- 學位論文
探討與心臟再生能力相關之長鏈非編碼核醣核酸
Identifying long noncoding RNAs linked to cardiac regeneration capacity
摘要
根據美國心臟學會的統計，心血管疾病的盛行率非常高，在年紀大於60歲的美國人中就佔了高達70%到80%的比例。而心血管疾病，特別是冠狀動脈疾病和心衰竭，更是美國的首位死因。這麼高的致死率大多是因為心臟受損後的修復能力十分的差。因此，探討如何促進心臟的再生能力成為心血管研究領域中的重要議題。面對壓力與傷害時，成年哺乳類心肌細胞的細胞分裂與再生能力很有限；相反的，新生哺乳類的心臟細胞卻依然保有心臟再生的能力。例如，出生不超過7天的新生小鼠，其心臟在經過心尖切除手術後可以完全的再生。不過造成新生小鼠和成鼠心臟細胞間再生能力之差別的分子機制並不清楚。長鏈非編碼核醣核酸(Long non-coding RNAs, lncRNAs)，為轉錄長度超過200個核苷酸的非編碼核醣核酸，但不會轉譯成蛋白質。lncRNAs藉由轉錄、附基因調控和轉譯後等調控來參與各種生理及病理現象，然而lncRNAs在心臟再生中所扮演的角色還尚未明瞭。我們假設lncRNAs的表現變化量造成了成鼠及新生小鼠心臟再生能力的差異，因此我們想要探討和心臟再生能力相關的lncRNAs及其相關機轉。 實驗中的細胞RNA分別純化自出生一天（P1, n=3）、三天 (P3, n=4) 之新生小鼠和成鼠的心肌細胞；以及出生一天 (n=2), 三天 (n=2) 的新生小鼠和成鼠 (n=2) 的心臟纖維母細胞。在RNA雙端定序 (Paired End RNA sequencing) 的結果中共發現了18527個信使核糖醣核酸（mRNA）和4775個長鏈非編碼核醣核酸 (lncRNAs)，其中有2895個mRNA（2822上升；73下降）和162個lncRNAs（149上升；13下降）在新生小鼠(P1和P3)和成鼠的心肌細胞中表現量不同。Gene Ontology 分析顯示出有許多基因在細胞分裂以及細胞週期中扮演重要角色，而這也與新生小鼠具有較佳的心臟再生能力這個主張相符合。除此之外，KEGG訊息傳遞路徑分析顯示出這些在出生一天、三天心肌細胞中表現量較高的基因也確實都在細胞週期的傳遞路徑中有所參與。 LncRNA Gm15328 在出生一天之新生小鼠的心肌細胞中高度表現，但是其表現量隨著年紀增加而遞減。Gm15328的表現量和cis-mRNA Cdh2的表現呈現高度的正相關，而Cdh2又曾被報導過能促進細胞生長及增生。 我們進而藉由HL-1心肌細胞以及分離自P1新生小鼠的心肌細胞來探討Cdh2和Gm15328在心臟再生中所扮演的角色。我們觀察到在Cdh2 基因敲低(knockdown)的HL-1細胞中，許多和細胞週期相關的基因表現量下降(包含Cdc16, Cdk4, Pcna)，還有G2/M 細胞週期停滯和蛋白Cyclin B1的表現量下降。另外，在Cdh2 knockdown的P1新生小鼠的心肌細胞中，和細胞週期相關的基因Cdc16的表現量下降，細胞的增生能力也降低。另一方面，Cdh2 過度表達則造成和細胞週期相關基因的表現量上升包含Cdc16, Cdk4, Pcna基因，而Ki-67的染色結果也顯示P1新生小鼠的心肌細胞中Cdh2 過度表達會促進細胞增生。 為了解Gm15328在心臟再生中所扮演的角色，我們進而探討Gm15328 在細胞中的表現位置，RNA螢光原位雜合技術顯示出Gm15328多表達在HL-1和P1新生小鼠心肌細胞的細胞核和核周圍。所以我們認為Gm15328可能會藉由調控Cdh2的表現量來調節細胞的增生。 在動物實驗的部分，我們對出生一天大的小鼠進行心尖切除手術，在被切除心尖的心臟中可以觀察到較高程度的細胞增生，特別是在心尖部位，除此之外，Cdh2 和Gm15328的表現量也都在增生的細胞中比較高，意味著Gm15328可能藉由調控Cdh2而影響心臟的再生。 在小鼠心臟中，Cdh2 和Gm15328 的表現和心臟再生的程度呈高度相關，knockdown Cdh2 造成心肌細胞增生能力下降和細胞週期的停滯，而Cdh2 的過度表現則可以促進心肌細胞的增生。因此藉由探討Cdh2和Gm15328 在心臟再生上所扮演的角色，或許可以為心臟疾病的病人提供新的治療標的。
並列摘要
Background The prevalence of cardiovascular diseases (CVD) is among the highest, up to 70% to 80% for Americans aged ≥ 60 years according to the American Heart Association. In addition, cardiovascular diseases, especially cardiac diseases including coronary artery disease and heart failure, is the number one cause of death in United State. The high mortality of cardiac diseases is largely owing to the poor cardiac regeneration capacity after injury. Therefore, finding means to boost cardiac regeneration has been an important topic in the field of cardiovascular research. Adult mammalian cardiomyocytes (CM) have limited mitotic activity to replenish CM loss in response to stress or injury. Neonatal mammalian CMs, in contrast, retain the capacity to regenerate. Neonatal mouse heart, for example, can fully regenerate from apical resection by 7 days old. The molecular determinants underlying the disparate regenerative capacity between neonatal and adult CM, however, remain unknown. Long non-coding RNAs (lncRNAs), a heterogeneous group of non-coding transcripts longer than 200 nucleotides, have been shown to be functional and involved in specific physiological and pathological process through epigenetic, transcriptional and posttranscriptional mechanisms. However, the roles of lncRNAs in cardiac regeneration remain unclear. We hypothesized that differences in long non-coding RNA (lncRNA) expression in neonatal and adult CM could account for the observed disparity in their ability to regenerate. Purpose This study aimed to identify lncRNAs that are linked to cardiac regeneration and the underlying molecular mechanisms. Methods and Results Total cellular RNA was isolated from adult cardiomyocytes (CM) (n=7), neonatal day 1 (P1, n=3), and day 3 (P3, n=4) CM, as well as adult cardiac fibroblasts (CF, n=2), P1 CF (n=2) and P3 CF (n=2). Pair-end RNA sequencing was performed, where 18527 mRNAs and 4775 lncRNAs were detected. A total of 2895 mRNA (2822 up & 73 down) and 162 lncRNA (149 up & 13 down) were differentially expressed in adult vs P1 & P3 CM. Gene ontology analysis shows strong enrichment in genes involved in cell cycle and mitosis, which is consistent with the notion that neonate heart has better regeneration capacity. Moreover, KEGG pathway analysis revealed that the differentially expressed genes are enriched in components of cell cycle pathway. LncRNA Gm15328 was identified to be strongly expressed in P1 CM but decreased gradually with age; the expression level of Gm15328 show a strong positive correlation with its cis-mRNA Cdh2, which has been reported to promote cell growth and proliferation. We further identified the potential role of Cdh2 and Gm15328 in cardiac regeneration by using HL-1 mouse cardiomyocytes and neonatal day 1(P1) mouse ventricular CM as an in-vitro model. We found that knockdown of Cdh2 in HL-1 mouse cardiomyocytes led to reduced transcript expression of cell cycle related genes such as Cdc16, Cdk4 and Pcna as well as G2/M cell cycle arrest and decreased the protein expression of cyclinB1. In addition, decreased transcript expression of Cdc16 and reduced cardiomyocyte proliferation rate were observed in Cdh2 knockdown P1 CM. On the other hand, overexpression of Cdh2 in P1 CM led to increased expression level of cycle related-genes including Cdc16, Cdk4, Pcna and Ccnb1. Moreover, overexpression of Cdh2 promotes cell proliferation in P1 CM, as evidenced by increased Ki-67 staining. In order to understand the potential roles of LncRNA Gm15328 in cardiac regeneration, the cellular localization of Gm15328 was investigated in HL-1 cells and P1 CM. RNA Fluorescence in situ hybridization confirmed that higher expression level of Gm15328 was observed in the nucleus and perinuclear space. We hypothesized that Gm15328 could regulate cardiomyocyte proliferation via modulating Cdh2 expression. For in vivo study, we performed apical resection of neonate day 1 mice. Resected hearts showed higher proliferation level compared to sham group. Among resected hearts, we found more proliferating cell in the regenerating apical area. Moreover, the transcript expression levels of Cdh2 and Gm15328 were also found to be higher in the apical area of resected hearts, implying a potential role of Cdh2 as well as Gm15328 in cardiomyocyte proliferation. Conclusion Expression of Cdh2 and LncRNA Gm15328 showed positive correlation with cardiac proliferation level in the mouse heart. Knockdown of Cdh2 led to reduced cardiomyocytes proliferation and resulted in cell cycle arrest, whereas overexpression of Cdh2 promote cardiomyocytes proliferation. Cdh2 and Gm15328, therefore, could be potential therapeutic targets to promote cardiac regeneration in heart diseases.