微型核醣核酸調控牙齦增生的病理機制

Abstract

牙齦體積的增加稱為牙齦增生或牙齦腫大，是一種常見的口腔徵狀，但是自發性或藥物誘發性牙齦增生的患者，疾病復發的風險卻成為牙周病科醫師在臨床治療上的一大難題。此種疾病在組織學上的特徵為：上皮細胞層數有增厚的發現，並呈現明顯的棘狀凸出延伸至結締組織中，在結締組織中則有大量膠原蛋白堆積而產生纖維化；然而引發牙齦增生的病理機制至今仍不明確。微型核醣核酸，它是一類全長具有約22個核苷酸的單鏈非編碼小分子核醣核酸，主要透過與target基因3’UTR的完全或不完全配對，降解target基因的mRNA或抑制其翻譯成蛋白質，進而調控個體發育、增殖、細胞凋亡…等重要功能。本實驗的目的是探討是否有特定的微型核醣核酸介入調控牙齦增生的病理機制，以及影響下游相關target mRNA的表現。首先，在初步實驗中利用遺傳性牙齦纖維瘤病患者的牙齦組織進行了微型核醣核酸以及基因表現的微陣列分析，建立了龐大的資料庫，並且證明在增生的牙齦組織中，某些特定的微型核醣核酸及基因有特異的表現；因此，我們加入另外兩位患有牙齦增生的組織檢體來分析，共同進行數據的比對，減少個體表現差異的干擾；結果顯示：共有六種微型核醣核酸在牙齦增生的實驗組中具有一致的表現趨勢，包括miR-203, -143, -145, -200a, -200b, -923。進一步，我們利用同部定量-反轉錄聚合酶連鎖反應來驗證，共包含九位受試者，其中實驗組有五位患者，因服用抗癲癇的藥物合併牙齦增生的發生；另外四位對照組的患者，因患有全口慢性牙周病需要進行切除性牙周手術，並無服用任何可能誘發牙齦增生的藥物。在簽署檢體分析的同意書後，取得牙齦檢體，考量微型核醣核酸在不同細胞中的表現差異大，為避免上皮層與結締組織之間不同細胞表現的干擾，我們將檢體分離成上皮層與結締組織層後，才分別進行微型核醣核酸的分析比較；並且利用初步實驗中微陣列分析所建立基因表現的資料庫，來檢視下游target mRNA的表現是否有變化。實驗結果顯示：臨床發炎程度相近的牙齦組織中，實驗組的miR-203, -143, -145, -200a, -200b在上皮層的表現，都是較對照組來的降低，並且達到統計上的顯著意義；而在結締組織層中，則有miR-143, -145，在實驗組的表現較對照組降低，並達到統計上的顯著意義。此外，miR-203調控的下游target p63，無論在mRNA的表現或是組織蛋白質的染色，在實驗組中均有顯著的上升，證明miR-203的表現下降，的確具有生物功能調控的能力及意義；然而anti-oncomir：miR-143及miR-145的表現抑制，也帶動細胞數量的增加，呼應牙齦增生此疾病在臨床上及組織學上的特徵；miR-200 family藉由調控ZEB1以及SIP1介入上皮-間質轉化過程，包括E-cadherin表現的下降，SIP1的上升，使得細胞型態產生變化，都與本實驗結果獲得一致性的結論。雖然微型核醣核酸對於牙齦增生的影響，在生物功能的實際調控機制上，仍需要更多的實驗來證明；但是本實驗創新的以微型核醣核酸來分析牙齦增生的病理機制，並建立牙齦組織中微型核醣核酸表現的資料庫，的確為未來的研究開拓了一個新的方向。

The increase of size in gingiva, named gingival overgrowth or gingival enlargement, is a common feature of gingival disease. Risk of recurrence in the patients with idiopathic or drug-induced gingival overgrowth is really a challenge to a periodontist. And the overgrowth lesions are characterized by epithelial acanthosis, with long, branched rete peg formation, and the excessive accumulation of radiating collagen fibers in the connective tissue layer. But the etiology and pathogenic mechanism of gingival overgrowth are still unclear. microRNAs (miRNAs) are a family of small non-coding RNA molecules of about 22 nucleotides in length, which involved in post-transcriptional gene regulation and regulated development, proliferation, apoptosis…etc. miRNAs control the expression of target genes by inhibiting translation or degradating target mRNAs through binding to their 3’UTR. The present study demonstrated the gingival overgrowth is characterized by specific miRNAs expression profile. In the preliminary study, we established a databank by using microarray analysis of miRNAs and gene expression from one patient with inherited gingival fibromatosis. Now, we expanded the sample size to three to decrease the interference of individual predisposition, and we successfully identified 6 specific miRNAs with consistent expression tendency in the overgrowth gingival tissues, including miR-203, -143, -145, -200a, -200b, -923. Furthermore, we used real time RT-PCR to verify the microarray data. Considering about the expression predominance of miRNAs in different cell types, we separated the gingiva to epithelial and connective tissue layers. We quantified the exression levels of 6 miRNAs in 9 patients (5 patients in experimental group; 4 patients in control group) after informed consent. miR-203, -143, -145, -200a, -200b in the epithelial layer and miR-143, -145 in the connective tissue layer are significantly downregulated in gingival overgrowth samples. The putative target of miR-203 is p63, whose mRNA and protein level expression are significantly increased. Therefore we postulate that miR-203 involves the biological function by regulating p63. The downregulation of anti-oncomir, miR-143 and miR-145, drives the increasing of cell number, which corresponds to the clinical and pathological features of gingival overgrowth. miR-200 family regulates the epithelial-mesenchymal transition (EMT) by targeting ZEB1 and SIP1. In our study, miR-200 family downregulated, TGF-β upregulated, E-cadherin decreased, SIP1 increased, and cell morphology changed, which were similar to EMT process. Although we need more future studies to clarify the exact biological function of miRNAs, we still explore a new direction to interpret the pathogenesis of gingival overgrowth.