IGFBP-3基因表達在卵巢類子宮內膜腺癌中之分子調控機制

胰島素生長因子結合蛋白-3(IGFBP-3)藉由和胰島素生長因子之結合,在體內執行抑制細胞生長的功能.在本實驗室之前的研究中,我們利用卵巢癌侵襲模型(Ovarial cancer invasion model)來建立卵巢類子宮內膜腺癌細胞株(ovarian endometrioid carcinoma) OVTW59的分株(P0~P4)。藉著互補去氧核糖核酸微矩陣(cDNA microarray)和及時定量聚合酶鍊反應(Real-time RT PCR),我們發現由P0到P4其細胞株之IGFBP-3的基因表現由高到低有顯著差異。在一系列體外(in vitro)及動物實驗的功能性分析顯示,IGFBP-3能夠抑制卵巢類子宮內膜腺癌細胞的移動、侵襲以及轉移的能力,並可誘發癌細胞的凋亡,證明IGFBP-3有抑癌基因的特色。在本實驗中,我們利用卵巢類子宮內膜腺癌細胞株P0以及P4作為模型,去探討IGFBP-3 的表現是否受到其基因啟動區(promoter region)的甲基化(methylation)而抑制其基因的表現。經過去甲基化藥物5’-aza-dC的處理過後,P4的mRNA以及蛋白的表現量有顯著回升的情形。同時,我們利用亞硫酸定序及專一性甲基化聚合酶鍊反應來分析卵巢類子宮內膜腺癌細胞株以及60個臨床檢體的IGFBP-3基因啟動區(promoter region)之甲基化情形。發現在卵巢類子宮內膜腺癌中, 40.43% 病人IGFBP-3基因啟動區(promoter region)有過度甲基化的情形;並且在細胞模型中,我們發現IGFBP-3甲基化靜默的現象是透過IGFBP-3基因啟動區上p53轉錄因子(transcription factor)結合位置的過度甲基化,進而影響了p53 調控IGFBP-3的轉錄活性所導致。以上研究顯示, IGFBP-3基因啟動區(promoter region)過度甲基化為影響其基因表達的一個調控機制。

關鍵字

IGFBP-3 ；卵巢癌

並列摘要

Insulin growth factor binding protein-3 (IGFBP-3) functions as a carrier of insulin-like growth factors (IGFs) in vascular circulation and has been postulated to be a mediator of growth suppression signal in cells. In our previously study, IGFBP-3 was selected from the cDNA microarray analysis of the progressive increase in invasion capability of ovarian endometrioid carcinoma cancer cell line OVTW59 sublines P0 to P4, its expression is very low in the advanced cancer cells such as P4 cell line. It has been proved as a tumor suppressor to inhibit tumor cell migration, invasion and metastasis and induce apoptosis in ovarian cancer pathogenesis. Since promoter CpG island hypermethylation can silence gene expression, we investigated whether hypermethylation of the IGFBP-3 promoter is involved in loss of IGFBP-3 expression in ovarian endometrioid carcinoma. After treatment with DNA methyltransferase inhibitor 5’-aza-2-deoxycytidine for eight days, restored expression of IGFBP-3 mRNA transcript was found in ovarian cancer cell line P4. In addition, we examined the methylation status of IGFBP-3 by methylation-specific PCR (MSP) assay and bisulfite genomic DNA sequencing, The methylation status correlated with IGFBP-3 mRNA and protein expression levels in endometrioid carcinoma cell lines. Furthermore, we analyzed 60 clinical samples and found that IGFBP-3 promoter was hypermethylated in 40.43% of ovarian endometrioid carcinoma surgical specimens. Moreover, we also found the hyprermethylated CpG sites of -210,-206,-183 and -179 in p53 consensus binding region in IGFBP-3 promoter may play a critical role in the effect of its promoter activity, These findings indicate that the silenced IGFBP-3 expression in advanced P4 line and patients is primarily regulated IGFBP-3 promoter region by hypermethylation in the p53 binding region.

並列關鍵字

IGFBP-3 ； methylation ； ovarian cancer

參考文獻

Baxter, R.C. (2001). Inhibition of the insulin-like growth factor (IGF)-IGF-binding protein interaction. Hormone research 55 Suppl 2, 68-72.

Baylin, S.B., and Herman, J.G. (2000). DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet 16, 168-174.

Bell, K.A., and Kurman, R.J. (2000). A clinicopathologic analysis of atypical proliferative (borderline) tumors and well-differentiated endometrioid adenocarcinomas sof the ovary. The American journal of surgical pathology 24, 1465-1479.

Bourdon, J.C., Deguin-Chambon, V., Lelong, J.C., Dessen, P., May, P., Debuire, B., and May, E. (1997). Further characterisation of the p53 responsive element--identification of new candidate genes for trans-activation by p53. Oncogene 14, 85-94.

Buckbinder, L., Talbott, R., Velasco-Miguel, S., Takenaka, I., Faha, B., Seizinger, B.R., and Kley, N. (1995). Induction of the growth inhibitor IGF-binding protein 3 by p53. Nature 377, 646-649.

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IGFBP-3基因表達在卵巢類子宮內膜腺癌中之分子調控機制

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