研究Peripheral myelin protein 22基因對人類肺癌細胞株生長的影響

PMP22 (Peripheral myelin protein22)基因也就是Growth arrest – specific-3 (GAS3)基因，位於染色體17p11.2，包含6個exons，為周邊神經系統髓鞘的結構蛋白。PMP22在細胞的增生、分化、細胞與細胞之間和細胞與基質之間的交互作用，扮演一個很重要的角色。PMP22 mRNA在肺癌病人之正常肺組織的表現量較肺腫瘤組織高約2~45倍不等(附圖一)。在CL1系列的細胞株中，CL1-0之PMP22的表現量最低。因此我們在CL1-0肺癌細胞中過度表現PMP22基因，觀察PMP22蛋白對細胞生長、死亡、細胞群落的形成、細胞週期的分佈及細胞週期調控蛋白的表現有什麼影響。由實驗結果發現，當PMP22基因過度表現時，會使CL1-0細胞有多核的現象產生。由MTT assay和細胞計數結果得知，過度表現PMP22會抑制CL1-0細胞的生長。由流氏細胞儀結果顯示，過度表現PMP22會使CL1-0細胞比CL1-0 parental及vector control的G1、G2/M期減少，S phase增加。進而利用西方點墨法偵測細胞週期調控蛋白的表現量，結果發現過度表現PMP22會使細胞調控蛋白CDKs (cyclin-dependent kinases)表現量減少，其中CDK1的表現量會隨著PMP22表現量的增加而降低。暗示PMP22會直接或間接調控CDK1的表現。接著利用RT-real time PCR得知PMP22對CDK1 mRNA的表現並無影響，因此PMP22對CDK1的影響並不是在mRNA level。此外，也發現過度表現PMP22時，會使細胞p21、p53的表現量增加及pRb的表現量減少。當過度表現PMP22時，會使得CL1-0細胞形成群落的初期比CL1-0 parental及vector control鬆散，不貼附生長( anchorage-independent growth )的能力降低，並可由西方點墨法發現β- catenin表現量的下降。可見PMP22會影響細胞的cell-cell interaction或cell-cell junction，而其作用機制還需要進一步探討。由以上的結果推論，PMP22基因在肺癌細胞CL1-0中可能扮演一個抑癌(tumor supreesor)的角色，其詳細的作用機轉及影響路徑仍有待探討。之後，可以從過度表現PMP22對CDK1後轉譯修飾的影響，更進一步研究之後的作用機轉及影響路徑來觀察PMP22對肺癌的影響。

關鍵字

PMP22

並列摘要

Peripheral myelin protein 22 (PMP22), also known as Growth arrest-specific-3 (GAS3) gene, is located at chromosome 17p11.2. PMP22 is composed of six exons and is a structural protein of the myelin sheath in the peripheral nervous system (PNS). The PMP22 gene plays an important role in cell proliferation, differentiate, cell-cell interactions and cell-matrix interaction. The mRNA level of PMP22 was 2 to 45 fold higher in normal lung than in lung cancer. The PMP22 expression level of CL1-0 is the lowest in CL1 series cell lines. We overexpressed PMP22 in CL1-0 to observe the effect of PMP22 on cell growth, apoptosis, colony formation, cell cycle distribution and expression of cell cycle regulators. When PMP22 is overexpressed in CL1-0 cells, 500μg/ml of G418 resistant cells developed mutinuclei, while this phenomenon disappeared in stable clones. MTT assay and cell counting data showed that overexpression of PMP22 decreased cell growth. Flow cytometry analysis showed that overexpression of PMP22 decreased the percentage of G1 and G2/M phase cells while increased S phase cells. We used Western blot analyses to examine the expression level of cell cycle regulators (CDKs). Overexpression of PMP22 decreased protein levels of CDKs. Among them, the expression of CDK1 decreased with the increasing expression level of exogenous PMP22, suggesting that PMP22 reduces CDK1 expression directly or indirectly. To assess whether PMP22 overexpression would decrease CDK1 by decreasing its mRNA level, RT-real time PCR was performed and showed that overexpression of PMP22 did not affect the mRNA level of CDK1. In addition, overexpression of PMP22 increased the protein level of p21, p53 and decreased the level of phosporylated-Rb. When PMP22 was overexpressed, CL1-0 cells formed losser colonies, and decreased the anchorage-independent growth. By Western blot analysis, the expression of β-catenin was decreased in cells overexpressed PMP22, suggesting that PMP22 may affect cell-cell interaction or cell-cell junction. Together, these results indicated that PMP22 might acts as a tumor suppressor in lung cancer. The detail mechanisms and pathways involved in tumor suppressor activity of PMP22 are required for further investigation.

並列關鍵字

PMP22

參考文獻

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研究Peripheral myelin protein 22基因對人類肺癌細胞株生長的影響

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