Association Of TMPRSS2 And KLK11 Gene Expression Levels With BPH And Prostate Cancer

Background and objective:Prostate cancer (PCa) and benign prostate hyperplasia (BPH) are two commom diseases for the elder men. In China 13.6% of the elder men suffered from BPH, and PCa accounts for approximately 13% of all cancer deaths in America. In clinical, PSA is used for diagnosis of PCa, and Gleason score or the tumor malignancy degree to evaluate the prostate cancer's pathologic classes. The TNM clinic classify system is used to define the cancer class. However, as a result of the insufficent specifity of PCa, elevated serum PSA levels may be the outcomes of prostatitis and BPH, even breast cancer. In particularly, it is difficult to distinguish PCa from BPH when serum PSA level is at 4-10 ng/ml. Affected by the subjective and empirical opinion of the pathologist, the Gleason score and the tumor malignancy may be inaccurate. So searching more specific biomarkers for PCa and the pathology staging is pressing.In these years, the human tissue kallikrein family is to be highly thought of by the researches. The gene of KLK11 specify expresses in the tissue of prostate. The androgen initiates cascade reaction via the activation of androgen receptor to lead to form the prostate cancer. On the other hand, the androgen response element in the KLK11 gene 5'termination enhanser combines with the androgen. They activate the gene to express KLK11 molecule to. The KLK11 gene amount expresses up-regulation. So it hints that we can evaluate the prostate cancer diagnosis early and the malignancy degree.The gene TMPRSS2, a transmembrane serine protease, expresses in the normal prostate cell and prostate cancer cell. It is regulated by androgen. The TMPRSS2 gene plays an important role in many physiology and pathology process by regulating the cell hyperplasy, differentiation and apoptosis or interaction between cells.Fluorescence quantitative realtime polymerase chain reaction(FQ-RT-PCR) is a technique which is adding fluorophore or dye agent in the PCR reaction system. It utilizes the fluorescence signal changes to monitor PCR reaction in real time. Eventually the unknown temple is analyzed by standard curve. The principle of quantization is the liner correlation between the fluorescence signals achieving the domain and the logarithm of the template onset copy number. The more number the template onset copy has, and less the Ct value will be. When the fluorescence quantitative realtime polymerase chain reaction begins to inverse reaction, the PCR will be carried out soon. The sample onset template numbers will be obtain as soon as the reaction result compared with the standard curve.In this study, fluorescence quantitative realtime polymerase chain reaction is utilized to detect the quantity of the gene of TMPRSS2 and KLK11 expression in the BPH and PCa. To approach the meaning gene of TMPRSS2 and KLK11 expression in the prostate cancer diagnosis and pathology ranking, the BPH and PCa's Gleason score, tumor malignancy grade and clinical stage, progression-free survival time are combined to analyze. The genes of TMPRSS2 and KLK11 biology ethology and clinical guidance come to be known.Material and method:74 cases of PCa tissue and 80 BPH tissue were collected from December 2004 to December 2009. The cases were from open operation or TURP or puncture biopsy tissue. The normal prostate tissue were obtained from radical bladder cancer operation. The tissue were put into a liquid vase as soon as they were obtained. The average age of PCa patients was 75 years old, the average age of BPH patients was 67 years old, the average age of normal prostate patients was 53 years old. The pathology malignancy grade and Gleason score detected from paraffin section were to recorded as well as the clinical stage and progression-free survival time. The end of the follow up was defined as:When the PSA value continuous arise twice, the first rise time was the end of follow up; osseous metastasis were detected by nuclide bone scan; the prostate tumor region progressed were detected by transrectal ultrasound, CT,MR; the patients died. The time of deadline was 31th December 2009.Fluorescence quantitative realtime polymerase chain reaction was used to determine the genes expression of TMPRSS2 and KLK11 in BPH and PCa. (1) The sample of tissue were obtained from clinical operation.(2) The Takara Taq HS agent was used. (3) The implements were:fluorescent quantization PCR meter RotorGene 2000(Corbett Co.), fluorescent quantization PCR analyze software was Rotor-gene v5. (4) mRNA was extracted. (5) The gene uniGene data was obtained from NCBI gene bank. The normal mRNA sequence was obtained. Vector NT6.0 software was used to locate the correlated uniSTS amplification fragment in Normal mRNA. The uniSTS sequence about 100-300 bp long and the location closes to mRNA 3'were selected. In the end, the fragments specificity were analyzed by NCBI Blast software and e-PCR software. The uniSTS primer of poor specificity and duplicate with e-PCR were rejected. According to the genes of TMPRSS2 and KLK11's mRNA sequence and UniSTS sequence, the better sequences were obtained. The probes of TMPRSS2 and KLK11 and the reciprocal UniSTS primer sequence.(6) Fluorescence quantitative realtime polymerase chain reactions were undertook for the sample of mRNA. (7) The standard curve was mede. (8) The date were dealed with relative quantitative method.Statistical treatment:Independence sample t test was undertook to compare the mRNA expression quantity of TMPRSS2 and KLK11. The malignancy grade of tumor was classified into G1-G2 and G3; Gleason score was classified into exceed 6 and smaller or equal to 6; clinical pathology staging was classified intoⅠ-ⅡandⅢ-Ⅳ. The mRNA expression quantity of TMPRSS2 and KLK11 in these groups was undertook independence sample t test. The progression-free survival time of malignancy grade(G1-G2 and G3), Gleason score (exceed 6 and smaller or equal to 6) and clinical pathology staging(Ⅰ-ⅡandⅢ-Ⅳ) were undertook log-rank test. The survival curves were made by Kaplan-Meier approach. The Cox proportional hazards regression was used to analyze multiplicity. The Gleason score, clinical staging, TMPRSS2/KLK11 value were included in Cox proportional hazards regression. Variance filting was adopted by step by step forward regression. P≤0.05 was internalized standard, P>0. 10 was rejected standard.Statistical treatment:The mean number of two sets of specimen measurement data was analyzed by t test. Log-rank test was deployed in the different factor of progression-free survival time. The Kaplan-Meier method was utilized to draw the survival curve; Cox proportional hazards regression models was utilized in the multiplicity.Result:1. Independence sample t test was undertook to compare the mRNA expression quantity of TMPRSS2 and KLK11. We found that the mRNA expression quantity of TMPRSS2 and KLK11 had significant difference between BPH and PCa (P<0.05). mRNA expression quantity of TMPRSS2 and KLK11 in PCa were higher than BPH.2. The mRNA expression quantity of TMPRSS2 and KLK11 had significant difference between clinical stage, Gleason score and tumor malignancy grade (P<0.05). When the clinical stage, Gleason score and tumor malignancy grade stepped up, the mRNA expression quantity of TMPRSS2 stepped up too. When the clinical stage, Gleason score and tumor malignancy grade stepped up, the mRNA expression quantity of KLK11 stepped down.3. The progression-free survival time of malignancy grade(G1-G2 and G3), Gleason score (exceed 6 and smaller or equal to 6),clinical pathology staging(I-II and III-IV) and ratio of TMPRSS2/KLK11 Ct value(exceed 2 and smaller or equal to 2) were undertook log-rank test. We found that the clinical stage, Gleason score, tumor malignancy grade and ratio of TMPRSS2/KLK11 Ct value were influential factors in prostate cancer progression-free survival time. The progression-free survival time survival curve were made according to malignancy grade, Gleason score and clinical pathology staging. In the cases, the progression-free survival time was longer in Gleason score smaller or equal to 6; clinical pathology staging ofⅠ-Ⅱwas longer; malignancy grade of G1-G2 was longer, TMPRSS2/KLK11 Ct value of smaller to 2 was longer(P<0.01, log-rank).4. Gleason score, ratio of TMPRSS2/KLK11 Ct value and clinical pathology staging were included in Cox proportional hazards regression. Gleason score, ratio of TMPRSS2/KLK11 Ct value were found to be the main factor of prostate cancer progression-free survival time. Clinical pathology staging were found to be lost statistical significance in multiplicity.Conclusion:1. The mRNA expression quantity of TMPRSS2 and KLK11 had significant difference between BPH and PCa. BPH and PCa can be discriminated by detected The mRNA expression quantity of TMPRSS2 and KLK11 in the tissue of BPH and PCa.2. The mRNA expression quantity of TMPRSS2 and KLK11 change with the Gleason score, clinical stage and tumor malignancy grade. The mRNA expression quantity of TMPRSS2 stepped up when Gleason score, tumor malignancy grade and clinical stage stepped up. The mRNA expression quantity of KLK11 stepped down when Gleason score, tumor malignancy grade and clinical stage stepped up. The ratio of TMPRSS2/KLK11 Ct value can act as a clinical index for the prostate cancer progression-free survival time.3. The ratio of TMPRSS2/KLK11 Ct value helped to indicate the prostate cancer progression-free survival time. The prostate cancer progression-free survival time will be longer when the ratio less than 2. While the time will be shorter when the ratio exceed of equal to 2.