MicroRNA-3906藉由微調Homer-1b使斑馬魚快肌內鈣離子濃度維持恆定

Abstract

研究發現，在myf5第一內含子中有了一個miRNA，稱為miR-3906或miR-In300，其標的基因為dickkopf-related protein（dkk3r/dkk3a）。miR-3906藉由抑制Dkk3a蛋白質表現，而抑制宿主基因myf5的啟動子活性，最終成熟體節內的myf5表現生成漸漸減少，使體節細胞繼續走向肌肉分化的命運。然而當宿主基因myf5已經不表現於軀幹部體節內，miR-3906仍然持續表現於體節。另外，以32 hpf斑馬魚胚胎進行LAMP assay並以in vitro/ 及in vivo Dual luciferase reporter system，發現miR-3906能夠結合於homer-1b 3‘UTR。這些結果暗示miR-3906和homer-1b在斑馬魚胚胎肌肉發育過程中可能是具有上下游的關係，但是miR-3906調控homer-1b進而影響晚期肌肉發育的功能仍然不清楚。因此本篇研究主要針對以下兩點進行探討：（1）miR-3906是否能獨立於myf5 transcript且擁有獨立的啟動子；（2）miR-3906和homer-1b對於斑馬魚肌肉發育晚期的生物意義。 為探討miR-3906是否能獨立於myf5 transcript且擁有獨立的啟動子，利用不同長度的myf5外顯子接於luciferase基因之前，並於斑馬魚一細胞時期注射。接著分別收取16、24、32 hpf觀察報導基因的活性，以判別myf5外顯子是否擁有轉錄miR-3906的能力。我們發現myf5外顯子具有啟動子活性，且在myf5不表現時期，其活性更大為增加。這顯示當myf5不表現時期，miR-3906能夠以myf5外顯子作為啟動子開啟表現。另外，針對miR-3906和homer-1b對於斑馬魚肌肉發育晚期的生物意義，我們首先以WISH、q-PCR和Western blot確定miR-3906能夠抑制Homer-1b的表現量。而由於曾有研究指出，Homer family能與鈣離子通道結合，在功能性探討上，我們推測斑馬魚miR-3906能夠過調節Homer-1b表現量參與Ca+2 dependent signal pathways。實驗先在mRNA和蛋白質層次探討miR-3906對於Homer-1b的調控，並針對斑馬魚胚胎24 hpf時，觀察miR-3906和Homer-1b表現量的不正常對於細胞內鈣離子濃度的影響。發現抑制內生性miR-3906或過量表現外源性Homer-1b，會使得細胞內鈣離子濃度上升82.9-97.3%。而過量表現外源性miR-3906或抑制內生性Homer-1b，會使得細胞內鈣離子濃度下降22-29.6%。最後，miR-3906和Homer-1b甚至以影響細胞內鈣離子濃度調控斑馬魚的肌肉發育。在WISH和q-PCR實驗中發現，抑制內生性miR-3906或過量表現外源性Homer-1b，會使得專一快肌分化基因表現量上升。而過量表現外源性miR-3906或抑制內生性Homer-1b，會使得專一快肌分化基因表現量下降。而由於改變miR-3906和Homer-1b表現量所造成的快肌基因的表現缺失，都能夠以彌補或減少鈣離子濃度來進行回覆。顯示miR-3906和Homer-1b確實是以透過影響鈣離子濃度來調控快肌分化基因的表現。除了在基因表現層次上的觀察，我們也發現若是抑制內生性miR-3906或過量表現外源性Homer-1b，會導致肌肉結構的不正常。因此綜合上述結果，當胚胎發育晚期myf5不表現時期，miR-3906能從自身啟動子轉錄出，並接著使Homer-1b維持於正常的表現量，使快肌細胞內鈣離子濃度維持恆定，最終確保快肌的發育過程能夠正常。

Previously we reported an intronic microRNA (miR), miR-In300 or miR-3906, which locates at the first intron of zebrafish myf5 gene, suppresses the transcription of myf5 through silencing dickkopf-related protein 3 (dkk3r/dkk3a) during early development such as 16 hpf when myf5 is highly transcribed. However, when myf5 mRNAs are gradually reduced to undetectable level in mature somites at late developmental stage, miR-3906 is still predominant. We had been performed LAMP assay and in vitro/in vivo luciferase reporter assay, and found that miR-3906 enabled to silence reporter gene fused by homer-1b-3’UTR. The line of these evidences give a clue that (1) miR-3906 may have its own promoter which can transcribe miR-3906 at late stage; and (2) miR-3906 plays an unknown function through regulating homer-1b in muscle development at late stage since Dkk3a is not detected anymore in muscle at that stage. Firstly, we constructed plasmids in which the luciferase gene was driven by various lengths of upstream segment of miR-3906. After luciferase activity assay was performed in embryos, we found that a motif located at +303/+402 was able to activate luciferase activity, suggesting that +303/+402 cis-element possesses a promoter activity when myf5 mRNA does not exist. Secondly, using WISH, q-PCR and western blotting, we confirmed that miR-3906 negatively modulates the expression levels of homer-1b mRNA and Homer-1b protein. Thirdly, we found that the expression levels of fast muscle-specific gene fmhc4 and calcium-sensitive gene atp2a1 were increased, but slow muscle-specific gene smhc1 was unchanged, in the miR-3906-MO-injected and homer-1b-mRNA-injected embryos. The downregulated expressions of fmhc4 and atp2a1 induced by excessive miR-3906 or absent homer-1b could be rescued by adding exogenous homer-1b mRNA. Fourthly, we observed that the sarcomeric actin arrangement and Z disk were disorganized in the fast muscle of miR-3906-MO-injected and homer-1b-mRNA-injected embryos. They also exhibited abnormal swimming abilities. Furthermore, we found that the [Ca2+]i in the fast muscle cells of either knockdown of miR-3906 embryos or overexpression of homer-1b embryos was increased 82.9-97.3% higher than that of control embryos. In contrast, the [Ca2+]i of the excessive miR-3906 and absent homer-1b embryos was decreased 22-29.6% lower than that of control group. Taken together, we concluded that miR-3906, which is transcribed from its own promoter at late developmental stage, controls [Ca2+]i homeostasis in fast muscle through fine tuning homer-1b expression during differentiation in order to maintain normal muscle development during embryogenesis.