The Molecular Diagnosis Of PSA And Expression And Significance Of NFKBIA/SREBP2 In Prostate Cancer

BackgroundAccording to age stage, the disease caused by prostate has different characterization, with that prostatitis in middle and young men, BPH and PCa in old men, moreover, there is increasing incidence of PCa with aging. Because of complex histological morphology and bioactivity of PCa, the route examination such as digital rectal examination, imageology and biopsy have some problems to diagnose PCa. The development of PCa results from combating between cancer gene and suppressor cancer, so we can detect PCa in the level of protein and gene, and identify biomarker of diagnosis and prognosis of PCa by gene chip, immunohistochemistry and RT-PCR. As molecular marker of PCa, PSA has been used in clinic for long time, but there are arguments about its limitation of specificity and sensitivity.Currently, PSMA, AMACR, CK34BE12, P63 and GSTP1 have been studied for diagnosis of PCa, but no one of them are suitable molecular marker for diagnosis of PCa in clinic, so it is important to find new reliable multiple molecular marker of PCa.RT-PCR has been used for molecular biological study in many areas including medicine, zoology, agriculture, national defense and the quarantine of sanitation.In the recent years, the bioactivity of NFKBIA/SREBP2 gene in prostate and cholesterol metabolism in PCa have been acknowledged, which may become a molecular marker to diagnose PCa, moreover, the PCa could be treaded in molecular level as a try by the further study of regulating cholesterol and fatty acid of SREBP in the PCa. In this paper, the diagnosing value of PSA in PCa would be analysed additionally, and detect the expression of NFKBIA/SREBP2 gene in PCa using RT-PCR, in order to reveal the diagnosis value of NFKBIA/SREBP2 in PCa as molecular marker.Part1 The diagnostic value of prostate specific antigen to prostate cancerObjectives:PSA have become important marker of PCa for its reasonable sensitivity and specificity, but with the increasingly using, there are some problems about its limitation of sensitivity and specificity. the diagnostic value of prostate specific antigen to prostate cancer will be further revealed through clinical analyzing patients with normal or abnormal PSA, and concluding the causes that Prostate canner can't been exactly marked by prostate specific antigen, including that patients couldn't be diagnosed with prostate canner but having PSA level higher than 4.0ng/mL, and as well the reverse.Methods:The outpatients' and inpatients' data with PSA had been analyzed retrospectively. Among totally 1475 patients,919 patients were with theirs PSA between 4.0 to 14152ng/ml, and 10 patients' PSA were normal with PCa. All the patients with PCa were diagnosed pathologically. The patients'blood samples were centrifuged after collecting, and separated the plasma, then measured the PSA with electrogenerated immunoassay chemiluminescence. The normal reference range of PSA was 0 to 4.0 ng/ml. the data was collected into excel table, and analyzed descriptively statistically about its sensitivity, specificity, and so on. The date such as patients' age and the volume of prostate was calculated through test variance analysis, independent test for analysis of difference, then chi square test. P≤0.05 was considered as having difference statistically and significance statistically.Results:Among the 919 patients with PSA higher than 4.0 ng/ml,149 were diagnosed with PCa, with positive rate 16.2%, of which,11 of 424 patients with PSA 4.0-10.0 ng/ml had PCa with positive rate 2.59%,138 of 495 PSA higher than 10.0 ng/ml with positive rate 27.9%. Of the 159 patients with PCa,10 patients'PSA was normal, with negative rate 6.3%. The sensitivity of this group was 93.7%, specificity 41.5%, and positive predict value 16.2%. The higher the PSA is, the more PCa there are. Age is an important factor to result in PSA increasing. In comparison with the black, white people, korean and Jananeses, the impact of age is the least to Chinese. Also, the bigger the patients' prostate volume, the higher the PSA was. Some other factors can also cause PSA to increase such as prostatitis, intraepithelial neoplasia, aute urinary retention, operation through urethra, drug and ejaculation. Of 10 patients with normal PSA level,7 belong to A and B stage,70.0%. The PSA level of PCa is relevant to the pathological stage:the higher the PSA, the higher the Gleason score was. Of those patients,8 patients have theirs PSA between 2 to 5, and 1 between 6 to 7.Conclusions:although having been used in clinic widely as an important marker to PCa, PSA has only the prostate tiuss specificity, not high specifical toumer marker, in that some factors could result in increasing of PSA such as age, BPH, prostatitis, aute urinary retention, operation through urethra, drug and even ejaculation sperm; on the other hand, on the condition of little volume, good pathological etc, the patients with PCa have normal PSA. Had the clinical doctor ingnored the deficiency of sensitivity and specificity, there would be some trouble for doctor to diagnose the diseases. To full up the deficiency of PSA, f/tPSA, PSAV, PSAD have been used and have help in some degree, but can't solve the problem fundamentaly. All of those inplus people to find more effective marker to improve the early diagnosis of PCa.The objective of the second study is to find more effective molecular marker of PCa to full up the deficiency of PSA, as an important early diagnosial marker of Ca.Part 2 The expression and significance of NFKBIA/SREBP2 in PCaObjective:PSA has widely been used in clinic as a significant marker for detecting prostate cancer (PCa). While, the specificity and sensitivity is controversial as it can be affected by benign prostatic hyperplasia (BPH), inflammation, ages and drugs, etc. Recently lots of researchers have been seeking other specific molecular markers for diagnosis of prostate cancer. According to the recently studies on gene transcription pattern and immunohistochemistry of PCa and BPH we choose NFKBIA/SREBF2 for study, expecting to find new indicators for diagnosis and malignant degree evaluation of PCa, and more efficient marker (or token object) to increase early diagnosis rate of prostate cancer. Methods:the clinical removed prostatic cancer tissues, hyperplasia tissues and needle biopsical tissue were collected and then preserved by Liquid Nitrogen Flash Freezer. Totally 127 patients are selected,67 of which are PCa-group,60 are BPH group. The average age is 66.5. We choose 52 PCa samples and 57 BPH samples identified by HE-staining to detect the levels of NFKBIA expression and SREBF2 with quantitative PCR.The melting curve of products in fluorescence quantitative PCRwas analyzed and then the 15 g/L Agarose Gel Electrophoresis analysis randomly, so it can be certain whether the products are target fragments amplified by PCR.With ultraviolet spectrophotometer, we test the OD260 value of gene plasmids DNA and calculate the concentration of the plasmid sample, then convert the DNA concentration of plasmids into copy numbers. We get the Ct value and the content of plasmids DNA added, describe the concentration-relation curve, the abscissa denotes Ct value, the ordinate denotes the content of DNA added. Then draw standard curve with ABI 7900HT SDS 2.0 software.β-actin standard curve was imported with SDS2.0 software and then madeβ-actin gene expression in different samples quantitative by Ct values. BPH is as baseline, then the relative value gotten.Standard curves of all target genes was maken to quantitative the expression of target gene of different samples on the standard curve.The quantitative outcome got from such standard curve was divided by the relative value got from the BPH baseline, then adjust the error of RNA content used in study. By the adjusted quantitative outcome, we can get the relative value of target genes expression in different samples, which is the relative quantitative of mRNA expression. The BPH gene expression is the baseline for all the genes, then expression changes ratio was calculated for one gene in other samples.The NFKBIA/SREBP2 expressions in BPH/PCa (Ct value) is under independent sample T-test. Tumor malignacy was classified by G1-G2 and G3 group and Gleason grades by≤7.0 and > 7.0. The results were depicted in x±s, checked the statics by the SPSS 13.0 statistic software, when P≤0.05, the difference has statistic significance.Results:Comparing the immunohistochemistry positive expression in PCa and BPH, we find that NFKBIA protein-expression is higher in BPH than that in PCa. while SREBF2 expression is lower in BPH. It is significant difference with statistical test P<0.001.Comparing the positive-expression values of protein of NFKBIA/SREBF2 Gleason grades and tumor malignancy evaluation, we find that the level of SREBF2 positive-expression in BPH/PCa raised with the increase of Gleason grades and tumor malignancy, which has significant difference (P≤0.05), while the NFKBIA of which has no significant difference (P>0.05).The BPH as the compared group, the expression rate of NFKBIA and SREBF2 gene was calculated in PCa. Comparing the Ct average and standard difference(SD) of these two gene-expressions, it has statistic differences by t-test, (P<0.05). The mRNA expression of NFKBIA gene is higher in BPH than in PCa, while mRNA expression of SREBF2 is lower in BPH.Relative expression differences of NFKBIA/SREBF2 in tissue sample of each PCa and BPH patient was calculated, finding that expression of NFKBIA in PCa is 0.099 times as in BPH, which is down-regulated, while the expression of SREBF2 in PCa is 34.54 times to BPH, up-regulated, (P<0.05), and the NFKBIA and SREBP2 have significantly negative corelation. Meanwhile, the sensitivity of combining of SREBP2 and NFKBIA is higher than that of SREBP2 and NFKBIA respectively.Conclusions:The down-regulation of NFKBIA expression in PCa indicates positive expression of NF-Kappa B in cells and fast cell multiplication. The low expression of NFKBIA in prostate tissues can be a marker for early diagnosis and prognosis of PC a.The expression level of SREBP2 in PCa (prostate cancer tissue) is markedly higher than in BPH, and the difference has statistic significance. The study shows that expression of SREBP2 in PCa cells is up-regulated, which implies that increased production of lipids destroy the dynamic balance of cholesterol. The high expression of SREBP2 in prostate tissues can be a marker for early diagnosis and prognosis of PCa.The NFKBIA/SREBF2 is a group of factors related to gene transcription level. The study shows that we might not get exact differential-diagnosis between BPH and PCa only by single gene-expression detection. But combination with NFKBIA/ SREBF2 as a comprehensive evaluation, the gene-combination expression could predicts the increasing probability of tumor malignancy, which can reflect the genesis and development of tumor more rationally and give better guidance for clinical diagnosis. Therefore combination of NFKBIA with SREBF2 can increase the accuracy of detecting single gene-expression, which helps clinical early diagnosis and prognosis evaluation for PCa.