| Prostate cancer is the second malignant tumors in Europe and United States. Its incidence is rising rapidly in China as the average lifespan increasing and the living standards improving. Early diagnosis is the most important factor for patient's recovering. Diagnosing methods now include serum prostate-specific antigen (PSA) test, leukocyte adherence inhibition test, digital rectal examination, imaging (ultrasound, CT, MRI) diagnostic methods such as needle biopsy. But they can not meet the needs of clinic because of high false positive and missed diagnosis. Searching for new biomarkers and establishing effective early diagnosing method are necessary. Epigenetics is a new field in molecular biology and is considered to be related with some cancer development. Abnormal DNA methylation is emerging as a class of promising biomarkers. So, our purpose is to investigate the relationship of RARβ2, P16, CDH1 and GSTP1 gene promoter methylations with the development of prostate cancer.There are many methods on DNA methylation detection. Such as MSP (Methylation Specific PCR), RLGS (Restriction Landmark Genome Scanning), Sanger's dideoxy DNA sequencing etc. However, they are limited in application because of lacking objectivity, poor reproducibility. Find new method for assay DNA methylation is necessary. Pyrosequencing is a new technology for sequencing the DNA sequence. It has been applied to study DNA methylation recently.Pyrosequencing was employed in our study. First of all, the method of methylation specific pyrosequencing was established on RARβ2 gene promoter analysis. Genomic DNA is extracted from human normal peripheral whole blood. Targeted gene fragment was amplified by composite nested PCR and cloned into pMD 18-T vector for constructing control plasmid. Five methylation loci are detected in RARβ2 gene promoter CpG Island by pyrosequencing after bisulfite treatment, and the results were confirmed by dideoxy sequencing. The percentage of methylation is detected in different proportion admixture of control PCR products and then the calibration curve is ploted. Single-stranded DNA template preparation and pyrosequencing analysis of the parameters were optimized. The methylation uniformity was detected by pyrosequencing from different amount of paraffin section of prostatic hyperplasia tissues. Results showed that the percentage of methylation in negative control is 0%; in positive control is 100%. By detecting mixed samples, we found that there is methylation-linear relationship between the frequencies of complex, the linear correlation is 0.99, the slope is almost 1, and the standard deviation in all sites is about 1%. The methylation specific pyrosequencing method for detecting gene promoter methylation is successfully established.To investigate the meaning of promoter methylation in prostate cancer, bioinformations including CpG islands position, DNA sequences, possible methylation regions of RARβ2, P16, CDH1 and GSTP1 genes were analyzed. Then PCR primers and pyrosequencing primers were designed, PCR amplification conditions were optimized. According to the method established above, methylation specific pyrosequencing were carried out in 4 genes CpG islands from 20 BPH (Benign Prostate Hyperplasia) and 20 PCa (Prostate cancer) patients. Results showed that at least one or more genes were hypermethylated in PCa patients. The methylation frequency in PCa was higher than BPH. The statistic differences of promoter methylation between PCa and BPH in RARβ2 and CDH1 genes were significant (P< 0.05). In other two genes of P16 and GSTP1, the percentages of promoter methylation in selected regions in cancer group were only slightly higher than BPH, and had no significant statistic differences.In conclusion, a methylation specific pyrosequencing method was modified to our study. The promoter methylation statuses in four genes were analyzed. Among them, RARβ2 and CDH1genes were found to have higher methylation frequency in PCa samples than BPH samples, which may become a biomarker in prostate cancer diagnosis and treatment in the future.
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