Shahi Mehdi H, Schiapparelli Paula, Afzal Mohammad, Sinha Subrata, Rey Juan A, Castresana Javier S
Brain Tumor Biology Unit-CIFA, University of Navarra School of Sciences, Pamplona, Spain.
Tumour Biol. 2011 Feb;32(1):113-27. doi: 10.1007/s13277-010-0105-x. Epub 2010 Sep 10.
It is well known that sonic hedgehog signaling pathway plays a vital role during early embryonic development. It is also responsible for stem cell renewal and development of several cancers like colorectal and breast carcinoma and major brain tumors as medulloblastoma and glioblastoma. The role of sonic hedgehog signaling in the development of neuroblastoma has not been thoroughly investigated. In this study, we attempted to determine the expression of Bmi-1 stem cell marker and of Shh pathway downstream target genes glioma-associated oncogene homolog 1 (GLI1), protein patched homolog 1 (PTCH1), Cyclin D2, plakoglobin (γ catenin), NK2 homeobox 2 (NKX2.2), paired box gene 6 (PAX6), secreted frizzled-related protein 1 (SFRP1), and hedgehog interacting protein (HHIP) in 11 neuroblastoma cell lines and 41 neuroblastoma samples. Also, inhibition of the pathway was performed genetically by GLI1 knockdown siRNA or chemically by cyclopamine. After inhibition, low transcript expression was detected in downstream target genes like PTCH1, in the cell lines. We further preformed promoter methylation studies of Cyclin D2, PTCH1, HHIP, and SFRP1 genes by melting curve analysis-based methylation assay (MCA-Meth) and methylation-specific PCR (MSP). Results revealed no methylation in Cyclin D2 gene promoter in neuroblastoma samples or in cell lines; one cell line (MHH-NB-11) showed PTCH1 methylation; 3/11 (27%) cell lines and 9/41 (22%) neuroblastoma samples showed HHIP methylation; and 3/11 (27%) cell lines and 11/41 (27%) samples showed SFRP1 methylation. Taken together, our results suggest the possibility of two levels of control of the sonic hedgehog signaling pathway: transcriptional and epigenetic, which might offer new therapeutic possibilities to modulate the pathway and try to suppress tumor growth.
众所周知，音猬因子信号通路在胚胎早期发育过程中起着至关重要的作用。它还负责干细胞更新以及多种癌症的发展，如结直肠癌、乳腺癌以及主要的脑肿瘤，如髓母细胞瘤和胶质母细胞瘤。音猬因子信号在神经母细胞瘤发展中的作用尚未得到充分研究。在本研究中，我们试图确定11种神经母细胞瘤细胞系和41个神经母细胞瘤样本中Bmi-1干细胞标志物以及Shh通路下游靶基因神经胶质瘤相关癌基因同源物1（GLI1）、蛋白 patched 同源物1（PTCH1）、细胞周期蛋白D2、连环蛋白（γ连环蛋白）、NK2同源盒2（NKX2.2）、配对盒基因6（PAX6）、分泌型卷曲相关蛋白1（SFRP1）和刺猬因子相互作用蛋白（HHIP）的表达。此外，通过GLI1敲低小干扰RNA对该通路进行基因抑制，或通过环杷明进行化学抑制。抑制后，在细胞系中检测到下游靶基因如PTCH1的转录本表达降低。我们进一步通过基于熔解曲线分析的甲基化检测（MCA-Meth）和甲基化特异性PCR（MSP）对细胞周期蛋白D2、PTCH1、HHIP和SFRP1基因进行启动子甲基化研究。结果显示，神经母细胞瘤样本或细胞系中细胞周期蛋白D2基因启动子未发生甲基化；一个细胞系（MHH-NB-11）显示PTCH1甲基化；3/11（27%）的细胞系和9/41（22%）的神经母细胞瘤样本显示HHIP甲基化；3/11（27%）的细胞系和11/41（27%）的样本显示SFRP1甲基化。综上所述，我们的结果表明音猬因子信号通路可能存在转录和表观遗传两个层面的调控，这可能为调节该通路并试图抑制肿瘤生长提供新的治疗可能性。
