探討RAI3基因對人類肺癌細胞株的影響

Investigate the effects of RAI3 gene in human lung cancer cell lines

陳文哲(Wen-Che Chen)

指導教授 ： 吳文俊

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2006年)

https://doi.org/10.6834/CSMU.2006.00007

摘要

本實驗室及其他研究團隊以cDNA microarray發現RAI3(retinoic acid induced-protein 3)基因在正常肺臟組織中表現量較高而在肺癌組織中表現量較低，另外也以RT-real time PCR證實此結果，因此推測其為抑癌基因。但有文獻指出，RAI3基因在乳癌細胞中為一致癌基因，因此本研究欲探討RAI3基因在肺癌細胞中扮演何種角色。首先，我們以shRNA干擾方式將RAI3抑制時會增加肺癌細胞的生長能力，而轉染HA-RAI3以促進RAI3表現則會降低肺癌細胞的生長能力。而anchorage-independent growth assay的結果顯示，干擾RAI3表現後的細胞株轉型較好而促進RAI3表現後的細胞株則轉型較差。但RAI3的表現對H1299及F4細胞株的生長能力及轉型都沒有影響。我們觀察對生長能力有影響的H1355、CL 1-0及CH27細胞株之細胞週期，發現RAI3不會影響細胞凋亡且增加生長能力的細胞其G2/M期百分比會隨之上升，而生長能力被抑制的細胞其G2/M期百分比則隨之下降，而其中干擾RAI3表現的CL 1-0細胞株的S期百分比另有下降的情形。接著以西方墨點法偵測細胞週期調控蛋白的表現，結果隨著細胞株的不同影響G2/M期的蛋白表現也跟著有所差異。由以上結果可推論RAI3在肺癌細胞株中可能扮演抑癌基因的角色。我們繼續探討RAI3對藥物敏感性及細胞轉移的影響，我們先以臨床常用抗癌藥物paclitaxel及gemcitabine處理細胞，結果不論是干擾RAI3或促進RAI3的細胞株對此二抗癌藥的敏感性都沒有改變；而在細胞移行試驗及細胞侵潤試驗中，我們發現RAI3的存在可以抑制肺癌細胞的移行能力及侵潤能力，但只在H1355及CH27細胞株觀察到此現象。最後我們觀察MAPKs及Akt的活化情形，發現在不同的細胞株其活化的情形也不一樣，這些現象與RAI3的抑癌功能之間的關係仍需再進一步探討。總括言之，RAI3基因在部份肺癌細胞株中可能扮演抑制細胞生長、轉型及轉移能力的角色。

關鍵字

G蛋白偶合受體 ； 肺癌細胞株 ； 非附著性細胞生長 ； 細胞週期 ； 細胞移行 ； 細胞侵潤

並列摘要

We and other research teams have discovered that RAI3 (retinoic acid induced-protein 3) gene has higher expression level in normal lung tissue than in lung tumor tissue by cDNA microarray. Also, we confirmed this result with RT-real time PCR. For this reason, we suppose RAI3 gene is a tumor suppressor gene. But some references pointed out that RAI3 is a oncogene in breast cancer cells. In this research, we will investigate the role of RAI3 gene in lung cancer cells. First of all, we found out when we use shRNA RAI3 to suppress RAI3 expression, it would increase cell growth. On the other hand, when we use HA-RAI3 to promote RAI3 expression, it would decrease cell growth. According to anchorage-independent growth assay, reduced RAI3 expression has better effect in cell transformation and enhanced RAI3 expression has worse effect in cell transformation. However, RAI3 does not have any effect on the cell growth and cell transformation in H1299 and F4 cell lines. We observed cell cycle of H1355, CL 1-0 and CH27 cell lines which RAI3 gene could affect their cell growth. RAI3 gene does not have any effect on the apoptosis, but the percentage of G2/M phase would increase in shRNA RAI3 transfected cells and it would decrease in RAI3 overexpressed cells. In addition, the percentage of S phase would reduce in CL 1-0 cell line transfected with shRNA RAI3. And then, we continually observed the expression of cell cycle regulated proteins. Unfortunately, the expression profile of these cell cycle regulated proteins was different in three RAI3 regulated lung cancer cell lines. Moreover, the effects of RAI3 on the sensitivity to chemotherapy and metastasis were investigated. In the anti-tumor drug sensitivity study, lung cancer cells were treated with paclitaxel and gemcitabine. The results indicated that RAI3 won’t affect the drug sensitivity to paclitaxel and gemcitabine. In the experiments of metastasis, we found that RAI3 could reduce migration and invasion of lung cancer cells. But we only observed these phenomena in H1355 and CH27 cell lines. Finally, the activities of MAPKs and Akt were also different in RAI3 regulated cell lines. The relationship between above phenomena and function of RAI3 need further study. In conclusion, RAI3 may play a role in reducing cell proliferation, cell transformation and metastasis in some lung cancer cell lines.

並列關鍵字

RAI3 ； G protein-coupled receptor ； lung cancer cell lines ； anchorage-independent growth ； cell cycle ； migration ； invasion

參考文獻

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