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目的:探讨雄激素和雄激素受体(AR)对小鼠附睾Caveolin-1表达的调控机制。方法:通过搜索前期染色体免疫共沉淀-测序(ChIP-seq)实验得到的小鼠附睾AR结合位点的数据库,寻找Caveolin-1基因相关联的AR结合位点。分别取正常小鼠、睾丸阉割小鼠以及睾丸阉割后补充外源雄激素的小鼠的附睾组织,一方面,抽提总RNA,利用逆转录PCR以及逆转录荧光定量PCR检测Caveolin-1基因的mRNA表达水平;另一方面,利用AR抗体进行染色体免疫共沉淀(ChIP),采用ChIP-PCR以及ChIP-qPCR的方法,检测Caveolin-1基因相关联的AR结合位点在体内与AR的结合情况。结果:从小鼠附睾AR结合位点的数据库中找到两个Caveolin-1相关联的AR结合位点,均位于第二内含子区域。睾丸阉割后,Caveolin-1基因的表达显著升高(P<0.05),表达量是对照组的(1.8±0.17)倍;两个AR结合位点的富集倍数分别由正常状态下的(13.5±1.47)倍和(10.5±1.03)倍降至(1.05±0.17)倍和(1.4±0.14)倍(P<0.01)。补充雄激素后,Caveolin-1基因的表达又降至正常水平(P<0.05),为正常对照组的(1.03±0.06)倍。两个AR结合位点的富集倍数则分别升至(16.4±2.6)倍和(10.0±0.92)倍(P<0.01)。结论:Caveolin-1在小鼠附睾内是AR的一个直接靶基因,其表达受雄激素的负调控。该研究为理解雄激素/AR在小鼠附睾内的调控网络提供了新的视角。
AIM: To investigate the regulatory mechanism of androgen and androgen receptor (AR) on the expression of Caveolin-1 in mouse epididymis. METHODS: The AR binding site associated with Caveolin-1 gene was searched by searching database of mouse epididymal AR binding sites obtained by ChIP-seq experiment. The normal mice, testis castrated mice and testicular castration of exogenous androgen supplemented mice epididymis tissue, on the one hand, total RNA was extracted, reverse transcription PCR and reverse transcriptional quantitative PCR detection of Caveolin-1 gene On the other hand, AR antibody was used to perform ChIP. ChIP-PCR and ChIP-qPCR were used to detect the binding of Caveolin-1-associated AR binding site to AR in vivo . RESULTS: Two Caveolin-1-associated AR binding sites were found in the database of mouse epididymal AR binding sites, both located in the second intron region. After testis castration, the expression of Caveolin-1 gene was significantly increased (P <0.05), and the expression level of Caveolin-1 was (1.8 ± 0.17) times that of the control group. The enrichment multiple of the two AR binding sites was ± 1.47 fold and 10.5 ± 1.03 fold to 1.05 ± 0.17 fold and 1.4 ± 0.14 fold respectively (P <0.01). After the addition of androgen, the expression of Caveolin-1 gene was reduced to the normal level (P <0.05), which was 1.03 ± 0.06 times of the normal control group. The enrichment fold of the two AR binding sites increased to (16.4 ± 2.6) and (10.0 ± 0.92) times (P <0.01), respectively. Conclusion: Caveolin-1 is a direct target gene of AR in mouse epididymis and its expression is negatively regulated by androgen. This study provides a new perspective for understanding the regulatory network of androgen / AR in mouse epididymis.