Rs10993994对前列腺癌细胞MSMB基因转录的影响

摘要

Background and purpose:Rs10993994 at 10q11 was found to be associated with prostate cancer risk by genome-wide association studies. This study tried to reveal its mechanism and to explore the role of rs10993994 for the transcriptional activity of microseminoprotein beta (MSMB) gene in prostate cancer cell lines. Methods:Promoter fragments were generated by chemical synthesis. Due to the two possibility of rs10993994 (T/C) 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 transfected into prostate cancer cell lines PC-3 and LNCaP, ifnally, relative level of lfuorescence was detected by lfuoresce 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 to pGL3-MSMB promoter-T and pGL3-MSMB promoter-C. In PC-3, the relative level of lfuorescence of pGL3-MSMB promoter-C was signiifcant higher than that of pGL3-MSMB promoter-T(2.27±0.39vs 0.57±0.13,P<0.05); In LNCaP, the relative level of lfuorescence of pGL3-MSMB promoter-C was signiifcant higher than that of pGL3-MSMB promoter-T (1.70±0.32vs 0.37±0.09,P<0.05).Conclusion:The transcriptional activity of pGL3-MSMB promoter-C was stronger than that of pGL3-MSMB promoter-T. Rs10993994 could inlfuence the transcriptional activity ofMSMB gene promoter in prostate cancer cell lines PC-3 LNCaP, allele C in rs10993994 could facilitate transcription than T.%背景与目的：全基因组相关联研究发现了染色体10q11区域的单核苷酸多态性(single nucleotide polymorphism，SNP)位点rs10993994与前列腺癌的遗传易感性相关,本研究探讨rs10993994影响前列腺癌发病风险的可能机制，即研究rs10993994对前列腺癌细胞微精液蛋白β(microseminoprotein beta，MSMB)基因转录活性的影响。方法：化学合成MSMB基因的启动子序列，由于位于序列中间的SNP位点 rs10993994有2种等位基因(T/C)可能，合成2条启动子MSMB promoter-T和MSMB promoter-C。将2条启动子通过酶切的方法导入荧光素酶报告基因载体(pGL3-basic vectors)中，筛选阳性克隆后，将携带启动子的质粒转染入前列腺癌细胞PC-3和LNCaP中，最后通过荧光检测仪检测重组质粒中启动子的转录活性。结果：成功合成2条启动子序列：MSMB promoter-T和MSMB promoter-C；分别将启动子序列与荧光素酶报告基因载体重组，形成重组质粒pGL3-MSMB promoter-T和pGL3-MSMB promoter-C。在前列腺癌细胞PC-3中，重组质粒MSMB promoter-C组的相对荧光度为2.27±0.39，显著高于重组质粒MSMB promoter-T组(0.57±0.13)，差异有统计学意义(P＜0.05)；在前列腺癌细胞LNCaP中，重组质粒MSMB promoter-C组的相对荧光度为1.70±0.32，显著高于重组质粒MSMB promoter-T组(0.37±0.09)，差异有统计学意义(P＜0.05)。结论：重组质粒pGL3-MSMB promoter-C的转录活性强于重组质粒pGL3-MSMB promoter-T的转录活性；rs10993994可以影响到MSMB基因启动子的启动转录活性，rs10993994位点上等位基因胞嘧啶(C)较胸腺嘧啶(T)更能促使MSMB基因转录。