探討miR-145-5p在非小細胞肺癌產生愛寧達抗藥性機制所扮演的角色

肺癌長年居於國人癌症死亡首位，其中非小細胞肺癌 (non-small-cell lung cancer, NSCLC)更是佔了肺癌的85％。Pemetrexed (Alimta)是一種葉酸抑制劑被用於治療局部晚期或轉移性非小細胞肺癌的二線以上化療藥物。然而長期使用pemetrexed治療可能會造成癌細胞對藥物產生抗藥性。其中，Thymidylate synthase (TS)的高表達被認為是肺癌細胞對pemetrexed藥物產生抗藥性的主要機制，不過有臨床發現可能有其他途徑會導致抗藥性的產生。 在本研究中發現miR-145-5p在pemetrexed抗藥性A549細胞 (稱為A400細胞)中表現較A549細胞低。透過siRNA轉染方式將miR-145-5p mimic送入A400細胞使細胞中的miR-145-5p高表達，抑或是透過miR-145-5p抑制劑抑制A549細胞中miR-145-5p的表現皆會使細胞對於pemetrexed藥物的敏感性受到影響。另外，我們也發現在pemetrexed抗藥株A400細胞中，細胞的BMI1 (B Lymphoma Mo-MLV insertion region 1 homolog)、Sp1與TS蛋白表現都比A549細胞來的高。透過TargetScan微小RNA預測網站，我們發現miR-145-5p可能會與Sp1的3端非轉譯區(3’-UTR)結合。而利用西方墨點法分析非小細胞肺癌中當miR-145-5p表現受到抑制或高表達時，Sp1蛋白表現會受到反向調控。除此之外，透過Sp1 3’UTR報導基因分析，最終我們確立在非小細胞肺癌中，miR-145-5p會與Sp1結合並調控其表現。 另一方面，在A549細胞中當Sp1高表達時，細胞對於pemetrexed藥物的敏感性下降，同時誘導細胞產生上皮間質轉換 (epithelial-mesenchymal transition, EMT)，並上調控EMT相關轉錄因子，如: Snail1、ZEB1與Twist11，並增加細胞轉移能力。更重要的是，我們也發現，當A549細胞BMI1高表達時，Sp1會被上調控，而BMI1的高表達也會誘導A549細胞產生EMT，並且細胞的增生及轉移能力也會增加。然而，透過Sp1 inhibitor (Mithramycin A)抑制Sp1表現或是外送miR-145-5p類似物使miR-145-5p高表達，則都可逆轉此一現象。 總而言之，本研究結果顯示，在非小細胞肺癌中，BMI1表現提升會藉由下調控miR-145-5p以提升Sp1的表現，促使細胞發生EMT，並且減低細胞的藥物敏感性，最終導致pemetrexed的抗藥性。這些結果表明，透過RNA藥物增加細胞中miR-145-5p表現可能可以做為pemetrexed抗藥性非小細胞肺癌患者的敏化劑。

Lung cancers are the leading cause of death among cancer patients in Taiwan and non-small-cell lung cancer (NSCLC) accounts for 85% of all lung cancers. Pemetrexed is an antifolate drug and is the second line chemotherapy drug for NSCLCs. However, the long-term pemetrexed treatment may cause resistance. The high expression of thymidylate synthase (TS) has been suggested to be one of the mechanisms in pemetrexed resistance in cell study but the clinical observations indicate the involvement of other factors.Here, we firstly found that miR-145-5p was decreased in pemetrexed resistant A549 cells (called A400 cells). The overexpression of miR-145-5p by a specific mimic to A400 cells or the knockdown of miR-145-5p by a specific inhibitor to A549 changed the pemetrexed sensitivity of the corresponding cells. We also found the increased expression of BMI1 (B Lymphoma Mo-MLV insertion region 1 homolog), Sp1, and TS in pemetrexed A400 cells. Using TargetScan database, we found that there was a putative miR-145-5p binding site within the 3’UTR (3’-untranslated region) of Sp1 gene. The Sp1 3’UTR reporter assay and western blot analysis of Sp1 protein expression after manipulation of miR-145-5p expression by the transfection of mimic or inhibitor further confirmed that Sp1 is a direct target of miR-145-5p in NSCLC cells.On the other hand, the Sp1 overexpression in A549 cells decreased their pemetrexed sensitivity and induced epithelial-mesenchymal transition (EMT) process by upregulation of EMT related transcription factors, such as Snail1/Twist11/ZEB1, as well as the increased cell migratory ability. We further found that Sp1 could be upregulated and the miR-145-5p expression could be downregulated by BMI1 overexpression in A549 cells. The BMI1-induced cell proliferation, migration or EMT in A549 cells could be abolished by the treatment of Mithramycin A, a Sp1 inhibitor or overexpression miR-145-5p through transfecting miR-145-5p mimic. Taken together, our data suggest that the increased expression of BMI1 in NSCLC cells causes the upregulation of Sp1 through the downregulation of miR-145-5p followed by the promotion of EMT process, which may cause the resistance of pemetrexed. These data also suggest that the RNA drug to increase cellular expression of miR-145-5p is potentially to be developed as a sensitizer for NSCLC patients with pemetrexed resistance.