| Part one:Fine mapping study of 10q11 for prostate cancer risk in Chinese population[Background]:Nowadays, more than 20 genome-wide association studies identified more than 70 prostate cancer risk loci at 16 chromosomes, which included rs10993994 at 10q11 identified by three GWAS projects. Due to the unknown mechanism of SNPs identified by GWAS in the pathogenesis of prostate cancer, there are many difficulties in the transformation from GWAS results to clinical benefits. Generally speaking, it needed two steps to explore the mechanism of SNPs identified by GWAS in the pathogenesis of prostate cancer, firstly, a re-sequencing study was needed to deal with the interference of linkage disequilibrium and find the real one at certain area, this step was called fine mapping; secondly, once the real risk loci was found, the functional analysis was carried out according to the genome background of the loci.[Objective]:This part was a fine mapping study of 10q11 for prostate cancer in Chinese population, tried to identify the real factors which influence prostate cancer risk.[Methods]:We gathered 1755 cases of prostate cancer and 1523 cases of healthy control individuals from ChinaPCa project, DNA was extracted from blood samples. SNPs were selected from HapMap Phase 2 CHB subjects, the region was chr10:51194000-51259000, based on r2≥0.8 and minor allele frequency (MAF)≥0.05,12 SNPs were identified to capture; By the analysis of potential functional area of the gene MSMB and NOCA4 in dbSNPs database, three SNPs were selected, two of which duplicated with 12 SNPs above, a total of 13 SNPs were selected from lOqll. Samples were genotyped using Sequenom genotyping technology platform. Statistical analysis of the relationship between SNPs and prostate cancer risk was done at last. [Results] One SNP (rs4630240) in 13 SNPs was failed to get date in the genotyping, the call rate of the remaining 12 SNPs was 98.3%-99.5%. At the level of P<0.01, all 12 SNPs in control group are in line with Hardy-Weinberg balance. There are three SNPs associated with prostate cancer risk (P<0.05):rs10993994 (risk allele:T,P= 0.012, OR=1.14), rs10821609 (risk allele:T, P= 0.030,OR=1.15) and rs10821610 (risk allele:G, P= 0.008,OR=1.16); after adjustment of age, the three SNPs were still associated with prostate cancer risk (P values were 0.011, 0.028 and 0.009, respectively). The r2 were 0.18 (rs10993994 vs rs10821609),0.05 (rs10993994 vs rs10821610) and 0.44 (rs10821609 vs rs10821610). We adjusted the influence of one SNP to the others by condition logistic regression model, only rs10821610 after adjustment of rs10993994 still significant at P<0.05, the other five P values were no longer significant.[Conclusions]:Rs10994994, rs10821609 and rs10821610 at 10q11 were prostate cancer risk loci in the Chinese population, rs10821610 was the most significant one; there was rarely linkage disequilibrium between rs10993994 and rs10821610, while there was mild linkage disequilibrium between the other two combinations; they might influence each other and participate in the pathogenesis of prostate cancer together, not independently, the reason of interaction might be linkage disequilibrium or something else.Part Ⅱ:The role of rs10993994 for the transcriptional activity of MSMB promoter in prostate cancer cell lines[Backgroud]:A SNP, rs10993994, at 10q11 was associated with prostate cancer risk in Chinese, American and European. More importantly, the SNP was located at the promoter region of MSMB (microseminoprotein beta) gene which encoded the prostatic secretary protein 94(PSP94), it was reported that the decreased expression of PSP94 was associated with the onset and progression of prostate cancer.[Objective]:To reveal the role of rs10993994 for the transcriptional activity of MSMB promoter in prostate cancer cell lines PC-3 and LNCaP. [Methods]:Promoter fragments were generated by chemical synthesis, corresponding to the promoter region from-299bp to +36bp relative to the start codon of MSMB gene. Due to the two possibility (T/C) of rs10993994 in the region, we generated two promoter fragments:MSMB promoter-T and MSMB promoter-C. The fragments were then cloned into pGL3-basic vectors, Positive clones were transfectd into prostate cancer cell lines PC-3 and LNCaP, finally, relative level of fluorescence was detected by fluoresce detector.[Results]:We generated two promoter fragments of MSMB, MSMB promoter-T and MSMB promoter-C. The two promoter fragments were cloned with pGL3-basic vectors and formed pGL3-MSMB promoter-T and pGL3-MSMB promoter-C. In PC-3, the relative light unit of pGL3-MSMB promoter-C was significant high than that of pGL3-MSMB promoter-T(2.27+ 0.39 vs 0.57 ±0.13, P<0.05); In LNCaP, the relative light unit of pGL3-MSMB promoter-C was significant high than that of pGL3-MSMB promoter-T(1.70 ±0.32 vs 0.37±0.09, P<0.05).[Conclusions]:The transcriptional activity of pGL3-MSMB promoter-C was higher than that of pGL3-MSMB promoter-T in PC-3 and LNCaP. Rs10993994 could influence the transcriptional activity of MSMB promoter; compared with allele T, allele C in rs10993994 could facilitate transcription. |
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