探討白血病抑制因子於口腔鱗狀細胞癌進展過程中所扮演之角色及機制研究

Investigation of The Role And Mechanism of Leukemia Inhibitory Factor in Oral Squamous Cell Carcinoma Progression

林庭安(Ting-An Lin)

指導教授 ： 章浩宏

國立臺灣大學/醫學院/口腔生物科學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201701411

摘要

目的：白血病抑制因子是IL-6 型細胞因子群的一個成員，其可藉由調控重要的訊息途徑包括 JAK / STAT3，PI3K，和ERK1 / 2 等訊息傳導途徑來調控細胞的增殖與存活。文獻回顧顯示白血病抑制因子在許多癌症的癌化過程中扮演重要且複雜的角色，其與間質組織有交互作用而影響癌化的過程及腫瘤的擴散；但白血病抑制因子在腫瘤發生中的確切作用仍然未知，特別在口腔癌中仍缺乏深入探討。
實驗設計：本研究包含了以免疫組織染色技術標定臨床口腔癌患者病理組織標本與相關之臨床參數，進行相關性分析，進一步更利用口腔癌細胞株進行白血病抑制因子可能的機制及角色探討，我們利用SAS、CA9-22、HSC3與Cal27四株口腔鱗狀上皮細胞癌細胞株來探討白血病抑制因子表現量被小髮夾型核糖核酸抑制或轉殖表現質體過度後之移行能力、侵襲能力以及生長能力是否受到影響。在細胞移動能力的測定上，利用博登細胞移行器實驗進行分析；生長能力使用細胞存活率分析進行測定。藉由核糖核酸微列陣與基因組富集分析主要訊息傳遞途徑。
結果：臨床病患之組織病理顯示在發生淋巴轉移及患者，其白血病抑制因子染色表現亦較明顯(p=0.022)，腫瘤分期較為嚴重的第三及四期患者與染色表現亦明顯相關(p=0.002)，進一步在細胞實驗發現白血病抑制因子與細胞移動和侵襲能力呈現正相關，抑制SAS與Cal27細胞中白血病抑制因子表現量後會使細胞移動能力明顯降低，但不影響細胞增生能力；相反地過量表現白血病抑制因子在口腔癌細胞株中則會促進癌細胞之轉移與侵襲能力。經由核糖核酸微陣列分析以及基因組富集分析認為A次單位抑制素(Inhibin beta A subunit, INHBA)為白血病抑制因子調控癌症進程之重要下游分子。過度表現INHBA後可顯著恢復細胞因白血病抑制因子抑制而造成之移動能力的下降。
結論：白血病抑制因子在口腔癌的進展過程中，可藉由侵襲及移動能力達到影響病人局部淋巴擴散及造成較嚴重的臨床分期結果，若能調控作為下游的INHBA訊息傳遞，白血病抑制因子有潛力成為抑制口腔癌惡性腫瘤局部擴散之治療目標。

關鍵字

口腔鱗狀上皮細胞癌 ； 移行 ； 侵襲 ； 白血病抑制因子 ； INHBA

並列摘要

Purpose:
Oral squamous cell carcinoma (OSCC) is the fifth common cancer worldwide. Distant metastasis is rare, but presents extremely poor prognosis clinically. Leukemia inhibitory factor (LIF) has been demonstrated an oncogene in several cancers, and we hypothesis that LIF might play crucial role in OSCC progression and metastasis.
Methods:
LIF expression was detected in OSCC samples by Immunohistochemistry. The ectopic LIF expression and silencing LIF were performed by LIF expression plasmids and short-hairpin RNA transfection in vitro, respectively. Boyden chamber assay was used to check migration and invasion abilities. Proliferation ability was analyzed by MTT. High throughput microarray analysis and GSEA analysis were also included. Statistical analyses were performed with unpaired Student’s two-tailed t test. A P value of <0.05 was considered significant.
Results:
Investigations on clinical parameters of OSCC patient and the expression of LIF revealed that patients with positive lymph node metastasis showed high expression of LIF protein (p = 0.022). Also, patient suffered from advanced cancer stage show high expression of LIF (p =0.002). In vitro, transient and stable knockdown LIF significantly decreased migration and invasion abilities. Moreover, overexpressed LIF could enhance OSCC cell motility (P < 0.05), but showed no effects on proliferation ability. In addition, using high throughput mRNA analysis and GSEA analysis, we identified INHBA as a crucial downstream effecter of LIF-promoted OSCC progression. Transfected with INHBA expression plasmid could significantly restore the migration and invasion abilities in shLIF stable transfectants (P < 0.05).
Conclusion:
Leukemia inhibitory factor contributes to cancer progression by enhancing regional lymphatic spread therefore leads to advanced cancer stage clinically. Regulation of the LIF downstream molecule such as INHBA thus can prohibition the invasion or migration ability of cancer cell, targeting on the leukemia inhibitory factor can be a potential strategy in preventing cancer progression and spreading in future.

並列關鍵字

Oral squamous cell carcinoma ； Migration ； Invasion ； LIF ； INHBA

參考文獻

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