探討AHR-KO 促使A549細胞再編程具有抗纖維化效果的作用機制

Abstract

誘導多能性幹細胞 (iPSCs) 為細胞較原始的狀態，可以重啟細胞再編程 (cell reprogramming) 分化成不同胚層。先前研究發現將細胞過量表達轉錄因子OSKM (Oct3/4、Sox2、Klf4、c-MYC) 後，已分化細胞重啟多能性，使其成為具有幹細胞特性之iPSCs。多環芳香烴受體 (Aryl hydrocarbon receptor, AHR是受戴奧辛等配體調控的轉錄因子，在藥物代謝和致癌反應中，扮演關鍵的角色。內生性AHR調控許多生理功能，像是細胞增生、細胞型態、細胞附著以及細胞移行，近來則發現AHR具有負調控細胞多能性的新穎功能，但其機制尚未清楚。我們在A549肺癌細胞株中將AHR基因以CRISPR/Cas9技術進行基因編輯加以剔除後，發現細胞具有類似iPSC的特性：在評估多能性 (Pluripotency) 的代表試驗─類胚體 (embryoid bodies) 的產生中，發現AHR-KO後會產生直徑較大且形態完整的類胚體；進一步在免疫螢光染色實驗中發現，AHR-KO細胞中代表多能性標誌的醣蛋白SSEA4和TRA-1-60，不論是在細胞質和細胞核中都較野生型A549表現多，且在多能分化性基因Oct4、Sox2、Klf4、c-Myc和Nanog的表現都有顯著增加的情況。由於細胞處於幹細胞狀態時，分化能力會受到限制，接著以MTT試驗方法觀察細胞生長的現象：發現AHR-KO細胞增生速率下降。此外在調控細胞多能性和細胞生長的TGF/Smad路徑中發現：在AHR基因剔除後，促纖維化的Smad3及磷酸化表現抑制；抗纖維化的Smad2 及磷酸化表現活化，在給予TGF纖維化刺激因子的情況下，AHR-KO細胞的移行能力和膠原蛋白的轉錄活性皆較野生型A549大幅減弱，顯示具有抗纖維化的特性。我們的研究結果顯示：A549經過CRISPR/Cas9方法AHR基因剔除後，細胞會再編程，成為具誘導性多能幹細胞特性的細胞，且具有抗纖維化的特性。

Induced pluripotent stem cells (iPSCs) were initially generated by introducing reprogramming factors via integrating viral vectors. Nonetheless, there was a clinical concern about iPSCs on account of the potential for insertional mutangenesis caused by the integration on transgenes into the genome of host cells. To make iPSCs more clinically available, various non-inegrating reprogramming methods have been developed to circumvent the risk of insertional mutagenesis and genetic alterations. While sensing the outer cell environment, the aryl hydrocarbon receptor (AHR) involved in embryonic development has a fine-tuned pattern to regulate the stemness in cell. Here, we present that AHR-Knockout (AHR-KO) by CRISPR/Cas9 system triggers cell reprogramming into iPSCs. The use of integration-free methods could avoid genomic insertion, therefore reducing risk when human iPSCs are used for clinical applications. In the first place, we employed CRISPR/Cas9 system to establish the AHR-KO clone from A549. Intriguingly, we found that AHR-KO cells had the characteristic of IPSCs. With the classical cell pluripotency methods, embryoid bodies (EB) formation assay, It showed that AHR-KO cells formed more complicated EB. We also found that AHR-KO presented more pluripotency marker, SSEA-4 and TRA-1-60 in the cell membrane or cytosol. Moreover, pluripotency gene expression level increased in AHR-KO cells. Owing to stem cell specific characteristics imply low cell growth rate, we found the cell proliferation rate decreased in AHR-KO cells. Furthermore, we analyzed the cell behavior, migration ability decreased in AHR-KO cells. Smad2 protein expression urged in AHR-KO cell, and Smad2 phosphorylation protein expression also increased; however, Smad3 and Smad3 phosphorylation protein expression declined. In the previous studies showed that Smad3 was participated in lung fibrosis progression. In order to assess fibrosis amelioration capability, we used wt-A549 and A549-AHR KO cells treated with transforming growth factor- (TGF-) to induce fibrosis. We suggest that Smad 2 and Smad2 phosphorylation protein expression decreased in AHR-KO cells. We may develop reprogrammed cell by CRISPR/Cas9 against tissue fibrosis.