褪黑素通过减弱催乳素基因增强子的突变抑制大鼠垂体催乳素瘤的增生

本工作旨在探讨褪黑素(melatonin,MLT)抑制17-β-雌二醇(17-β-estradiol,E2)诱发的Sprague-Dawley大鼠垂体催乳素(prolactin,PRL)瘤增生的分子机制。结果表明,每只大鼠每日定时皮下注射一定剂量的MLT(0.25、0.50mg)能显著抑制E2诱发的大鼠垂体PRL瘤的增生;偏低(0.05mg)或过高剂量(1.00、2.00mg)的MLT也抑制PRL瘤的增生,但无统计学意义。采用PCR和DNA直接测序显示,与正常垂体对照组比较,PRL瘤中PRL基因增强子出现五处突变,-1885bp位点由C突变为G,-1857~-1855由ACA替换为G,-1792~-1791插入G,-1383~-1382插入GGTGTGTG片段,-1265~-1250缺失GTGTGTGTGTGTGTGT片段。0.25mg/dMLT处理组,PRL瘤中的PRL基因增强子上述个别突变部位仍然存在(-1885由C突变为G),突变消失(-1792~-1791无插入G),大部分表现为突变减弱(-1856~-1855缺失AC,-1385~-1384缺失TG,-1250~-1253缺失GTGT)。采用荧光素酶报告基因检测PRL基因增强子活性显示,正常垂体、PRL瘤和0.25mg/dMLT处理的PRL瘤三组中,PRL基因增强子的活性分别为(13448.17±3012.74)、(161831.67±60996.01)和(10212.17±2634.71)OD单位。PRL瘤组增强子活性较正常垂体升高11倍(P<0.001),MLT处理组增强子活性较PRL瘤组降低93.69%(P<0.001)。上述三组PRL基因增强子空间结构的分析表明,PRL基因增强子DNA的曲折程度为PRL瘤组>MLT处理组>正常垂体。以上结果证实,MLT抑制大鼠垂体PRL瘤增生的重要分子机制之一可能是减弱PRL基因增强子的突变,也提示MLT可减弱PRL基因增强子的突变,从而下调PRL基因的高表达,可能与降低DNA的曲折程度有关。

Melatonin inhibits the proliferation of pituitary prolactin-secreting tumor by suppressing the enhancer elements mutation of PRL gene in the rat

In order to investigate the molecular mechanisms of the inhibition of the proliferation of 17-beta-estradiol (E(2))-induced pituitary prolactin-secreting tumor (prolactinoma) by melatonin (MLT) in the rat, we examined the inhibitory effects of MLT on the proliferation of E(2)-induced prolactinoma of the rat and the suppressing effects of MLT on the enhancer elements mutation of PRL gene in vivo and in vitro. The results showed that the weights of prolactinomas in MLT groups, in which 0.25 mg or 0.50 mg per day per rat of MLT was administered subcutaneously at 18:00, were decreased significantly. Out of the dosage of MLT, such as 0.05, 1.00 mg and 2.00 mg per day per rat, the antitumor action of MLT is less or disappointing. Polymerase chain reaction (PCR) and DNA sequencing showed five mutations in the enhancer elements of PRL gene in prolactinoma, such as -1885 point mutation (C --> G), -1857 - -1855 substitution (ACA --> G), -1792 - -1791 insertion G, -1383 - -1382 insertion (GGTGTGTG), -1265 - -1250 deletion (GTGTGTGTGTGTGTGT). Excluding of -1885 point mutation (C --> G), the mutation in the prolactinoma treated with 0.25 mg per day per rat MLT was decreased, such as -1792 - -1791 without insertion of G, -1856 - -1855 deletion AC, -1385 - -1384 deletion TG, -1250 - -1253 deletion GTGT. Firefly luciferase reporter gene systems showed that the luminosity of enhancer elements-luciferase reporter fusion gene in normal pituitary, prolactinoma treated without or with 0.25 mg per day per rat MLT were (13448.17+/-3012.74), (161831.67+/-60996.01), and (10212.17+/-634.71) OD units. Compared with the normal pituitary, the activity of PRL gene enhancer elements in prolactinoma was increased by 11 times (P<0.001). Compared with the prolactinoma, the activity of PRL gene enhancer elements in prolactinoma treated with MLT was decreased by 93.69% (P<0.001). Analysis of the space structure of PRL gene enhancer elements showed that the bending index in prolactinoma was higher than that in prolactinoma treated with MLT, which was higher than that in the normal pituitary. These results demonstrate that one of the important molecular mechanisms of MLT inhibiting the proliferation of prolactinoma is related to the reduction of enhancer elements mutation of PRL gene. These data also suggest that MLT-induced attenuation of enhancer elements mutation of PRL gene is involved in decreasing the bending index and attenuating the higher expression of PRL gene.