探討兒童急性淋巴性白血病相關之微核醣核酸-181A及微核醣核酸-151的功能

Investigating the function of childhood acute lymphoblastic leukemia associated microRNAs: miR-181A and miR-151

顏靜慈(Ching-Tzu Yen)

指導教授 ： 林淑華

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.10345

摘要

第一部分:
急性淋巴性白血病為最常見的兒童癌症，而造成 ETV6/RUNX1 融合基因表現的 t(12;21) 染色體轉位則是在兒童急性前 B 細胞淋巴性白血病中最常見的染色體異常。微核醣核酸是一非編碼的小核醣核酸，長度僅 18–23 個核苷酸，是由 70-100 個核苷酸的微核醣核酸前驅物切割而來，其作用主要是在後基因轉錄階段抑制基因表達。幾乎所有的生理機制都受到微核醣核酸的影響，包括造血細胞分化，甚至已知有部分微核醣核酸參與在白血病癌化過程中。為探討與 ETV6/RUNX1 相關之微核醣核酸，本研究分析 50 個兒童前 B 細胞淋巴性白血病檢體中微核醣核酸的表達情形，其中包含 10 個 ETV6/RUNX1 陽性病例。透過與 ETV6/RUNX1 陰性病人細胞相比較，本研究找到 17 個在 ETV6/RUNX1 陽性病人細胞表達量顯著下降的微核醣核酸。在這些具顯著差異的微核醣核酸之中，由微核醣核酸 181a-1 前驅物 3ʹ 端衍生而來的成熟產物微核醣核酸 181a-1（miR-181a-1），其表現量的改變最具統計意義（下降近百分之七十五，P值 < 0.001）。此外，MIR181A1 基因中具有 RUNX1 蛋白的 DNA 結合位（TGTGGT），因此本研究選擇針對 MIR181A1 進行更深入的探討。
REH 細胞為 ETV6/RUNX1 陽性的前B細胞急性淋巴性白血病細胞株，本研究利用小片段干擾 RNA 抑制 REH 細胞的 ETV6/RUNX1 表達可使 miR-181a-1 表現量上升，此外在人類胚胎腎臟 293FT 細胞中過量表達 ETV6/RUNX1 融合蛋白則可抑制 miR-181a-1 表現量。本研究並以免疫染色質沉澱法（chromatin immunoprecipitation）在 REH 細胞株驗證被預測的 RUNX1 結合位，證明 MIR181A1 直接受到 ETV6/RUNX1 融合蛋白調控。相較於 miR-181a-1，被報導具有功能的是另一股微核醣核酸 181a（miR-181a），為尋找其下游標的，本研究將 REH 細胞轉染 miR-181a 並檢測 PLAG1 表達，已知在慢性淋巴性白血病（chronic lymphoblastic leukemia）中 PLAG1 為 miR-181a 標的。在過量表達 miR-181a 的 REH 細胞中，PLAG1 蛋白表達量下降；而在 ETV6/RUNX1 陽性臨床檢體中 PLAG1 mRNA 表達明顯增加。上述證據皆指出，如同慢性淋巴性白血病研究結果，PLAG1 基因在兒童急性淋巴性白血病亦為 miR-181a 標的。此外，在 miR-181a 轉染之 REH 細胞中 ETV6/RUNX1 蛋白表達量亦顯著下降，且與 RUNX1 3ʹ 端未轉譯區域（3ʹ -untranslated region）上的 miR-181a 辨識序列（UGAAUGU）相關。本研究並利用 REH 細胞證明過量表達 miR-181a 會促進表現 ETV6/RUNX1 之前 B 細胞急性淋巴性白血病細胞由前 BI 階段（pre-BI stage）分化為未成熟 B 細胞（immature B cells）。且 miR-181a 亦可誘導 ETV6/RUNX1 陽性臨床病人檢體 CD10 抗原表達量減少，意即細胞有部分分化的現象。
統整上述研究成果，本研究顯示 MIR181A1 及 ETV6/RUNX1 可相互調控，並推論一涉及 MIR181A1 與 ETV6/RUNX1 的雙向負調控迴圈機制可能參與在由 ETV6/RUNX1 驅動之前B細胞急性淋巴性白血病分化停滯。
第二部分:
微核醣核酸在後轉錄階段可藉由負向調控編碼基因的表現，影響細胞的各種生理層面。目前已知微核醣核酸151會與宿主基因PTK2一起表達，在先前研究中則顯示微核醣核酸151與癌症及心臟肥大症相關，但整體來說，關於微核醣核酸151的報導相當少。儘管已有數個目標基因被發表，包括RhoGDIA、CCNE1、和 ATP2A2基因，微核醣核酸151的生理功能及病理角色仍不清楚。先前針對60個兒童急性淋巴性白血病檢體分析微核醣核酸表達，發現微核醣核酸151在前 B 細胞急性淋巴性白血病中表現量遠高於T細胞急性淋巴性白血病，為了解微核醣核酸151在此現象中扮演的角色，本研究利用基因重組工程技術產製微核醣核酸151 （Mir151）基因剔除小鼠並已確認在DNA及RNA表現上皆為Mir151缺失。本研究並進一步探討Mir151基因剔除鼠的表現型，發現年輕小鼠（小於1歲）的紅血球生成增加並伴隨腎臟紅血球生成素轉錄的上升，此現象在缺氧環境下更為顯著且可能與Hif-α的調控機制相關。在長期觀測結果中，我們發現Mir151缺失並未影響長期存活，但年紀較長的小鼠（大於1.5歲）中，有33% Mir151+/- 及23% Mir151-/-小鼠產生自發性肺癌。本研究並更進一步利用urethane在年輕小鼠誘導肺癌產生，結果卻與預期不同，Mir151 缺乏在年輕小鼠身上具有保護作用。
統整上述結果，本研究揭露了微核醣核酸151先前未被發現的生理及病理角色，並為之後的研究提供新的研究方向。

關鍵字

前B細胞急性淋巴性白血病 ； t(12；21)轉位 ； ETV6/RUNX1 ； 微核醣核酸 181a-1 ； 微核醣核酸 181a ； 微核醣核酸 151 ； 基因剔除小鼠 ； 紅血球生成素 ； 慢性缺氧 ； 自發性肺癌

並列摘要

The first part:
Acute lymphoblastic leukemia is the most common pediatric cancer, and the chromosomal translocation t(12;21), which resulting in expression of ETV6/RUNX1 fusion gene, is the most frequent chromosomal lesion in childhood B-cell precursor (pre-B) ALL. MicroRNAs (miRNAs) are small noncoding RNAs with 18–23 nucleotides arisen from cleavage of 70-100 nucleotide precursors and mostly down regulate gene expression at post-transcriptional level. They have been implicated in virtually all aspects of biology including hematopoietic cell differentiation and some of them are also known to participate in leukemogenesis. To investigate the miRNAs that are associated with regulation of ETV6/RUNX1 expression, we performed miRNA expression profiling on fifty leukemic samples from children with pre-BALL, including 10 cases positive for ETV6/RUNX1. We identified 17 miRNAs that were down-regulated in ETV6/RUNX1-positive, compared with ETV6/RUNX1-negative B-ALL. Of these miRNAs, miR-181a-1, one of the mature form derived from the 3ʹ arm of precursor hsa-mir-181a-1, gives the most significant fold-change (reduced by ~75%, P<0.001). In addition, MIR181A1 contains a potential RUNX1 binding site (TGTGGT), thus we selected MIR181A1 for further investigation.
In REH cells, an ETV6/RUNX1-positive B-ALL line, siRNA knockdown of ETV6/RUNX1 resulted in increased miR-181a-1 expression, while overexpression of ETV6/RUNX1 fusion protein in HEK-293FT cells resulted in reduction of miR-181a-1 level. The predicted RUNX1 binding site was also confirmed in REH cells by chromatin immunoprecipitation analysis, indicating MIR181A1 is a direct target of the ETV6/RUNX1 fusion protein. To search for downstream targets of miR-181a, the functional counterpart of miR-181a-1, REH cells were transfected with miR-181a and the expression of PLAG1, shown to be a target of miR-181a-1 in chronic lymphoblastic leukemia (CLL) was examined. The PLAG1 protein level was decreased in miR-181a over-expressed REH cells. In addition, the PLAG1 mRNA level was increased in ETV6/RUNX1-positive clinical samples. This indicated that PLAG1 gene might be the down-stream target of miR-181a in childhood ALL as in CLL. Furthermore, we found ETV6/RUNX1 protein was also decreased in miR-181a-transfected REH cells, correlating with the existence of a miR-181a recognition sequence (UGAAUGU) at the 3’-untranslated region of RUNX1. Using REH cells, we showed ectopic expression of miR-181a could enhance ETV6/RUNX1-expressing B-ALL cells differentiate from pre-BI stage to immature B cells. In addition, miR-181a could induce partial differentiation of ETV6/RUNX1-positive clinical patient samples by diminishing CD10 expression.
Taken together, our results demonstrate that MIR181A1 and ETV6/RUNX1 regulate each other, and we propose that a double negative loop involving MIR181A1 and ETV6/RUNX1 may contribute to ETV6/RUNX1-driven differentiation arrest in B-ALL.
The second part:
MicroRNAs (miRNAs) are the major key players to negatively regulate the expression of coding genes in post-transcriptional level and control almost all aspects of biology of the cells. MIR151A expresses a miRNA that co-expresses with PTK2 gene and has been reported to be involved in cancers and cardiac hypertrophy. The description about MIR151A in previous reports is rare. Although several target mRNAs including RhoGDIA, CCNE1, and ATP2A2 have been identified, the physiological function and pathological role of MIR151A1 remains a puzzle. We have performed a miRNA expression profiling on 60 childhood ALL patients and identified miR-151 to be differentially expressed in B-ALL. To elucidate the role of miR-151 involved in this phenomenon, we generated and the Mir151 conventional knockout mice using recombineering technology and confirmed the deficiency of Mir151 gene on both DNA and RNA level. We further characterized phenotypes of Mir151 knockout mice and found in young mice (< 1year) an increased erythropoiesis concordant with the elevated renal Epo transcription, which was more prominent under hypoxic and may associate with a Hif-α-regulated mechanism. In elder mice (>1.5 year), we found that Mir151 deficiency did not affect the long-term survival, whereas spontaneous lung tumors were developed in 33% Mir151+/- and 23% Mir151-/- mice. We further performed a urethane-induced lung cancer model on young N10F2 mice, however, an unexpected protective effect of was exerted in young mice deficient in Mir151.
Taken together, our results reveal the undiscovered physiological and pathological role of MIR151A, which may provide new aspects for future research.

並列關鍵字

pre-B ALL ； t(12；21) translocation ； ETV6/RUNX1 ； miR-181a-1 ； miR-181a ； miR-151 ； genetic knockout mice ； erythropoietin ； chronic hypoxia ； spontaneous developed lung cancer

參考文獻

第一部分

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