Study subjects
Newly diagnosed 25 UBC patients, 10 BPH patients, along with 25 healthy controls, were introduced in the study. The demographic details of the participants are given in Table 1.
Table 1
Demographic and clinical data for the UBC patients and the control subjects
|
Patient Sample
|
Control Samples
|
C1
|
C2
|
C3
|
Serum
|
25
|
25
|
10
|
––
|
Urine
|
15
|
15
|
10
|
––
|
Tissue
|
25
|
––
|
10
|
12
|
Male/Female
|
21/4
|
20/5
|
10/0
|
10/2
|
Age (in yrs)
|
59 ± 8
|
49 ± 8
|
60 ± 9
|
55 ± 9
|
**C1: Healthy subjects; C2: BPH patients; C3: Adjacent healthy tissue from same patient |
Values are represented as mean ± SD |
Relative microRNA expression in serum
The relative expression of CD44 associated miRNAs (miR-9, miR-34a and miR-203) has been examined in serum of UBC, BPH and healthy subjects using qRT-PCR. The relative expression of miR-9 in serum of UBC patients was significantly upregulated in contrast to healthy (p < 0.001) and BPH controls (p < 0.01) (Fig. 1A, Table 2, 3). The relative expression of miR-34a in serum of UBC patients was significantly downregulated in UBC patients in opposition to healthy controls (p < 0.05) but was non-significant in BPH controls (Fig. 1B, Table 2, 3). The relative expression of miR-203 was significantly upregulated in UBC patients in contrast to healthy controls (p < 0.05) but was non-significant in BPH controls (Fig. 1C, Table 2, 3).
Table 2
Relative miRNA expression in blood urine and tissue samples of UBC patients and their respective BPH and healthy controls
Relative microRNA expression
Median (Range)
|
|
|
Control
|
BPH
|
UBC
|
miR-9
|
Serum
|
5.097e-005
(8.745e-007– 0.008)
|
1.274e-005
(3.035e-006 – 2.479e-005)
|
0.001
(0.0001 – 0.939)
|
Urine
|
0.0002
(4.069e-005 – 0.004)
|
0.0007
(1.053e-005 – 0.003)
|
0.003
(0.0005 – 0.048)
|
Tissue
|
----
|
0.153
(0.039 –0.303)
|
UBC NT
0.088
(0.008 – 0.88)
|
UBC T
0.468
(0.013 – 8.056)
|
miR-34a
|
Serum
|
0.006
(0.0002 – 0.646)
|
0.011
(0.005 – 0.175)
|
0.002
(2.968e-005 – 0.036)
|
Urine
|
0.011
(0.0008-0.982)
|
0.001
(0.0001– 0.008)
|
0.005
(0.001 – 0.282)
|
Tissue
|
----
|
2.034
(0.384 – 14.93)
|
UBC NT
1.159
(0.013 – 8.574)
|
UBC T
0.406
(0.025– 3.106)
|
miR-203
|
Serum
|
6.867e-005
(1.066e-006 –0.009)
|
1.025e-005
(4.146e-006 – 0.0001)
|
0.001
(1.168e-007 – 0.216)
|
Urine
|
0.001
(6.726e-005 – 0.010)
|
0.0004
(1.079e-005– 0.009)
|
0.045
(0.002 – 3.434)
|
Tissue
|
----
|
0.271
(0.039- 2.694)
|
UBC NT
0.243
(0.011 – 2.723)
|
UBC T
2.021
(0.016– 11.75)
|
UBC T: UBC tumor tissue; UBC NT: UBC adjacent normal tissue |
Table 3
The level of significance of microRNAs between different groups in serum, urine, and tissue samples
|
miR-9
|
miR-34a
|
miR-203
|
|
Control/UBC
|
BPH/UBC
|
Control/UBC
|
BPH/UBC
|
Control/UBC
|
BPH/UBC
|
Serum
|
<0.0001
|
<0.001
|
<0.05
|
<0.05
|
<0.05
|
ns
|
Urine
|
<0.05
|
ns
|
ns
|
Ns
|
<0.001
|
<0.05
|
|
BPH/UBC T
|
UBC NT/UBC T
|
BPH/UBC T
|
UBC NT/UBC T
|
BPH/UBC T
|
UBC NT/UBC T
|
Tissue
|
Ns
|
<0.05
|
<0.05
|
Ns
|
Ns
|
ns
|
UBC T: UBC tumor tissue; UBC NT: UBC adjacent normal tissue; ns: non-significance |
Relative microRNA expression in urine
The relative expression of CD44 associated miRNAs (miR-9, miR-34a and miR-203) has been studied in the urine of UBC patients along with healthy and BPH controls using qRT-PCR. The relative expression of miR-9 in the urine of UBC patients was significantly upregulated in relation to healthy control (p < 0.05) but was non-significant in BPH controls (Fig. 2A, Table 2, 3). The relative expression of miR-34a in the urine of UBC patients was non-significant as opposed to BPH and healthy controls (Fig. 2B, Table 2, 3). The relative expression of miR-203 was significantly upregulated in UBC patients in relation to healthy (p < 0.01) and BPH controls (p < 0.05) (Fig. 2C, Table 2, 3) which indicates its non-invasive diagnostic ability.
Relative microRNA expression in tissue
The relative expression of CD44 associated miRNAs (miR-9, miR-34a and miR-203) has been studied in the tissue specimens of UBC patients along with BPH controls using qRT-PCR. The relative expression of miR-9 in tumor tissue of UBC patients was significantly upregulated as opposed to adjacent non-tumor tissue of UBC patients (p < 0.05) but was non-significant in relation to BPH controls (Fig. 3A, Table 2, 3). The relative expression of miR-34a in tumor tissue of UBC was downregulated in comparison to BPH (p < 0.05) but non-significant as opposed to the adjacent non-tumor issue of same UBC patients (Fig. 3B, Table 2, 3). The relative expression of miR-203 in tumor tissue of UBC patients was non-significant in comparison to adjacent non-tumor tissue of UBC patients and BPH controls (Fig. 3C, Table 2, 3).
Diagnostic potential of microRNAs in serum, urine, and tissue of UBC
The diagnostic potential of CD44 associated miRNAs (miR-9, miR-34a and miR-203) was evaluated by plotting the ROC curve, which provides sensitivity, specificity, area under the curve (AUC) and cut-off values of these biomolecules. miR-203 showed optimum sensitivity and specificity in the serum (Fig. 4. A-F), urine (Fig. 5A-F) and tissue specimen (Fig. 6A-F) of UBC patients in contrast to miR-9 and miR-34a.
Correlation
The expression of microRNAs (miR-9, miR-34a and miR-203) in serum, urine and tissue specimen of UBC patients was correlated by Spearman correlation analysis. It has been observed that expression of miR-203 in urine showed significant positive correlation with expression in tumor tissue of UBC patients (r= 0.5832, p=0.0393). This finding, thus, suggests that urinary miR-203 could be exploited further for diagnosis of UBC in future.