IGFBP-6經由調控EGR-1的表現在鼻咽癌細胞中扮演抑癌基因的角色

Abstract

鼻咽癌在東南亞頗為盛行，尤其是中國南方、新加坡及台灣。本研究的目的是藉由實驗發現鼻咽癌進行時關鍵基因的變化。之前我們以利用 microarray 比較鼻咽癌和正常鼻黏膜細胞篩選出 18 種基因，再經由 Q-RT-PCR 和組織免疫染色發現 IGFBP-6 在鼻咽癌細胞的表現和正常鼻黏膜細胞比較有顯著的減少。為探討 IGFBP-6 在鼻咽癌腫瘤生長時的作用，我們建立 IGFBP-6 轉染的鼻咽癌穩定並可被 Doxycycline 調控 IGFBP-6 表現的細胞株，進行試管及活體功能性分析，發現 IGFBP-6 表現增加時鼻咽癌細胞的增生、侵犯及轉移能力均顯著降低且讓細胞凋亡數目明顯增加；進一步分析發現 EGR-1、 Caspase-1及 TSP-1 等基因表現也明顯降低。利用 siRNA 抑制 EGR-1 的基因表現，Caspase-1及 TSP-1 表現也降低，但 IGFBP-6 的表現及鼻咽癌細胞的移動能力則不受影響。 Chromatin immunoprecipitation 和 luciferase reporter 實驗證明 IGFBP-6 能黏結在 EGR-1的 promoter 上並啟動 EGR-1 基因表現。總結來說， IGFBP-6 能經由調控 EGR-1 的表現來影響鼻咽癌的進行，因此， IGFBP-6 可能被利用來作為治療鼻咽癌的標的。

Nasopharyngeal carcinoma (NPC) is prevalent in southeastern Asia, particularly southern China, Singapore and Taiwan. The aim of this study was to identify the pivotal genes that may be altered during NPC progression. Using cDNA microarray analysis, we compared the expression of 18 genes between NPC and normal nasomucosal cells. Q-RT-PCR analysis found the expression of IBFBP-6 in NPC cell lines and immunolocalization of IGFBP-6 in NPC to be very weak. To explore the effects of IGFBP-6 on NPC tumour growth, we constructed inducible plasmids containing full length of IGFBP-6 c-DNA (pBIG2i-IGFBP-6) and established pBIG2i-IGFBP-6 transfected NPC stable cell lines (NPC-TW01-pBIG2i-IGFBP-6). We then performed functional analysis of the IGFBP-6 in cell lines in vitro and in vivo. Overexpression of IGFBP-6 significantly suppressed the proliferation, invasion, and metastatic activity of NPC cells and increased their apoptosis. We found EGR-1, caspase-1 and TSP-1 genes to be markedly upregulated when NPC-pBIG2i-IGFBP-6 was treated with doxycycline. Knocking down EGR-1 with EGR-1 siRNA resulted in a decrease in expression of caspase-1, TSP-1 and EGR-1, but not the expression of IGFBP-6. However, in knockdown cells the unchanged expression of IGFBP-6 did not inhibit the migration of NPC cells. Chromatin immunoprecipitation and luciferase reporter assay experiments showed that IGFBP-6 bound the EGR-1 promoter regions and activated EGR-1 promoter. We conclude that IGFBP-6 can regulate the progression of NPC by regulating the expression of EGR-1. These results suggest that IGFBP-6 could be used as a new target in NPC therapy.