MiR-192/194在急性肝損傷中的生物學功能

Abstract

藥物過量、膽汁滯留、酒精過量、病毒感染是急性肝損傷的危險因素。目前，治療的功效取決於受損後肝細胞是否能成功再生與正常發揮功能。本論文我們針對miR-192/194在肝損傷中的功能特別感興趣。MiR-192和miR-194高度表達於肝臟中，且與肝臟成熟、非酒精性肝炎的形成有關，並且可當作肝損傷後的血清生化指標；過去的研究指出miR-192/194為P53之下游基因且扮演抑癌基因的角色。此外，miR-192/194已知可透過抑制Mdm2與NF-κB訊息傳遞來抑制癌細胞的生長。臨床研究亦發現，miR-192的降低常見於損傷的肝臟，且細胞株實驗證實可以保護因過氧化壓力造成的損傷。綜上，我們假設調降miR-192/194可以保護肝臟免受肝損傷，並利用miR-192/194基因突變小鼠以證實此假設並研究相關分子機制。 首先，我們發現miR-192/194基因突變小鼠發育正常且肝臟具有正常型態和功能，顯示miR-192/194的缺失不影響正常肝臟功能。接著，我們利用突變小鼠測試因膽汁淤積或藥物誘導造成的肝損傷模型。在膽汁淤積模型中，我們在小鼠身上進行膽管結紮手術(Bile Duct Ligation, BDL)引起膽汁滯留和不可逆之門靜脈病變。在藥物誘導模型中，我們則透過腹腔注射乙醯胺酚(N-acetyl-para-aminophenol, APAP)誘導肝細胞壞死和中央靜脈(central vein)周圍短暫可逆之病變。在這兩種傷害模型中，我們皆發現突變小鼠之肝臟壞死區域較正常小鼠範圍小，且經乙醯胺酚誘導處理後24及48小時的突變小鼠之肝損傷血清生化指標也顯著降低。此結論顯示，降低肝細胞miR-192/194可保護肝臟免受急性肝損傷。 為進一步了解其機制，我們透過西方墨點法及即時定量RT-PCR (qPCR)檢測基因的表現量。首先，在急性肝損傷下，已知為miR-192/194的目標基因TRIM23和NR1H4，其mRNA或蛋白質表現呈現上升的趨勢；有趣的是，此狀況不見於正常之突變小鼠中，顯示miR-192/194的缺失僅在肝臟損傷下影響下游基因表現。此外，許多與細胞週期相關的基因，如CCND1, CDC2等，以及與抗細胞凋亡的基因，如XIAP, ZEB2均於損傷後在突變小鼠肝臟中大量表現，顯示miR-192/194的缺失促進細胞再生與存活，此可解釋為何突變小鼠的肝臟損傷較控制組為輕微。為進一步證實此發現，我們過度表達miR-194於HepG2細胞，發現細胞核內的P65表現量下降，且同為miR-194目標基因及活化NF-κB的上游基因TRIM23和NR1H4，以及與細胞存活正相關的基因，均有不同程度的下降。總結以上的結果，我們認為miR-192/194的基因缺失可能透過上調NR1H4與NF-κB訊息傳遞路徑，以及下游之維持細胞存活的基因，降低小鼠肝臟損傷的程度。

Several risk factors were involved in the development of acute liver injury (ALI), including drug-overdose, bile retention, alcohol overdose, and viral infection. Currently, the efficacy of ALI treatment depends on whether the hepatocytes successfully regenerate and function properly after the injury. Here we investigated the biological role of miR-192/194 in ALI. MiR-192 and miR-194 are both highly expressed in liver and used as serum biomarker for ALI. Previous studies have shown that miR-192/194 function as critical tumor suppressors via stabilizing P53 and repressing NFB signaling in hepatocarcinogenesis. However, its role in the progression of ALI remains to be elucidated. Interestingly, clinical research have shown that down-regulation of miR-192 protects against ALI caused by oxidative stress. We hypothesized that miR-192/194 depletion may protect liver from ALI. MiR-192/194 mutant mice were used to test this hypothesis and study related molecular mechanisms. RNA analysis determined that hepatic miR-192/194 was significantly reduced to below ~99% in mutant mice compared to control. However, histochemical and serum analysis demonstrated that mutant liver without ALI preserved normal structures and metabolic functions in the context of miR-192/194 depletion. Next, mutant mice were tested with cholestasis- and drug-induced liver damages. In cholestasis-induced model, bile duct ligation (BDL) was conducted to induce bile retention and peri-portal lesion. In drug-induced model, necrosis around central vein was induced by intraperitoneal injection of N-acetyl-para-aminophenol (APAP). As a result, mutant mice exhibited lower mortality after BDL and smaller necrotic area after APAP challenge than control mice. Also, serum ALT was significantly less in mutant mice compared to that in control mice at 24 and 48 hours after APAP treatment. These data suggested that miR-192/194 depletion protected liver from acute liver injury. To further understand the mechanism, liver tissues were collected from both mutant and control mice at various time points after APAP treatment to profile gene expression. TRIM23 and NR1H4, two known miR-192/194 target genes, were both increased in mutant mice compared to control mice after APAP-induced injury. Previous studies have shown that TRIM23 and NR1H4 promoted survival of liver cells by positively regulating NFB signaling and cell cycle progression, respectively. Indeed, genes involved in cell cycle regulation, such as CCND1, CDC2, and in anti-apoptosis, such as XIAP and ZEB2, are expressed in a higher extent in mutant livers compared to control after injury, indicating that miR-192/194 depletion promotes cellular regeneration and survival. To verify if the upregulation of these genes in APAP-challenged mutant liver were resulted from miR-194 depletion, ectopic expression of miR-194 in HepG2 cells was performed by transfection and, as expected, leaded to global decrease of genes that were increased in mutant liver. Moreover, miR-194 overexpression reduced nuclear P65 expression in HepG2 cells, which again suggested that miR-194 serves as positive regulator of NFB signaling. Taken together, our study suggested that genetic depletion of miR-192/194 may elevate NR1H4 and NF-κB signaling to promote liver regeneration and survival after ALI.