Qin Liang He Zhaohong Ren Junkai Yang Tiejun Feng Chaojie Li Jing Zhao Pengcheng Ma Jing Li Jinglei
Prostate cancer is a common tumor among men and has the highest incidence rate in Europe and the United States, surpassing lung cancer in incidence and second only to lung cancer in death rate. In recent years, the incidence of prostate cancer in China is increasing and it has become a major factor threatening men’s health. At present, the early diagnosis of prostate cancer is mainly through prostate-specific antigen (PSA) screening combined with digital rectal examination (DRE), and the discovery of PSA is a milestone for the early diagnosis of prostate cancer. However, PSA is only specific for prostate tissue, but not for prostate tumors. A large number of studies have shown that early prostate cancer antigen (EPCA) immunohistochemical staining, especially ELISA for serum EPCA, has shown good specificity and sensitivity, and if EPCA is used in combination with PSA screening, it is bound to reduce the number of unnecessary prostate puncture biopsies and decrease the rate of missed diagnoses. It is possible that EPCA will become a new specific marker for screening and diagnosis of prostate cancer. He Zhaohong, Department of Urology, Henan Cancer Hospital
1. Materials and methods
1.1 Collection of tissue specimens
There were 41 prostate cancer (Pca) specimens, aged 54-86 years (66.4±8.5 years), 9 from transurethral resection specimens of prostate by electrolysis and 32 specimens obtained from systematic puncture biopsy of the prostate under transrectal ultrasound guidance, which were pathologically confirmed as prostate cancer (all adenocarcinomas), all without hormonal and surgical debulking and radiation therapy. Benign prostatic hyperplasia (BPH) tissue specimens were obtained from 34 specimens aged 55-71 years (62.7±5.6 years) by transurethral electrodesiccation of the prostate and suprapubic prostate removal; 5 normal controls were obtained from prostate tissue specimens removed by radical total cystectomy. Benign prostatic hyperplasia (BPH) tissue and normal prostate (NP) tissue specimens were pathologically confirmed. The specimens were stored in a -80°C refrigerator within 15 min after surgical resection and rectal puncture biopsy.
1.2 Collection of blood specimens
In order to minimize factors that may affect the serum EPCA and PSA levels, the samples were collected according to the following criteria for inclusion: (i) no prostate massage, transrectal prostate ultrasonography, prostate puncture biopsy and cystoscopy before admission; (ii) no acute or chronic prostatitis before admission; (iii) patients admitted for acute urinary retention were also excluded.
1.3 Experimental methods
1.3.1 Extraction of tissue mRNA
Tissue specimens were stored in -80℃ refrigerator within 15 min after surgical resection and transrectal ultrasound-guided systematic puncture biopsy of the prostate, and total RNA extraction was performed after confirmed by pathological examination.
1.3.2 Reverse transcription polymerase chain reaction (RT-PCR) analysis
The first strand was synthesized with reverse transcriptase MMLV. It was performed according to the instruction manual provided by Beijing All-Style Gold Biotechnology Co.
1.3.3 Agarose gel electrophoresis
Take 5ul of amplification product and mix with buffer and add into the spiked wells of agarose gel for electrophoresis. The electrophoresis conditions were 2% agarose gel, voltage 4-10v/cm. EB staining, observation of electrophoretic bands under UV projector, and analysis of electrophoretic bands by D-140 image recording and analysis system.
1.3.4 Detection of serum EPCA level by ELISA
The human (Human) early prostate cancer antigen (EPCA) test kit used in this experiment is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). Standards of known EPCA concentration and samples of unknown concentration were added to the microtiter enzyme standard plate for the assay.
2. Results
2.1 RT-PCR reaction results
After observation of RT-PCR reaction products by gel imaging system, all reverse transcribed cDNAs were PCR amplified with GAPDH primers to get 138bp target fragment, and PCR amplified with EPCA gene primers, in which prostate cancer tissues got 411bp size fragment in 34 out of 41 samples (as shown in Figure 3 and 4), indicating that EPCA mRNA was expressed in the tissues EPCA mRNA had a positive expression rate of 82.9% in prostate cancer tissues. The results indicated that EPCA mRNA was specifically expressed in prostate cancer tissues.
Figure 1 Electrophoresis of PCR products of prostate cancer and prostate hyperplasia
Figure 2 Electrophoresis of PCR products from prostate cancer, prostate hyperplasia and normal prostate tissues
2.2 Serum EPCA values of prostate cancer, incidental prostate cancer, BPH and healthy control group
The preoperative serum EPCA levels of 31 cases of PCa group, 9 cases of IPCa group, 181 cases of BPH group and 26 cases of healthy control group, as well as the preoperative serum EPCA levels of various clinicopathological features correlated with serum EPCA, the relevant subgroups, and the P values for the comparison between the relevant groups are shown in the following table.
Table 1 Serum EPCA values for PCa, IPCa, BPH, and healthy controls; P values for comparison between the relevant groups
Characteristics
Number of cases
Mean ± standard deviation
Median
Range
P-value
EPCA (ng/ml)
Clinical study subjects
247
7.07±6.98
4.76
0.28 to 32.46
PCa
31
19.35±7.86
21.11
7.89～32.46