Lu Z, Hardt J, Kim J J
Division of Reproductive Biology Research, Department of Obstetrics and Gynecology, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL 60611, USA.
Mol Hum Reprod. 2008 Jun;14(6):357-66. doi: 10.1093/molehr/gan023. Epub 2008 May 2.
Homeobox (HOX) A10 is essential for fertility as demonstrated in transgenic mice, specifically affecting implantation and decidualization. Its role in human decidualization, however, remains unknown. In this study, we used gene silencing followed by microarray analysis to decipher the role of HOXA10 during decidualization of human endometrial stromal cells (HESCs). HOXA10 was knocked down using siRNA oligonucleotide transfection and cells were treated with estradiol, medroxyprogesterone acetate and dibutyryl cAMP (H + cAMP) to induce decidualization. Genes significantly regulated were identified using the Affymetrix microarray chip. With this method, 2361 transcripts were significantly altered by 1.5-fold or higher (P < 0.05) with H + cAMP treatment only. Of these genes, 258 were significantly up-regulated by HOXA10 knockdown and 236 transcripts were significantly down-regulated by more than 1.5-fold, totaling 494 genes that were regulated by HOXA10 during decidualization. Data analysis using the Ingenuity System revealed that many of the genes regulated by HOXA10 knockdown during H + cAMP treatment were associated with cell cycle. Real-time PCR was used to confirm that HOXA10 knockdown decreased expression of the cell cycle genes CDC2 and CCNB2. In addition, a higher percentage of cells were arrested in the G2/M phase. Next, we observed that cell proliferation as measured by BrdU incorporation was decreased upon HOXA10 knockdown and H + cAMP treatment. Apoptosis, on the other hand, as measured by Annexin V staining was not influenced by siHOXA10 in decidualizing cells. Together, these data demonstrate that during decidualization of HESC, HOXA10 is actively involved in promoting cell proliferation through the regulation of hundreds of genes.
同源框（HOX）A10对生育能力至关重要，这在转基因小鼠中得到了证实，它特别影响着床和蜕膜化。然而，其在人类蜕膜化中的作用仍不清楚。在本研究中，我们采用基因沉默并结合微阵列分析来解读HOXA10在人子宫内膜基质细胞（HESC）蜕膜化过程中的作用。使用小干扰RNA（siRNA）寡核苷酸转染敲低HOXA10，并使用雌二醇、醋酸甲羟孕酮和二丁酰环磷腺苷（H + cAMP）处理细胞以诱导蜕膜化。使用Affymetrix微阵列芯片鉴定显著调控的基因。通过这种方法，仅H + cAMP处理就使2361个转录本显著改变了1.5倍或更高（P < 0.05）。在这些基因中，258个因HOXA10敲低而显著上调，236个转录本显著下调超过1.5倍，在蜕膜化过程中共有494个基因受HOXA10调控。使用Ingenuity系统进行数据分析表明，在H + cAMP处理期间，许多因HOXA10敲低而调控的基因与细胞周期相关。使用实时定量聚合酶链反应（Real-time PCR）来确认HOXA10敲低会降低细胞周期基因细胞周期蛋白依赖性激酶2（CDC2）和细胞周期蛋白B2（CCNB2）的表达。此外，更高比例的细胞停滞在G2/M期。接下来，我们观察到，通过5-溴脱氧尿嘧啶核苷（BrdU）掺入法测量的细胞增殖在HOXA10敲低和H + cAMP处理后降低。另一方面，通过膜联蛋白V染色测量的凋亡在蜕膜化细胞中不受小干扰RNA HOXA10（siHOXA10）的影响。总之，这些数据表明，在HESC蜕膜化过程中，HOXA10通过调控数百个基因积极参与促进细胞增殖。
