CLNS1A在肺癌細胞株H1299中引發細胞轉型的分子機制探討

The molecular mechanisms of CLNS1A-induced cell transformation in lung cancer cell line H1299.

王怡文(Yi-Wun Wang)

指導教授 ： 余長澤

國立暨南國際大學/科技學院/應用化學系生物醫學碩士班/碩士(2015年)

https://doi.org/10.6837/NCNU.2015.00243

摘要

CLNS1A (Chloride channel, nucleotide sensitive 1A)是一個多功能蛋白質，屬於volume-sensitive organic osmolyte/anion channel (VSOAC)的一員。CLNS1A 本身不但形成氯離子通道，調控細胞體積的大小，並且與 PRMT5 及 MEP50 形成複合體稱之為 Methylosome ，控制甲基化轉移酶對於其受質的選擇。近年來發現VSOAC 成員參與在多種細胞癌化的過程，如細胞增生、轉移、侵入、血管新生、抗細胞凋亡與不用貼附即可生長之癌細胞特性，而CLNS1A 又是VSOAC 的一員。並且在本實驗室先前的研究已證實 PRMT5 與 MEP50 為致癌蛋白，所以 CLNS1A 可能與癌症形成有關。因此欲瞭解CLNS1A 是否具有刺激細胞轉型之活性，首先利用分析CLNS1A 於肺癌病人組織中的表現量，發現CLNS1A 於肺癌組織中表現量較高。接著將CLNS1A 穩定高量表現及抑制其表現，發現CLNS1A 的確影響肺癌細胞株生長、非固著性細胞生長能力、細胞爬行和動物體內腫瘤大小，顯示CLNS1A 為潛在的致癌蛋白。接著，透過使用PRMT5 與氯離子通道抑制劑去確認CLNS1A 透過哪個途徑造成細胞轉型的能力。實驗結果發現CLNS1A促進細胞生長是透過氯離子通道的調節，至於CLNS1A促進細胞爬行及增進非貼附細胞生長能力則是透過氯離子通道以及PRMT5雙方的調控。

關鍵字

CLNS1A ； VSOAC ； 氯離子通道 ； 細胞轉型 ； 致癌蛋白

並列摘要

Chloride channel nucleotide sensitive 1A (CLNS1A), a multifunctional protein, belongs to a member of volume-sensitive organic osmolyte/anion channel (VSOAC) and is a chloride channel controlling the outward flow of chloride ion. Moreover, CLNS1A is suggested as a component of methylosome with PRMT5 and MEP50, specifying the substrate selection of the protein methyltransferase complex. Importantly, the members of VASOC family have been pointed out to be engaged in several carcinogenesis processes such as cell proliferation, migration, invasiveness, angiogenesis, apoptosis and cell adhesion; however, our published works have shown that PRMT5 and MEP50 work as oncoproteins. Accordingly, it is logically anticipated that CLNS1A should have a role in cancer formation. To rapidly understand whether CLNS1A is involved in cancer development, we have screened the expression of CLNS1A in collected panels of lung cancer tissues by analyzing the level of protein. Overexpression of CLNS1A increases, while silence of CLNS1A decreases, cell migration, cell growth in cultured dishes and nude mice, and anchorage-independent growth in lung cancer cell lines, implying CLNS1A as an oncoprotein candidate. Mechanistic analyses show that inhibition of CLNS1A, or knockdown of PRMT5, attenuated the cell transforming activities of CLNS1A, indicating CLNS1A transforms cells via separated pathways. We observed CLNS1A promotes cell proliferation through chloride channel regulated; while CLNS1A stimulated cell migration and enhance anchorage-independent-growth via Chloride channel and PRMT5 modulated.

並列關鍵字

CLNS1A ； VSOAC ； chloride channel ； cell transformation ； oncogene

參考文獻

參考文獻

Anguita J, Chalfant ML, Civan MM, Coca-Prados M. ( 1995) “Molecular cloning of the human volume-sensitive chloride conductance regulatory protein, pICln, from ocularciliary epithelium.” Biochem Biophys Res Commun. 208(1):89-95.

Azzouz TN, Pillai RS, Däpp C, Chari A, Meister G, Kambach C, Fischer U, Schümperli D. (2005) ”Toward an assembly line for U7 snRNPs: interactions of U7-specific Lsm proteins with PRMT5 and SMNcomplexes.” J Biol Chem. 280(41):34435-40. Epub 2005 Aug 8.

Barbarossa A, Antoine E, Neel H, Gostan T, Soret J, Bordonné R. (2014) “Characterization and in vivo functional analysis of the Schizosaccharomyces pombe ICLN gene.” Mol Cell Bio. 34(4):595-605.

Bond T, Basavappa S, Christensen M, Strange K. (1999) “ATP dependence of the ICl,swell channel varies with rate of cell swelling. Evidence for two modes of channelactivation.” J Gen Physiol. 113(3):441-56.