleotides and also identical (Fig.2C). Then, Kaplan–Meier overall survival curve was designed to show the effect of miR-1275 expression on the prognosis of cases with BC. Data was used from The Cancer Genome Atlas (TCGA) database and analyzed. Kaplan–Meier overall survival curve displayed that cases were separated into two classes according to its expression. The decreased miR-1275 (P-value=0.0401) was related to overall survival in cases with BC (Fig.2D).
3.3 Differentially expressed genes for adjacent normal and BC tissue by MACE-seq.
Differentially expressed genes in BC and NATs was displayed (Fig. 3A). 26843 differentially expressed genes (P≤0.05) were filtered out by a SAM software. In order to show genes that were more significantly different in their expressions, 7041 genes were standardized and log2-transformed to show on a scatter plot. 3624 genes were significantly overexpressed and 3417 genes were significantly down-expressed in tumoral cells compared to adjacent normal cells. The P value for that was ranged from smaller (Blue) to greater (Red). Each point on the scatter plot represents the gene. the x-axis denotes the data of the NAT and y-axis denotes the data of the cancerous tissue.
3.4 Candidate target genes regulated in BC by miRNA-1275
Table 3 showed that eleven computational prediction programs were applied for discovering the strongest candidate genes possessed miR-1275 binding sites in the 3’ -UTR. Six predicted genes genes (DVL3, PPP2R2D, THSD4, CREB1, SYT7, and PRKACA) were selected to have binding site to miRNA-1275. The information on these six predicted genes were summarized in table 4. Eleven databases showed that DVL3 and PPP2R2D possess the binding site to miRNA-1275; whereas, THSD4, and CREB1 were confirmed in ten prediction programs to be targeted by miRNA-1275, but SYT7 and PRKACA were confirmed by six tools to be predicted targets. These putative target genes are important for biological analysis of the BC tissues because the over- or down-expression of which can play a damaging role in several cellular processes and contribute to the cancer progression and tumorigenesis.
3.5 Determination of expression level of candidate target genes by MACE-seq approach
Among 3624 upregulated genes, the six predicted genes (DVL3, PPP2R2D, THSD4, CREB1, SYT7, and PRKACA) were pointed and named in the BC cells as compared to NAT (Fig.3B). Then the differential expression of which and their binding sites to has-miR-1275 were shown in figure 4. SYT7 gene was more overexpressed in BC, as compared to PRKACA gene. Overexpression level of THSD54 gene was higher than the up-regulation of PPP2R2D and DVL3 genes. CREB1 was upregulated but more over-expressed than ST73 gene. Table 5 demonstrates the information on P.value, False Discovering Rate (FDR), and Fold Change (FC) of these six predicted genes possessed miR-1275 binding sites in the 3’ -UTR. They were identified as potentially modulated by miR-1275 using computational prediction databases and TCGA algorithm.
Next, the relationship between the expression level of these 8 candidates and histopathological significance were examined based on data from TCGA database. Among 204 target genes, DVL3: P=6.98E-04, PPP2R2D: P=1.53E-03, THSD4: P=2.28E-12, CREB1: P=3.12E-01, SYT7: P=1.28E-48, and PRKACA: P=5.68E-02 were markedly relationship with worse prognosis in cases with BC. The Kaplan–Meier overall survival curve analyses of cases with BC were designed to be separated into 2 classes according to their expression (Fig.5).
3.6 The role of miR-1275 by targeting selected putative genes in BC
MiRNAs are implicated in silencing mRNA transcripts through matching or mismatching with target mRNAs. As hypothesis of microRNA biogenesis, major strands of miR-1275 come from miRNA duplex are joined into the RISC protein and modulate the mRNA transcripts, but minor strands are broken down and cannot modulate gene expression. miR-1275 can play an essential role in regulating several biological mechanisms; including cell growth, migration, differentiation, proliferation and apoptosis. In this study, the down-expression of which regulate a set of genes and regulators related with tumor development. Six genes (DVL3, PPP2R2D, THSD4, CREB1, SYT7, and PRKACA) were detected to be over-expressed in BC cells. Fig.6 shows the relationship between miR-1275 and these target genes. miR-1275 in breast cancer promotes cancer cell proliferation, cell differentiation, tumor growth, invasion and migration and also inhibits apoptosis through several gene targets. PPP2R2D acts as a tumor suppressor in signaling pathway in BC and is negatively regulated by miR-1275. The overexpression of which decreases AKT and RACK1 abilities. Then these regulators decrease cell survival and migration. DVL3 is implicated in the breast cancer pathways and negatively controlled by miR-1275. The up-regulation of this gene increases the cancer cell proliferation, migration and invasion. The cancer cell proliferation ability is increased when miR-1275 becomes overexpressed. another target gene. CREB1 and PRKACA show also negative correlation with miR-1275 level. Whereas CREB1 was found to reduce apoptosis process and increase cell proliferation in breast cancer, PRKACA plays a key role in tumorigenesis and development of BC. However, the function of THSD4 and SYT7, currently unidentified, may boost tumor growth in breast cancer.
Table 2. Comparison of marked down-expressed miRNAs in BC with NAT
miRNA
|
miRBase accession
|
Location
|
Log2FC
|
P. value
|
FDR
|
Hsa-miR-1
|
MIMAT0031892
|
20q13.33
|
-2.6291
|
0.3109
|
1.0
|
Hsa-miR-100-5p
|
MIMAT0004512
|
11q24.1
|
-1.2349
|
0.2824
|
1.0
|
Hsa-miR-10b-5p
|
MIMAT0000254
|
2q31.1
|
-1.2852
|
0.261
|
1.0
|
Hsa-miR-125a-5p
|
MIMAT0000443
|
19q13.41
|
-1.4153
|
0.213
|
1.0
|
Hsa-miR-125b-5p
|
MIMAT0000423
|
11q24.1
|
-2.0041
|
0.083
|
1.0
|
Hsa-miR-1275
|
MIMAT0005929
|
6p21.31
|
-1.4150
|
0.6 14
|
1.0
|
Hsa-miR-130a-3p
|
MIMAT0004593
|
11q12.1
|
-2.0168
|
0.104
|
1.0
|
Hsa-miR-133a-5p
|
MIMAT0026478
|
18q11.2
|
-2.3885
|
0.220
|
1.0
|
Hsa-miR-143-5p
|
MIMAT0004599
|
5q32
|
-1.2823
|
0.308
|
1.0
|
Hsa-miR-204-5p
|
MIMAT0000265
|
9q21.12
|
-4.0627
|
0.086
|
1.0
|
Hsa-miR-21-3p
|
MIMAT0004494
|
17q23.1
|
-0.0365
|
0.995
|
1.0
|
Hsa-miR-214-3p
|
MIMAT0000271
|
1q24.3
|
-0.8746
|
0.440
|
1.0
|
Hsa-miR-25-5p
|
MIMAT0004498
|
7q22.1
|
-1.2630
|
0.793
|
1.0
|
Hsa-miR-30a-3p
|
MIMAT0000088
|
6q13
|
-0.2358
|
0.838
|
1.0
|
Hsa-miR-30b-5p
|
MIMAT0000420
|
8q24.22
|
-1.3254
|
0.245
|
1.0
|
Hsa-miR-30d-5p
|
MIMAT0000245
|
8q24.22
|
-0.9730
|
0.389
|
1.0
|
Hsa-miR-374b-5p
|
MIMAT0004955
|
Xq13.2
|
-0.2420
|
0.8435
|
1.0
|
Hsa-miR-410-5p
|
MIMAT0026558
|
14q32.31
|
-0.6780
|
0.989
|
1.0
|
Hsa-miR-423-5p
|
MIMAT0004748
|
17q11.2
|
-0.5727
|
0.614
|
1.0
|
Hsa-miR-451a
|
MIMAT0001631
|
17q11.2
|
-0.41900
|
0.711
|
1.0
|
Hsa-miR-455-5p
|
MIMAT0003150
|
9q32
|
-2.5081
|
0.305
|
1.0
|
Hsa-miR-505-5p
|
MIMAT0004776
|
Xq27.1
|
-1.5956
|
0.611
|
1.0
|
Hsa-miR-532-3p
|
MIMAT0004780
|
Xp11.23
|
-0.7496
|
0.612
|
1.0
|
Hsa-miR-624-5p
|
MIMAT0003293
|
14q12
|
-0.0931
|
1.0
|
1.0
|
Hsa-miR-664a-3p
|
MIMAT0005949
|
1q41
|
-0.9249
|
0.539
|
1.0
|
Hsa-miR-664b-5p
|
MIMAT0022271
|
Xq28
|
-0.5081
|
0.899
|
1.0
|
Hsa-miR-92a-1-5p
|
MIMAT0004507
|
13q31.3
|
-0.2085
|
0.972
|
1.0
|
Hsa-miR-934
|
MIMAT0004977
|
Xq26.3
|
-3.0931
|
0.417
|
1.0
|
Hsa-miR-99b-5p
|
MIMAT0000689
|
19q13.41
|
-0.1667
|
0.883
|
1.0
|
Table 3. Brief information on target predicted databases was shown to find putative targets possessing binding sequence to miR-1275.
Target predicted
sites
|
Species
|
Tool properties
|
Website
Websites
|
miRTarBase
|
Human, Mouse, Rat
|
Conservation, seed location
|
http://mirtarbase.mbc.nctu.edu.tw/php/index.php
|
Target scan
|
Human, Mouse, Fly, Fish, and Worm
|
Conservation, seed location
|
http://www.targetscan.org/
|
TargetMiner
|
Human, Mouse, Rat, Fly
|
Conservation, seed location
|
https://www.isical.ac.in/~bioinfo_miu/TargetMiner.html
|
MirTar2
|
Human, Mouse, rat, Dog and Chicken
|
Conservation, seed location
|
http://www.mirdb.org/
|
DIANA
|
Any
|
Conservation, seed match, and free energy
|
http://www.microrna.gr/microT-CDS
|
miRWalk
|
Human, Mouse, and Rat
|
Conservation, seed match and free energy
|
http://mirwalk.uni-hd.de/
|
miRmap
|
Human, Chimpanzee, Mouse, Rat, Cow,Chicken, Zebrafish, and Opossum
|
Conservation, seed match, and free energy
|
https://mirmap.ezlab.org/
|
RNA22
|
Human, Fruit Fly, Mouse, and Worm
|
Seed match and free energy
|
https://cm.jefferson.edu/rna22/
|
PicTar - Tools4miRs
|
Human, Mouse, Rat, Fly
|
Conservation, seed location
|
https://tools4mirs.org/software/target_prediction/pictar/
|
mirPath
|
|
Human, Mouse, D. melanogaster, C. elegans, R. norvegicus, D. rerio and G. gallus
|
Conservation, seed match and free energy
|
http://snf-515788.vm.okeanos.grnet.gr/index.php?r=mirpath/geneList
|
Microrna. org
|
|
Human, mouse, Fruit Fly, and rat
|
Conservation, seed match, free energy
|
http://www.microrna.org/
|
Table 4. candidate target genes possessing binding sequence to miR-1275 was shown.
Target gene
|
Ensemble ID
|
Position on Chromosome
|
No. of sites predicted the gene as miR-1275 target
|
DVL3
|
ENST00000313143.3
|
3q27.1
|
11
|
PPP2R2D
|
ENST00000422256.2
|
10q26
|
11
|
THSD4
|
ENST00000355327.3
|
15q23
|
10
|
CREB1
|
ENST00000432329.2
|
2q34
|
10
|
SYT7
|
ENST00000263846.4
|
11q12.2
|
6
|
PRKACA
|
ENST00000308677.4
|
17q24.2
|
6
|
Table 5. Experimentally validated target genes of miR-1275 in BC
Targets
|
Gen ID
|
Description
|
P.value
|
FDR
|
Log2fc
|
DVL3
|
ENSG00000161202
|
Dishevelled segment polarity protein 3
|
6.98E-04
|
3.48E-03
|
0.58975
|
PPP2R2D
|
ENSG00000175470
|
Protein phosphatase 2, regulatory subunit B, delta
|
1.53E-03
|
6.95E-03
|
0.616981
|
THSD4
|
ENSG00000187720
|
Thrombospondin type 1 domain containing 4
|
2.28E-12
|
3.63E-11
|
0.938081
|
CREB1
|
ENSG00000118260
|
cAMP responsive element binding protein 1
|
3.12E-01
|
5.13E-01
|
0.269971
|
SYT7
|
ENSG00000011347
|
Synaptotagmin 7
|
1.28E-48
|
8.14E-47
|
1.84905
|
PRKACA
|
ENSG00000072062
|
Protein kinase cAMP-activated catalytic subunit alpha
|
1.77E-02
|
5.68E-02
|
0.296759
|