An integrated analysis of key microRNAs, regulatory pathways and clinical relevance in bladder cancer

Figures & data

Table 1 Characteristics of the microarray datasets from GEO database

Accession/ID	PMID	Platform	Number of BC tissues	Number of normal bladder tissues (control)	Gene/miRNA
GSE36121	22745731	GPL8227	n=16 (Ta–T1=13, T2–T4=3)	n=8	miRNA
GSE39093	23270926	GPL8786	n=10 (Ta–T1=5, T2–T4=5)	n=10	miRNA
GSE68594	26943042	GPL20120	n=33 (Ta–T1=18, T2–T4=15)	n=17	miRNA
GSE27448	21483740	GPL2895	n=10 (Ta–T1=6, T2–T4=4)	n=5	Gene
GSE40355	–	GPL13497	n=16 (Ta–T1=13, T2–T4=3)	n=8	Gene
GSE52519	–	GPL6884	n=9 (Ta–T1=3, T2–T4=6)	n=3	Gene

Abbreviations: BC, bladder cancer; GEO, Gene Expression Omnibus; miRNA, microRNA; PMID, PubMed ID.

Figure 1 Venn diagram of DEM/DEG selection in different datasets.

Notes: (A) DEMs; (B) upregulated DEGs; (C) downregulated DEGs.
Abbreviations: DEGs, differentially expressed genes; DEM, differentially expressed miRNA; miRNAs, microRNAs.

Table 2 Results of significant GO biological processes and the KEGG pathway analysis

Category	Description	Gene counts	Adjusted P-value
Upregulated gene
GO:0005654, CC	Nucleoplasm	49	4.40E-09
GO:0005634, CC	Nucleus	65	2.20E-06
GO:0000777, CC	Condensed chromosome kinetochore	7	8.70E-04
GO:0005819, CC	Spindle	8	9.30E-04
GO:0030496, CC	Midbody	8	1.10E-03
GO:0005515, MF	Protein binding	89	1.40E-06
GO:0003682, MF	Chromatin binding	16	6.00E-06
GO:0005524, MF	ATP binding	26	1.10E-03
GO:0051301, BP	Cell division	19	1.40E-08
GO:0007067, BP	Mitotic nuclear division	14	4.70E-06
GO:0007062, BP	Sister chromatid cohesion	9	1.30E-04
GO:0016925, BP	Protein sumoylation	8	3.00E-03
GO:0007052, BP	Mitotic spindle organization	5	8.40E-03
KEGG: hsa04110	Cell cycle	12	6.20E-08
Downregulated gene
GO:0043034, CC	Costamere	7	7.10E-06
GO:0043034, CC	Focal adhesion	18	1.00E-04
GO:0015629, CC	Actin cytoskeleton	12	1.60E-03
GO:0005576, CC	Extracellular region	35	3.90E-03
GO:0070062, CC	Extracellular exosome	51	4.30E-03
GO:0008201, MF	Heparin binding	11	3.20E-03
GO:0003779, MF	Actin binding	13	8.50E-03
GO:0007160, BP	Cell–matrix adhesion	8	4.30E-02
KEGG: hsa04110	MAPK signaling pathway	14	1.80E-03
KEGG: hsa05200	Pathways in cancer	15	2.10E-02
KEGG: hsa05205	Proteoglycans in cancer	10	3.80E-02

Abbreviations: BP, biological process; CC, cellular component; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function.

Figure 2 PPI network of DEGs.

Notes: (A) Upregulated DEGs; (B) downregulated DEGs.
Abbreviations: DEGs, differentially expressed genes; PPI, protein–protein interaction.

Table 3 The top 5% hub genes in the PPI networks

Ensembl gene ID	Gene symbol	Full gene name	Degree
Upregulated gene
ENSG00000170312	CDK1	Cyclin-dependent kinase 1	25
ENSG00000134057	CCNB1	Cyclin B1	21
ENSG00000072501	SMC1A	Structural maintenance of chromosomes 1A	16
ENSG00000117399	CDC20	Cell division cycle 20	14
Downregulated gene
ENSG00000035403	VCL	Vinculin	8
ENSG00000154229	PRKCA	Protein kinase C alpha	8
ENSG00000170345	FOS	Fos proto-oncogene, AP-1 transcription factor subunit	7
ENSG00000168610	STAT3	Signal transducer and activator of transcription 3	5

Abbreviation: PPI, protein–protein interaction.

Figure 3 The miRNA–mRNA regulatory network.

Note: Round nodes, miRNA; square nodes, mRNA; green line, from Targetscan database; blue line, from miRanda prediction.
Abbreviation: miRNA, microRNA.

Table 4 Results of the significant DEM expression validation and the relationship with bladder cancer clinical stages in TCGA dataset

miRNA ID	Higher expression	P-value (t-test)*	Pathological stages	P-value (chi-square)
hsa-miR-145-3p	Normal	1.73e-06	Pathological T/N/M status	4.76e-04/8.90e-05/9.62e-04
hsa-miR-21-3p	Tumor	1.79e-04	Pathological M status	7.27e-03
hsa-miR-487b-3p	Normal	1.92e-11	Pathological T status	3.87e-03
hsa-miR-494-3p	–	–	Pathological T status	2.65e-02
hsa-miR-222-3p	Tumor	4.10e-02	Pathological M status	5.54e-03
hsa-miR-335-3p	Tumor	1.64e-06	–	–
hsa-miR-212-3p	–	–	Pathological T/M status	2.21e-03/1.60e-02
hsa-miR-200a-3p	Tumor	8.14e-05	Pathological T/M status	1.41e-03/7.23e-06
hsa-miR-210-3p	Tumor	3.43e-08	Pathological T/N status	3.01e-02/4.59e-03
hsa-miR-214-3p	–	–	Pathological T/M status	1.84e-07/1.74e-03
hsa-miR-182-3p	Tumor	1.59e-02	Pathological M status	2.34e-06
hsa-miR-100-3p	–	–	Pathological T status	4.15e-02
hsa-miR-145-5p	Normal	1.33e-04	Pathological T/N/M status	7.14e-03/1.79e-03/7.26e-05
hsa-miR-21-5p	Tumor	2.42e-06	Pathological M status	5.83e-06
hsa-miR-222-5p	Tumor	1.43e-02	–	–
hsa-miR-335-5p	Tumor	5.22e-05	Pathological M status	1.85e-02
hsa-miR-200a-5p	Tumor	7.68e-05	–	–
hsa-miR-125b-5p	Normal	4.25e-02	Pathological T/N/M status	3.26e-05/8.07e-04/5.08e-06
hsa-miR-214-5p	Tumor	9.80e-03	Pathological T/N/M status	5.29e-09/2.15e-03/5.31e-07
hsa-miR-182-5p	Tumor	1.03e-06	Pathological M status	6.18e-05
hsa-miR-99a-5p	Normal	1.94e-02	Pathological T/N/M status	1.63e-07/1.10e-03/4.17e-04
hsa-miR-100-5p	Normal	7.01e-04	Pathological T/N/M status	6.86e-04/6.83e-04/5.99e-07
hsa-miR-138-5p	Tumor	9.07e-03	Pathological T/N status	1.24e-02/3.73e-02

Note:

* Independent two-sample Student’s t-test.

Abbreviations: DEMs, differentially expressed miRNAs; miRNAs, microRNAs; TCGA, The Cancer Genome Atlas.

Figure 4 The Kaplan–Meier plots of patients from TCGA dataset.

Notes: Survival plots of the bladder cancer patients with high and low expression of miR-145-3p (A), miR-126-5p (B), miR-99a-5p (C), miR-100-5p (D), miR-214-5p (E), miR-487b-3p (F), miR-200a-3p (G) and miR-138-5p (H).
Abbreviation: TCGA, The Cancer Genome Atlas.

Figure S1 Venn diagram of DEMs (A: Ta–T1 vs T2–T4; B: G3 vs G1) in different datasets.

Notes: (A) GSE36121: hsa-miR-135b, hsa-miR-508, hsa-miR-7, hsa-miR-21, hsa-miR-615, hsa-miR-124, hsa-miR-106b, hsa-miR-98, hsa-miR-132, hsa-miR-30e, hsa-miR-130a, hsa-miR-193a, hsa-miR-582, hsa-miR-151, hsa-let-7c. GSE39093: hsa-mir-1308, hsa-mir-192, hsa-mir-574, hsa-mir-720, hsa-mir-92b. GSE68594: hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7f, hsa-let-7i, hsa-mir-155, hsa-mir-196, hsa-mir-429, hsa-mir-579. (B) GSE36121: hsa-miR-1224-5p, hsa-miR-1225-5p, hsa-miR-1228, hsa-miR-125a-3p, hsa-miR-134, hsa-miR-135a, hsa-miR-135b, hsa-miR-136, hsa-miR-146a, hsa-miR-150, hsa-miR-188-5p, hsa-miR-192, hsa-miR-193a-3p, hsa-miR-194, hsa-miR-197, hsa-miR-21, hsa-miR-224, hsa-miR-31, hsa-miR-345, hsa-miR-362-5p, hsa-miR-363, hsa-miR-371-5p, hsa-miR-423-5p, hsa-miR-508-5p, hsa-miR-518c, hsa-miR-532-5p, hsa-miR-574-5p, hsa-miR-575, hsa-miR-582-5p, hsa-miR-598, hsa-miR-601, hsa-miR-622, hsa-miR-629, hsa-miR-663, hsa-miR-671-5p, hsa-miR-7, hsa-miR-801, hsa-miR-940. GSE68594: hsa-let-7d, hsa-let-7f, hsa-let-7i, hsa-mir-122a, hsa-mir-184, hsa-mir-196a1, hsa-mir-196a2, hsa-mir-203, hsa-mir-206, hsa-mir-223, hsa-mir-34a, hsa-mir-595, hsa-mir-601, hsa-mir-611-P.
Abbreviations: DEMs, differentially expressed miRNAs; miRNAs, microRNAs.

Table S1 Detailed information of patients in each included GEO datasets

Accession/ID	Ethnic group	Number of males/females	Range of age (years)	Number of BC tissues with different histological grades	Number of NMIBC/MIBC tissues
GSE36121	Caucasian	22/2	47–80	G1: 3, G2: 6, G3: 7	NMIBC: 13, MIBC: 3
GSE39093	Chinese Han	10/0	55–73	NR	NMIBC: 5, MIBC: 5
GSE68594	Caucasian	39/5	36–86	G1: 4, G2: 14, G3: 15	NMIBC: 18, MIBC: 15
GSE27448	Caucasian	15/0	44–86	G1: 0, G2: 3, G3: 7	NMIBC: 6, MIBC: 4
GSE40355	NR	20/2	47–80	G1: 3, G2: 6, G3: 7	NMIBC: 13, MIBC: 3
GSE52519	NR	11/1	43–77	G1: 0, G2: 4, G3: 5	NMIBC: 3, MIBC: 6

Abbreviations: BC, bladder cancer; GEO, Gene Expression Omnibus; MIBC, muscle-invasive bladder cancer; NMIBC, non-muscle-invasive bladder cancer; NR, not reported.