Department of Biological and Health Sciences, Texas A&M University- Kingsville, Kingsville, TX, USA.
J Ovarian Res. 2010 Feb 18;3(1):4. doi: 10.1186/1757-2215-3-4.
The Foxl2 transcription factor is required for ovarian function during follicular development. The mechanism of Foxl2 regulation of this process has not been elucidated. Our approach to begin to understand Foxl2 function is through the identification of Foxl2 regulated genes in the ovary.
Transiently transfected KK1 mouse granulosa cells were used to identify genes that are potentially regulated by Foxl2. KK1 cells were transfected in three groups (mock, activated, and repressed) and twenty-four hours later RNA was isolated and submitted for Affymetrix microarray analysis. Genesifter software was used to carry out analysis of microarray data. One identified target, the gonadotropin releasing hormone receptor (GnRHR) gene, was chosen for further study and validation of Foxl2 responsiveness. Transient transfection analyses were carried out to study the effect of Foxl2 over-expression on GnRHR gene promoter-luciferase fusion activity. Data generated was analyzed with GraphPad Prism software.
Microarray analysis identified 996 genes of known function that are potentially regulated by Foxl2 in mouse KK1 granulosa cells. The steroidogenic acute regulatory protein (StAR) gene that has been identified as Foxl2 responsive by others was identified in this study also, thereby supporting the effectiveness of our strategy. The GnRHR gene was chosen for further study because it is known to be expressed in the ovary and the results of previous work has indicated that Foxl2 may regulate GnRHR gene expression. Cellular levels of Foxl2 were increased via transient co-transfection of KK1 cells using a Foxl2 expression vector and a GnRHR promoter-luciferase fusion reporter vector. The results of these analyses indicate that over-expression of Foxl2 resulted in a significant increase in GnRHR promoter activity. Therefore, these transfection data validate the microarray data which suggest that Foxl2 regulates GnRHR and demonstrate that Foxl2 acts as an activator of the GnRHR gene.
Potential Foxl2 regulated ovarian genes have been identified through microarray analysis and comparison of these data to other microarray studies. The Foxl2 responsiveness of the GnRHR gene has been validated and provided evidence of Foxl2 transcriptional activation of the GnRHR gene promoter in the mouse ovary derived KK1 granulosa cell line.
Foxl2 转录因子在卵泡发育过程中对于卵巢功能是必需的。Foxl2 调控此过程的机制尚未阐明。我们通过鉴定卵巢中 Foxl2 调控的基因来开始理解 Foxl2 功能的方法。
瞬时转染 KK1 小鼠颗粒细胞以鉴定潜在受 Foxl2 调控的基因。KK1 细胞分三组（mock、激活和抑制）转染，24 小时后分离 RNA 并进行 Affymetrix 微阵列分析。使用 Genesifter 软件进行微阵列数据分析。选择一个鉴定的靶标，即促性腺激素释放激素受体（GnRHR）基因，进行进一步研究并验证 Foxl2 的反应性。进行瞬时转染分析以研究 Foxl2 过表达对 GnRHR 基因启动子-荧光素酶融合活性的影响。使用 GraphPad Prism 软件分析生成的数据。
微阵列分析鉴定了 996 个已知功能的基因，这些基因在小鼠 KK1 颗粒细胞中可能受 Foxl2 调控。本研究还鉴定了其他研究报道的 Foxl2 反应性的类固醇急性调节蛋白（StAR）基因，从而支持了我们策略的有效性。GnRHR 基因被选择进行进一步研究，因为它已知在卵巢中表达，并且先前的工作结果表明 Foxl2 可能调节 GnRHR 基因表达。通过瞬时共转染 KK1 细胞使用 Foxl2 表达载体和 GnRHR 启动子-荧光素酶融合报告载体，增加细胞内 Foxl2 的水平。这些分析的结果表明，Foxl2 的过表达导致 GnRHR 启动子活性显著增加。因此，这些转染数据验证了微阵列数据，表明 Foxl2 调控 GnRHR 并证明 Foxl2 作为 GnRHR 基因的激活剂起作用。
通过微阵列分析鉴定了潜在的 Foxl2 调控的卵巢基因，并将这些数据与其他微阵列研究进行比较。GnRHR 基因的 Foxl2 反应性已得到验证，并提供了在小鼠卵巢衍生的 KK1 颗粒细胞系中 Foxl2 转录激活 GnRHR 基因启动子的证据。
