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標題: 探討KLF4在非小細胞肺癌細胞之侵襲能力上所扮演的角色
The study on the role of KLF4 in NSCLC cell invasiveness
作者: 馬潔如
Ma, Chieh-Ju
關鍵字: KLF4
Lung cancer
出版社: 生物醫學研究所
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摘要: Non-small cell lung carcinoma (NSCLC) is the most predominant type of lung cancer and one of the leading causes of lethal malignancies in Taiwan. Widespread metastasis is common in NSCLC and accounts for high mortality and low cure rate. Nevertheless, the molecular mechanisms underlying the metastasis of NSCLC remain unclear. KLF4 (a.k.a. GKLF, Gut-enriched Krüppel-like factor) is a newly identified transcription factor which is expressed mainly in the epithelial cells of the gastrointestinal tract. Deregulation of KLF4 expression has been linked to several types of cancer, including gastric cancer, colon cancer and breast cancer. Its role in the tumorigenesis and progression of NSCLC, however, has never been examined and is therefore the subject of this study. We first unraveled that KLF4 expression is selectively increased in the highly invasive NSCLC cell line CL1-5, while nearly no expression of KLF4 was observed in its parental, low invasive cell line CL1-0. KLF4-stably expressing cell lines were subsequently established in CL1-0 cells to analyze the effect of KLF4 on the growth and invasion of NSCLC cells. Our results indicated that KLF4 overexpression suppressed cell proliferation, likely associated with p21 upregulation in these cells. Furthermore, serum starvation treatment and colony formation in soft agar demonstrated that KLF4 expression promotes cell survival. Moreover, by means of wound healing assay and BoydenTM chamber migration assay, we found that KLF4 expression increased cell migration, but this effect is not statistically significant. In addition, the effect of KLF4 on NSCLC cell invasion was further examined by cell adhesion assay and Transwell-based cell invasion assay. Our results indicated that KLF4 promotes the interaction of cells with ECM. Importantly, Transwell-based cell invasion assay indicated that KLF4 is a potential invasion-promoting gene. Mechanistic study revealed that KLF4 expression suppressed E-cadherin and CRMP-1 protein expression as well as the promoter activity of E-cadherin and CRMP1 genes, consistent with its role in promoting invasion. In summary, our studies clearly demonstrated, for the first time, the promoting role of KLF4 in NSCLC invasiveness, and this effect is likely associated with the suppression of E-cadherin and CRMP1 expression by KLF4. Furthermore, the anchorage-independent growth and the resistance to serum starvation in KLF4-stable cells suggested the oncogenic function of KLF4.
近年來,在台灣的肺癌發生比例有顯著增加的趨勢,尤其是女性的肺癌病例中甚至高居癌症死亡的首位。肺癌根據組織學以及臨床表現可分為小細胞肺癌及非小細胞肺癌,其中較常見的非小細胞肺癌主要包括腺癌、鱗狀細胞癌與大細胞癌三種類型。目前肺癌五年內的治癒率僅13%,而大部分病人治療失敗的主因是癌細胞產生遠處轉移,但是主要影響肺癌轉移的分子機制並不十分明瞭。KLF4是近年來被發現的轉錄因子,主要表現在腸胃道的上皮性細胞,許多文獻指出KLF4表現量的降低與胃癌、大腸癌、乳癌皆有關聯。而它在非小細胞肺癌腫瘤的生成與發展上之角色並未被探討,因此引發了本研究的動機。 本實驗中首先發現,KLF4選擇性表現在具有高轉移特性的細胞株CL1-5中,而較不轉移的母細胞株CL1-0中幾乎沒有表現。進一步研究KLF4在生理功能與細胞型態上的影響,在CL1-0細胞株中製備KLF4穩定且持續大量表現的細胞株,作為日後分析KLF4的表現對於腫瘤細胞生長、發展之材料。初步檢測顯示大量表現KLF4可以抑制肺癌細胞增生,在分子機制方面也發現p21蛋白層次的增加。透過無血清環境培養以及軟瓊脂集落形成實驗,發現KLF4表現下會促進細胞存活的能力。在腫瘤轉移研究中,針對細胞遷移性以及細胞侵襲能力兩方面;首先在細胞遷移性分析上,透過傷口癒合實驗以及博登細胞移行器分析,結果發現在KLF4的表現下,細胞遷移能力會有些微增加,但是並沒有統計上的顯著差異,另外檢測一些遷移指標基因Rac-1、cdc42、Rho或是FAK的表現量時,也沒有明顯趨勢。在細胞侵襲能力分析方面,透過細胞黏著能力實驗,發現KLF4表現下增強了細胞與細胞外基質之間的交互作用;進一步利用雙層通透培養盤分析細胞侵襲能力,發現在KLF4表現下確實增加肺癌細胞侵襲能力,指出了KLF4可能是一個促進侵襲能力的基因;進一步於分子機制上的研究顯示,KLF4的表現抑制了E-cadherin以及轉移指標基因CRMP1之蛋白層次與基因啟動子活性。 綜合上述結果可知,我們首先發現KLF4在非小細胞肺癌細胞中扮演促進轉移的角色,可能是與抑制E-cadherin以及CRMP1表現量有關,並且KLF4表現的細胞株顯現出不需貼附生長的特性以及對低血清的抗性,指出此分子可能具有致癌基因的特性。未來除了更進一步確認KLF4與轉移指標基因之間的關係外,也須進行體內動物試驗或非小細胞肺癌病人臨床的研究,以釐清KLF4在非小細胞肺癌中所扮演的角色。
其他識別: U0005-1408200615220800
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