- 學位論文
探討TGF-β-stimulated clone-22 基因對人類肺癌細胞生長及轉移的影響
Investigate the growth and metastasis effects of TGF-β-stimulated clone-22 gene in human lung cancer cells
摘要
TGF-β-stimulated clone-22 (TSC-22)是由TGF-β所誘導表現的基因，首先在老鼠造骨細胞MCT3T3E1中發現透過TGF-β的刺激會誘發TSC-22基因的表現。人類的TSC-22基因位於染色體13q14位置。文獻指出TSC-22能夠調控胚胎的發生，以及在部分癌細胞中，發現TSC-22基因的表現具有促進細胞凋亡(apoptosis)的趨勢，另外在人類唾液腺癌細胞中抑制TSC-22的表現能加速癌細胞的生長，因此推測TSC-22可能具有抑癌基因的潛力。本研究欲探討TSC-22基因在肺癌細胞中扮演何種角色。首先，由細胞計數和MTT assay結果得知，當我們在H358細胞株以shRNA干擾方式抑制TSC-22時會增加肺癌細胞的生長與細胞形成群落能力，而在CL1-5細胞株過度表現TSC-22基因則會降低肺癌細胞的生長及細胞形成群落能力。另外當抑制TSC-22表現時，會使得細胞轉型能力增強，相反的，過度表現TSC-22會抑制癌細胞的轉型能力。接著我們利用流式細胞儀分析TSC-22對細胞週期的影響，發現抑制TSC-22的表現會造成細胞週期之S期及G2/M期百分比上升。相反地，當過度表現TSC-22會造成細胞的細胞凋亡比例增加，並且在G1期、S期及G2/M期表現比例降低。此外利用短暫轉染計數將H226細胞株過度表現TSC-22亦會造成細胞凋亡比例增加的現象。進而以西方墨點法偵測細胞週期調控蛋白的表現，結果顯示將H358細胞株抑制TSC-22會明顯抑制p16的表現量，另一方面過度表現TSC-22造成CL1-5細胞株CDK1及CDK4表現量下降，並且p16的表現量有增加的趨勢，推測TSC-22的表現與p16基因之調控具有顯著相關性，並且由以上結果可以推論TSC-22在肺癌細胞株中可能扮演抑癌基因的角色。另一方面，當H358細胞抑制TSC-22後主要經由活化p38-MAPK路徑，而將CL1-5過度表現TSC-22會造成pAkt的表現量大幅降低，這些現象與TSC-22的抑癌功能之間的關係仍需再進一步釐清。接著我們繼續探討TSC-22對細胞轉移的影響，在細胞移行試驗及細胞侵潤試驗中，我們發現在CL1-0細胞株中抑制TSC-22的表現能促進細胞的移行能力，然而過度表現TSC-22會減弱CL1-5細胞株的侵潤能力。最後我們也利用動物模式觀察到抑制TSC-22時會促進腫瘤的生長能力。綜合以上結果，TSC-22基因在部份肺癌細胞株中可能扮演抑制癌細胞生長、群落形成及轉型能力的角色，但其詳細的作用機轉及影響路徑仍有待進一步探討。
並列摘要
TGF-β-stimulated clone-22(TSC-22) was originally reported as a TGF-β-inducible gene in mouse osteoblastic cells, MCT3T3E1. In 1992, TSC-22 was initially isolated from mouse osteoblastic cells as an immediate early response gene of TGF-β. The human TSC-22 gene was mapped to chromosome 13q14. Recently, several roles of TSC-22 have been proposed, including the regulation of embryogenesis, and the pro-apoptotic factor in several cancer cells. It has been reported that downregulation of TSC-22 enhances cell growth in the salivary gland cell line. Accumulated data suggest that TSC-22 is a potential tumor suppressor gene. In this research, we will investigate the role of TSC-22 gene in lung cancer cells. First of all, to address the function of endogenous TSC-22 in lung cancer cells, H358 and CL1-5 are known to express higher and lower levels of TSC-22, respectively. We established H358 and CL1-5 that stably expressed shRNA for TSC-22 and wt-TSC-22, respectively. Cell counting, MTT assay and colony formation assay data showed that suppression of TSC-22 in H358 increased cell growth and cell colony formation. On the other hand, overexpression of TSC-22 in CL1-5 would decrease cell growth and form smaller and looser colonies. According to anchorage-independent growth assay, reduced TSC-22 expression has better effect in cell transformation and enhanced TSC-22 expression has worse effect in cell transformation. We used flow cytometry to analyze cell cycle distribution in H358- and CL1-5-derived cell lines. Cells with suppressed TSC-22 gene would increase the cell population in S and G2/M phases. However, the percentage of sub-G1 phase was increased and the cell population in G1, S and G2//M phases was decreased in wild-type TSC-22 gene transfected cells. Moreover, the induction of sub-G1 population was also observed in the transient transfection of wild-type TSC-22 into H226 lung cancer cells. And then, we continually observed the expressions of cell cycle regulated proteins. The expression level of p16 was obviously decreased in shRNA TSC-22 transfected cells. On the contrary, overexpression of TSC-22 in CL1-5 could decrease the expression levels of CDK1 and CDK4. Besides, the protein level of p16 was elevated in wild-type TSC-22 gene transfected cells. According to our present results indicated that TSC-22 might acts as a tumor suppressor function in lung cancer. Next, we examined the expression of MAPKs and Akt by Western blot. We observed that the activities of MAPKs and Akt were also affected in TSC-22 regulated cell lines. The relationship between above phenomena and functions of TSC-22 need further study. In the experiments of metastasis, we want to determine if TSC-22 also affected the migratory and invasive capability of lung cancer cell lines. The increased migratory ability was observed in TSC-22 knockdown CL1-0 cells. But the decreased invasive ability was observed in TSC-22 overexpressed CL1-5 cells. The molecular mechanism involved in TSC-22 mediated cell migratory and invasive ability required further study. Finally, to assess whether TSC-22 plays a causal role in tumor growth, H358/vector control and H358/RNAi-4 cells were injected in subcutaneous into the right flank of SCID mice. Compared with vector control, suppression of TSC-22 may promote ability of tumor growth. In conclusion, our results suggest that TSC-22 may play a role in reducing cell proliferation, cell colony formation, cell transformation and metastasis in some lung cancer cell lines studied here. Nevertheless, the detailed mechanisms and pathways involved in tumor suppressor activity of TSC-22 are required for further investigation.