探討微核醣核酸-135b在肺癌幹性中所扮演的角色

Abstract

肺癌(主要為非小細胞肺癌)是國人及世界上因癌症造成死亡的主要原因之一。其治療的過程中常有許多困難及阻礙，例如：肺癌轉移、癌症復發。近期研究指出腫瘤中的癌幹細胞(CSCs)可能是主要造成原因。微核醣核酸(miRNAs)是小片段且非編碼的(non-coding RNA)的單股內生性RNA，藉由類似RNAi的方式抑制並調控下游基因的表現繼而影響細胞增生、發育及凋亡。特定的微核醣核酸同時也被發現在癌症中扮演抑癌基因或是致癌基因的角色，透過直接或間接調控下游基因群的表現，使正常細胞癌化或是腫瘤惡化。 在實驗室過去的研究發現微核醣核酸-135b(miR-135b)的表現不論在體外(in vitro)或體內(in vivo)的實驗中都與肺癌細胞的轉移能力呈現高度正相關。在本篇論文中，我們發現在非小細胞肺癌細胞株(NSCLC cell line)中過量表現miR-135b能夠增強其幹性及自我更新的能力。相對地，以miR-135b抑制劑(antogmiR-135b)來抑制其表現則減少幹性。透過驗證幹性標記：Oct-4、Nanog、Sox2，也發現這些幹性標記在miR-135b過量表現時會有不同程度的影響。抑制細胞激素訊號四(suppressor of cytokine signaling 4, SOCS4)為我們鑑定的潛在miR-135b標靶之一。我們利用螢光素酶檢測系統驗證SOCS4的下游不轉綠序列(3’-UTR)在miR-135b 存在時，其表現會被抑制。經由逆轉錄聚合酶鏈式反應(RT-PCR)發現內生性SOCS4的訊息核糖核酸(mRNA)並不會受到過量表現的miR-135b影響，然而其內生性蛋白質是否會受到miR-135b的調控仍尚待驗證。利用已建立的HA標記SOCS4的質體(HA-SOCS4 plasmid)，我們發現在NSCLC cell line中過量表現HA-SOCS4能夠抑制其幹性；並可能透過參與在miR-135b所調控的幹性路徑中來抑制其幹性及自我更新的能力。此外我們利用人類細胞激素陣列(human cytokine array)分析，發現在NSCLC cell line中過量表現miR-135b會改變癌細胞原本的細胞激素分泌譜(secreted-cytokine profile)。 綜合以上的研究結果，我們了解miR-135b在調控非小細胞肺癌的幹性中扮演重要的角色，並可能透過改變癌細胞的細胞激素分泌譜而影響其與腫瘤微環境(tumor microenvironment)的交互作用；未來若能更進一步釐清miR-135b調控癌細胞幹性的機轉將有助於發展癌幹細胞標靶治療(CSCs targeting therapy)並幫助肺癌患者。

Dysregulation of microRNAs (miRNAs) had been shown to play a critical role in tumor progression and metastasis. Our lab previously demonstrated an oncogenic miRNA, miR-135b, can promote lung cancer invasion and metastasis. In this study, we identified miR-135b as a promoting cancer stemness miRNA in NSCLC. Overexpression of miR-135b in non-small-cell lung carcinoma (NSCLC) cells promoted sphere-forming ability and its self-renewal ability in sphere formation assay and secondary sphere formation assay. Vice versa, knocked-down miR-135b decreased its sphere-forming ability. The expression of stemness markers: Oct-4, Nanog and Sox2 was affected by overexpression of miR-135b. The suppressor of cytokine signaling 4 (SOCS4) was predicted one of miR-135b putative target. In luciferase reporter assay, luciferase activity was decreased with present of miR-135b. In RT-PCR, endogenous SOCS4 mRNA expression was not affected by overexpression of miR-135b. Overexpression of SOCS4-expressing plasmid tag with HA (HA-SOCS4) in NSCLC cells suppressed sphere forming ability; and may suppress miR-135b-mediated stemness property and self-renewal ability as well. In addition, upon using human cytokine array, we observed that overexpressing miR-135b cancer cells showed different secreted-cytokine profile compared to the non-overexpressing group. Collectively, we understand that miR-135b play an important role in regulation of stemness in NSCLC and may affect the interaction of cancer cells with its microenvironment by modulation of cancer cells secreted-cytokine profile. Eventually, it should be helpful for lung cancer patients to clarify the detail mechanism of how miR-135b regulates cancer stemness and develop CSC targeting therapy in the future.