長鏈非編碼核糖核酸lnc-fibrogen在心臟纖維化與心臟肥厚的角色

Abstract

心臟疾病是全球致死率最高之疾病之一，由心臟疾病所引起心臟衰竭每年全球發生率超過2300萬人，五年的死亡率高達10%，甚至比許多癌症更嚴重。當心肌受損後會伴隨過多的細胞外間質(Extracellular matrix, ECM)累積，造成心臟纖維化(Cardiac fibrosis)使心臟功能喪失而最終導致心衰竭(Heart failure, HF)。然而目前並沒有能夠針對心臟纖維化做治療之藥物，因此目前急需要找到可以有效抑制心臟纖維化的治療方式。 隨著基因學的進步，目前已知的人類基因有百分之九十以上會被轉錄為RNA，然而其中能被轉譯為蛋白質的RNA只佔了大約百分之一。其餘無法被轉錄的RNA包含了小分子核糖核酸(microRNA, miRNA)以及長鏈非編碼核糖核酸(Long non-coding RNA, lncRNA)，目前已有許多研究指出長鏈非編碼核糖核酸與許多心臟疾病及心臟的發育相關。 因此透過RNA定序技術(RNA sequencing)，我們從人類心衰竭之樣本找出與心臟纖維化有高度相關的lncRNA—lnc-fibrogen，目前的研究成果發現lnc-fibrogen能夠藉由調節microRNA—miR-29a來影響心臟纖維化之病理過程。透過抑制lnc-fibrogen的表現減少心臟纖維化的機轉，未來有機會可以做為治療心臟纖維化之治療標的。 Lnc-fibrogen 是主要存在於人類心臟纖維母細胞(HCF)的長鏈非編碼核糖核酸，我們發現再給予TGF-β1刺激後，人類心臟纖維母細胞中lnc-fibrogen含量顯著增加。抑制lnc-fibrogen在HCF中的表現量可以減少TGF-β1刺激所引起的纖維母細胞活化，以及ECM 相關基因的表現量增加。另外，在HCF使lnc-fibrogen過過度表現會造成ECM相關基因，包含COL1A1以及ACTA2 的表現量增加、細胞增生、以及促進肌纖維母細胞(Myofibroblast)形成。Lnc-fibrogen主要分佈在細胞質(Cytosol)當中，並且我們發現lnc-fibrogen能夠使細胞質中ECM相關基因的穩定性降低。接著我們利用相關的計算分析方法預測出可能會有交互作用的小分子核糖核酸(microRNA, miRNA)，miR-29a。在先前的研究中指出miR-29a與心臟纖維化相關，因此後續便進一步針對miR-29a與lnc-fibrogen之間的機制做研究。利用小分子核糖核酸定序(MicroRNA sequencing)我們發現抑制lnc-fibrogen表現後miR-29a的表現量顯著下降。透過RNA pull-down assay 我們證明了lnc-fibrogen能與miR-29a直接結合，此外，我們藉由luciferase reporter assay的方法證明了lnc-fibrogen與miR-29a之間結合的專一性，並且miR-29a能夠有效抑制lnc-fibrogen的表現量。並且，當我們將lnc-fibrogen與miR-29a之間的專一結合序列改變，lnc-fibrogen與miR-29a的專一結合性便會消失。隨後，給予miR-29a的target site blocker 抑制miR-29a與lnc-fibrogen之間的結合，我們發現target site blocker 可以有效抑制lnc-fibrogen過度表現所造成的ECM相關基因的表現量下降。同樣地，為了瞭解lnc-fibrogen在生物體內(in vivo)的功能，我們利用CRISPR/Cas9的基因編輯技術製造了lnc-fibrogen-/-的小鼠。在動物實驗中也發現lnc-fibrogen-/-的小鼠可以有效減少血管收縮數血管收縮素(angiotensin II, AngII)所導致的心臟肥厚(cardiac hypertrophy)、心功能不全(cardiac dysfunction)、心臟纖維化及相關ECM基因的表現的表現量增加。更重要的是，我們也發現miR-29在野生型(wild-type, WT)C57BL/6J老鼠給予一個月的血管收縮素後會顯著減少，而lnc-fibrogen-/-小鼠的miR-29a減少情形有明顯改善。因此，我們驗證了lnc-fibrogen在生物體內也具有相同促進纖維化的作用。 總結來說，從我們的實驗結果可以發現大量表現在心臟纖維母細胞的長鏈非編碼核糖核酸—lnc-fibrogen 可以藉由扮演抗心臟纖維化小分子核糖核酸miR-29a之micro RNA sponge進而促進心臟纖維化。因此藉由探討lnc-fibrogen參與在心臟纖維化過程中的機轉，或許可以作為人類心臟纖維化疾病的治療標的。

Introduction: Heart failure (HF) is one of the leading causes of death worldwide, with a 5-year mortality rate of ~10%, worse than many cancers. The loss of myocardium following cardiac injury is compensated by the excessive production of extracellular matrix (ECM) and the formation of a collagen-rich fibrotic scar. Scar formation, tissue remodeling, and progressive interstitial fibrosis lead to a severe loss of function and ultimately HF. There is, however, no effective therapy to prevent or reverse the progression of cardiac fibrosis. There is a clear need to identify novel mediators/pathways underlying cardiac fibrosis to develop effective therapeutics. With the advances in genomic medicine, it is now known that more than 90% of the human genome is transcribed into RNA while the expression of messenger RNAs (mRNAs) and microRNAs (miRNAs) account for only~1％ of all transcribed species, up to 90％ of mammalian genome is transcribed as long non-coding RNAs (lncRNAs). Furthermore, studies have shown that lncRNAs are critically involved in cardiac development and diseases. Leveraging next-generation RNA sequencing on human failing myocardium, we have identified one cardiac fibroblast-enriched lncRNA, lnc-fibrogen, which is dysregulated in failing heart and its expression levels are highly correlated with that of cardiac fibrosis genes. Purpose: To test the hypothesis that lncRNA lnc-fibrogen plays a critical role for the development of cardiac fibrosis and to decipher the underlying molecular mechanisms Methods and Results: Lnc-fibrogen was specifically enriched in human cardiac fibroblasts (HCF) and was significantly upregulated in response to pro-fibrotic stimuli such as TGFβ1 treatment. Knocking down lnc-fibrogen in HCF prevented TGFβ1-induced fibroblast activation and ECM gene production. In addition, overexpression of lnc-fibrogen was sufficient to result in ECM gene, including COL1A1 and ACTA2, up-regulation, cell proliferation and myofibroblast transformation in HCF. Lnc-fibrogen was found to be mainly distributed in the cytosol, where it modulates ECM gene transcript stability. Computational analyses predict the interaction between lnc-fibrogen and miR-29a, a miRNA known to inhibit cardiac fibrosis. MicroRNA sequencing revealed that knockdown of lnc-fibrogen resulted in up-regulation of miR-29a. RNA pull-down assay using biotinylated miR-29a showed strong physical interaction between lnc-fibrogen and miR29a. Also, luciferase reporter assays in HCF using constructs with luc-WT lnc-fibrogen and luc-lnc-fibrogen mutant with miR-29a binding site deletion showed that miR-29a reduced the activity of luc-WT lnc-fibrogen, but not luc-lnc-fibrogen mutant, suggesting the functional interaction between lnc-fibrogen and miR-29a. Importantly, the treatment of miR-29a target site blocker prevented the increment of ECM and HCF proliferation induced by ectopic lnc-fibrogen expression. Furthermore, to understand the biological function of lnc-fibrogen in vivo, we generated lnc-fibrogen-/- mice using CRISPR/Cas9 genome editing technology. We found that AngII infusion for 28 days would marke cardiac hypertrophy and impaired contractility in wild-type C57BL/6J mouse. However, knockout of lnc-fibrogen significantly improved AngII-induced cardiac dysfunction and alleviated cardiac hypertrophy. Moreover, targeted deletion of lnc-fibrogen significantly repressed ECM gene such as Ctgf, Eln, Fn1 expression and cardiac fibrosis after AngII infusion for 28 days compared with wild-type C57BL/6J mouse. Consistent with the results observed in HCF, we observed that lnc-fibrogen-/- mouse had less reduction of miR-29a expression after AngII administration. Taken together, these data suggest that lnc-fibrogen modulates cardiac fibrosis by interacting with miR-29a. Conclusions: The present study identified a novel, cardiac fibroblast-enriched lncRNA lnc-fibrogen, which promotes cardiac fibrosis by sponging anti-fibrotic miR-29a. These results suggest that targeting lnc-fibrogen could be a potential novel therapeutic approach to treat or prevent cardiac fibrosis and heart failure.