Association Of SOX9and NM23Gene Expression Levels In Prostate Cancer

Background and objective:Prostate cancer (PCa) and benign prostate hyperplasia (BPH) are two commom diseases for the elder men. In China13.6%of the elder men suffered from BPH, and PCa accounts for approximately13%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 at4-10ng/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 SOX9specify 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 SOX9gene5’termination enhanser combines with the androgen. They activate the gene to express SOX9molecule to. The SOX9gene amount expresses up-regulation. So it hints that we can evaluate the prostate cancer diagnosis early and the malignancy degree.The gene NM23, a transmembrane serine protease, expresses in the normal prostate cell and prostate cancer cell. It is regulated by androgen. The NM23gene 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 NM23and SOX9expression in the BPH and PCa. To approach the meaning gene of NM23and SOX9expression 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 NM23and SOX9biology ethology and clinical guidance come to be known.Material and method:100cases of PCa tissue and80BPH tissue were collected from December2009to December2012. 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 was73.5years old, the average age of BPH patients was68.3years old, the average age of normal prostate patients was58years 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 was31th December2009.Fluorescence quantitative realtime polymerase chain reaction was used to determine the genes expression of SOX and NM23in 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 RotorGene2000(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 NT6.0software was used to locate the correlated uniSTS amplification fragment in Normal mRNA. The uniSTS sequence about100～300bp long and the location closes to mRNA3’were selected. In the end, the fragments specificity were analyzed by NCBI Blast software arid e-PCR software. The uniSTS primer of poor specificity and duplicate with e-PCR were rejected. According to the genes of NM23and SOX9’s mRNA sequence and UniSTS sequence, the better sequences were obtained. The probes of NM23and SOX9and 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 SOX9and NM23. The malignancy grade of tumor was classified into G1-G2and G3; Gleason score was classified into exceed6and smaller or equal to6; clinical pathology staging was classified into I-II and III-IV. The mRNA expression quantity of SOX9and NM23in these groups was undertook independence sample t test. The progression-free survival time of malignancy grade(Gl-G2and G3), Gleason score (exceed6and smaller or equal to6) and clinical pathology staging(I-II and III-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, SOX9/NM23value were included in Cox proportional hazards regression. Variance filting was adopted by step by step forward regression. P<0.05was internalized standard, P>0.10was 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 SOX9and NM23. We found that the mRNA expression quantity of SOX9and NM23had significant difference between BPH and PCa (P<0.05). mRNA expression quantity of SOX9and NM23in PCa were higher than BPH.2. The mRNA expression quantity of SOX9and NM23had 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 SOX9stepped up too. When the clinical stage, Gleason score and tumor malignancy grade stepped up, the mRNA expression quantity of NM23stepped down.3. The progression-free survival time of malignancy grade(Gl-G2and G3), Gleason score (exceed6and smaller or equal to6),clinical pathology staging(Ⅰ-Ⅱ and III-IV) and ratio of SOX9/NM23Ct value(exceed2and smaller or equal to2) were undertook log-rank test. We found that the clinical stage, Gleason score, tumor malignancy grade and ratio of SOX9/NM23Ct 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 to6; clinical pathology staging of I-II was longer; malignancy grade of G1-G2was longer, SOX9/NM23Ct value of smaller to2was longer(P<0.01, log-rank).4. Gleason score, ratio of SOX9/NM23Ct value and clinical pathology staging were included in Cox proportional hazards regression. Gleason score, ratio of SOX9/NM23Ct 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 SOX9and NM23had significant difference between BPH and PCa. BPH and PCa can be discriminated by detected The mRNA expression quantity of S0X9and NM23in the tissue of BPH and PCa.2. The mRNA expression quantity of SOX9and NM23change with the Gleason score, clinical stage and tumor malignancy grade. The mRNA expression quantity of SOX9stepped up when Gleason score, tumor malignancy grade and clinical stage stepped up. The mRNA expression quantity of NM23stepped down when Gleason score, tumor malignancy grade and clinical stage stepped up. The ratio of SOX9/NM23Ct value can act as a clinical index for the prostate cancer progression-free survival time.3. The ratio of SOX9/NM23Ct 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 than2. While the time will be shorter when the ratio exceed of equal to2.