The research of the application of a new urinary biomarker PCA-M of prostate cancer (PSA from 4 to 20 ng/ml)

Figures & data

Table 1. General data of pyrophosphate sequencing.

Reagents	Amount (µL)
10 x PCR Buffer	4
dNTP	2.4
Primer F	0.6
Primer R	0.6
TaKaRa Taq™ HS	0.5
Template	2
ddH2O	31.5

Pyrosequencing. Each PCR was performed in a 40 µl volume containing 0.6 µl of each primer, 4 µl 10XPCR buffer, 0.5 µl QIAGEN hotstart Taq, 31.5 µl distilled water, and 2.0 µl template DNA (treated by bisulfite, EpiTect Bisulfite Kit, Qiagen). Reactions were incubated at 95°C for 3 min, followed by 50 amplification cycles (95°C for 15 s, 54°C for 30s, 72°C for 30 s), and then a final elongation at 72°C for 5 min, with the temperature then held at 4°C. Amplicons were confirmed by agarose gel electrophoresis and purified using a QIAquick Gel Extraction Kit. After mixed with 40 µl sequencing buffer (contained 0.5 µM sequencing primer), degeneration was performed at 80°C for 2 min.

Figure 1. Bioinformatics analysis of PRKY and TGIF2LY expression in prostate cancer. The expression of PRKY was high in cancer tissues and low in normal tissues, and the difference was statistically significant (P < 0.001, Figure 1(A)). The expression of TGIF2LY in prostate cancer tissues was significantly lower than that in normal tissues (P < 0.001, Figure 1(B)).

Table 2. The clinical characteristics of the two groups of patients.

Variable	PCa	BPH	P/χ^2 Value
Age	68.50 ± 7.79	67.29 ± 6.46	0.1173
BMI	23.68 ± 2.69	23.10 ± 1.94	0.2281
TPSA	9.86 ± 4.90	9.67 ± 4.90	0.9956
f/tPSA	0.12 ± 0.06	0.17 ± 0.16	0.0185
PSAD	0.26 ± 0.20	0.18 ± 0.10	0.0167
DRE			0.1787
Positive	22	27
Negative	27	19
MRI			0.1103
Positive	24	30
Negative	25	16
PRKY	3.96 ± 1.05	2.47 ± 0.77	<0.0001
TGIF2LY	71.13 ± 13.16	91.96 ± 5.56	<0.0001

Characteristics of two groups. There was no significant difference in age, BMI, PSA, DRE and MRI between two groups. Group PCa had lower f/t PSA values while higher PSAD values compared to Group BPH. The methylation ratio of Gene PRKY was expressed higher in Group PCa and the methylation ratio of gene TGIF2LY was expressed lower in Group PCa.

Figure 2. The ROC for urine PCA-M DNA in diagnosis of PCa. The AUC of PRKY, TGIF2LY, f/t PSA, PSAD was 0.8636, 0.9352, 0.6395, 0.6417, respectively.

Data availability statement

All the original data of this study can be accessed from https://figshare.com/s/b2786bd44a844ba9b9c5. Publicly available data was also obtained from the UCSC Xena (https://xena.ucsc.edu/) TCGA database, reference [GDC TCGA Prostate Cancer (PRAD)].