| Prostate cancer (PCa) is one of the most common tumors in the western countries, accounting for the second leading cause of cancer mortality. As the increase of life expectancy and the improvement of diagnostic techniques, its incidence and detection rate show an upward trend. Although incidence of prostate cancer in China is lower than the western developed countries, it grows very fast in recent years. Prostate biopsy is the gold standard for diagnosis of prostate cancer, but the positive rate is only 1/3 when the serum PSA>4ng/ml. During recent years, human kallikrein 2 and prostate stem cell antigen were confirmed to be prostate cancer-specific gene, but they are not applicable to improve the early diagnosis rate of prostate cancer. Therefore, great attention have been attracted on finding a new marker for prostate cancer diagnosis and establishing a more specific diagnostic methods.DD3 gene is a newly-discovered prostate-specific gene. It is highly expressed in malignant prostate epithelial cells line (>95%) and is significantly increased at the incidence of prostate cancer. The sensitivity and specificity for prostate cancer diagnosis is greater than 90%. It is currently considered to be a potential clinical application marker for prostate cancer. In our study, we evaluated the significance of DD3 gene in prostate cancer diagnosis, treatment and prognosis.In this study, we firstly cloned a DD3 gene fragment. A pair of primers between exon 4 were designed and the total RNA was extracted from the LNCaP cell. LNCaP cell cDNA was used as template for PCR amplification. The products were purified by agarose gel electrophoresis and ligated with PMD18-T vector, and then introduced into the competent cells and then cloned. The recombinant plasmid was extracted, quantified in a UV spectrophotometer, and taken as a quantitative standard for DD3 detection. PCR amplification and sequencing indicated that PMD18-T-DD3 cDNA was cloned successfully. It could be used as standard for detection of prostate cancer by fluorescence quantitative RT-PCR and in quantitative mapping of the standard curve for prostate cancer-specific, which provided a guarantee for DD3 gene fluorescence quantitative PCR method.In this study, real-time fluorescence quantitative PCR for detecting DD3 mRNA in prostate cancer was established according to TaqMan probe method .Its lowest limitation was 1.64×101 copies/ml and its line scope ranged from 1.64×101 copies/ml to 1.64×1011 copies/ml with excellent sensitivity. The amplified products were identical by direct sequencing and Blast. The specificity, stability and repeatability were also approved.In clinical application, DD3 mRNA positive rate of 27 cases Prostate cancer group were significantly higher than that of non-prostate cancer and the healthy control group. In prostate cancer patients, DD3 mRNA could be detected in 18 whole blood specimens, 4 of the 5 urine specimens and 4 prostatic fluid. Within prostatic fluid from 30 patients with non-prostate cancer specimens, only one had the value of low-copy of 1.984 log copies/ml. The reasons needed further observation. DD3 mRNA was negative in all 30 healthy specimens. The difference between the results from patients with prostate cancer and those with non-prostate cancer were statistically significant. Correspondingly, pathological grade of prostate cancer and DD3 cDNA copies were observed at the same time. The results indicated that DD3 cDNA copies increased with the increased pathlogical grades.We also compared the positive rate of DD3 and PSA in prostate cancer patients. The DD3 mRNA was positive in all cases of 18 prostate cancer, while the positive of PSA was 16. On the other hand, DD3 mRNA were negative in whole blood specimens from 20 cases of non-prostate cancer and 10 healthy control, while PSA were positive in four cases of the non- prostate cancer group. It was indicated that detection of DD3 mRNA in whole blood was with the superior specificity and sensitivity than that of PSA. On the base of quantifying PSA mRNA, a new concept called DD3 scores was introduced, which mean ratio between absolute value of DD3 mRNA and that of PSA mRNA. The result was identical to our sole DD3 mRNA quantification.Therefore, using homemade Real-time fluorescence quantitative PCR system, we confirmed that DD3 gene was a specific marker for the early diagnosis of prostate cancer. It can be used to detect a few cancer cells in biological fluids such as blood, urine and prostate fluid. It provides a potential application value for the early diagnosis, prognosis, and molecular staging of prostate cancer.
|
PDF Full Text Request