TWI461540B - The use of micronucleic acid - 96a molecules and micronucleic acid - 196 molecules used in the detection of oral cancer method - Google Patents
The use of micronucleic acid - 96a molecules and micronucleic acid - 196 molecules used in the detection of oral cancer method Download PDFInfo
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本發明係提供一種以微核酸-196a(miR-196a)分子及微核酸-196b(miR-196b)分子運用於偵測口腔癌之方法，尤其係以miR-196a分子及miR-196b分子作為口腔癌的腫瘤標記，係分析潛在病患之生物檢體中miR-196a分子及miR-196b分子的表現量來推斷該潛在病患罹患口腔癌的方法。The present invention provides a method for detecting oral cancer using a micronucleic acid-196a (miR-196a) molecule and a micronucleic acid-196b (miR-196b) molecule, in particular, miR-196a molecule and miR-196b molecule as oral cavity. The tumor marker of cancer is a method for analyzing the expression of miR-196a molecule and miR-196b molecule in a biological specimen of a potential patient to infer the potential patient suffering from oral cancer.
血液腫瘤標誌被認為是一種侵入性低的方式，具有疾病偵測、預後及監控治療結果的價值。因此，一個臨床上應用的血液型腫瘤標誌可以提供更好的疾病控制。現行的血液型腫瘤標誌有偵測大腸直腸癌的CEA、偵測肝癌的AFP與偵測攝護腺癌的PSA。過去也有報導指出SCC抗原與Cyfra21可以作為頭頸癌的偵測標誌，其偵測敏感度分別為SCC 15.5%~38.1%，Cyfra21 38.1%~58.3%[參考文獻1-4]；然而在這些報導中尚未提到有臨床使用的口腔癌偵測標誌。The hematological oncology marker is considered to be an invasive, low-cost way to detect disease, prognose, and monitor treatment outcomes. Therefore, a clinically applied blood type tumor marker can provide better disease control. Current blood-type tumor markers include CEA for detecting colorectal cancer, AFP for detecting liver cancer, and PSA for detecting prostate cancer. In the past, it has been reported that SCC antigen and Cyfra21 can be used as detection markers for head and neck cancer. The detection sensitivity is 15.5%~38.1% for SCC and 38.1%~58.3% for Cyfra21 [References 1-4]; however, in these reports There are no mentions of clinically used oral cancer detection markers.
微核酸(miRNA)是內生性，不轉譯蛋白的小片段RNA，微核酸長度為18-25鹼基對，其利用結合到標的基因訊息RNA的3’不轉譯區域(3’UTR)，進而抑制標的基因轉譯而達到負向調控基因表現。目前已經有700多種人類微核酸被 標註於Sanger miRNABase資料庫中，而預測微核酸的數目會超過1000個。微核酸被認為是一群重要的基因調控分子，估計有1/3~1/2的人類基因會受微核酸調控，此外每個微小RNA亦可以同時調控不同的標的基因。已知微核酸參與多樣的生物功能，如細胞增生，分化，凋亡及移行功能，甚至微核酸表現異常也認為與細胞惡性轉化有關。近年來研究顯示，許多不同的癌症都有其特異的微核酸表現，而在頭頸癌中的微核酸也被分析過，然而，研究卻顯示共同的結果並不多，表示微核酸的複雜性及其為之的機制並未被真正了解。Micronucleic acids (miRNAs) are endogenous, non-translated small fragments of RNA, which are 18-25 base pairs in length and which bind to the 3' untranslated region (3'UTR) of the target gene message RNA, thereby inhibiting The target gene is translated to achieve negative regulation of gene expression. There are currently more than 700 human micronucleic acids Marked in the Sanger miRNABase database, the number of predicted micronuclei will exceed 1,000. Micronucleic acids are considered to be a group of important gene regulatory molecules. It is estimated that 1/3~1/2 of human genes are regulated by micronuclei, and each microRNA can simultaneously regulate different target genes. Micronucleic acids are known to be involved in a variety of biological functions, such as cell proliferation, differentiation, apoptosis and migration, and even microRNA performance is also thought to be associated with malignant transformation of cells. In recent years, studies have shown that many different cancers have their specific micronucleic acid expression, and the micronucleic acids in head and neck cancer have also been analyzed. However, studies have shown that the common results are not many, indicating the complexity of micronucleic acids and The mechanism for it is not really understood.
目前，口腔癌是十大癌症之一，每年約以五十萬人的新病例增加。為了改善醫療成效，提升口腔癌患者的存活率，除了改善治療的方式外，發現並建立一個臨床上可以用來偵測以及監控病程的腫瘤標誌，相信可以有效的減少口腔癌病人的死亡率。Currently, oral cancer is one of the top ten cancers, with an increase of approximately 500,000 new cases each year. In order to improve medical outcomes and improve the survival rate of patients with oral cancer, in addition to improving the treatment, it is found that a tumor marker that can be used to detect and monitor the course of the disease is found to be effective in reducing the mortality of patients with oral cancer.
1. Monteiro E, Varzim G, Crespo M, Araújo L. Cyfra 21.1, TPS and SCC in squamous cell carcinoma of larynx. An Otorrinolaringol Ibero Am 2003;30:467-791. Monteiro E, Varzim G, Crespo M, Araújo L. Cyfra 21.1, TPS and SCC in squamous cell carcinoma of larynx. An Otorrinolaringol Ibero Am 2003;30:467-79
2. Yen TC, Lin WY, Kao CH, Cheng KY, Wang SJ. A study of a new tumour marker, CYFRA 21-1, in squamous cell carcinoma of the head and neck, and comparison with squamous cell carcinoma antigen. Clin Otolaryngol Allied Sci 1998; 23: 82-6.2. Yen TC, Lin WY, Kao CH, Cheng KY, Wang SJ. A study of a new tumour marker, CYFRA 21-1, in squamous cell carcinoma of the head and neck, and comparison with squamous cell carcinoma antigen. Clin Otolaryngol Allied Sci 1998; 23: 82-6.
3. Feng XY, Li JH, Li JZ, Han ZX, Xing RD. Serum SCCA, Cyfra 21-1, EGFR and Cyclin D1 levels in patients with oral squamous cell carcinoma. In t J Biol Markers 2010; 25: 93-8.3. Feng XY, Li JH, Li JZ, Han ZX, Xing RD. Serum SCCA, Cyfra 21-1, EGFR and Cyclin D1 levels in patients with oral squamous cell carcinoma. In t J Biol Markers 2010; 25: 93-8 .
4.Kurokawa H, Yamashita Y, Tokudome S, Kajiyama M. Combination assay for tumor markers in oral squamous cell carcinoma. J Oral Maxillofac Surg 1997; 55: 964-6.4. Kurokawa H, Yamashita Y, Tokudome S, Kajiyama M. Combination assay for tumor markers in oral squamous cell carcinoma. J Oral Maxillofac Surg 1997; 55: 964-6.
口腔癌是世界十大癌症之一， 但目前並無適當之分子腫瘤標誌，可應用於口腔癌的臨床偵測。又，近年來研究顯示，許多不同的癌症都有其特異的微核酸表現，而在頭頸癌中的微核酸也被分析過，然而，研究卻顯示共同的結果並不多，表示微核酸的複雜性及其為之的機制並未被真正了解。Oral cancer is one of the top ten cancers in the world. However, there is currently no suitable molecular tumor marker for clinical detection of oral cancer. In addition, recent studies have shown that many different cancers have their specific micronucleic acid expression, and the micronucleic acids in head and neck cancer have also been analyzed. However, studies have shown that the common results are not many, indicating the complexity of micronucleic acids. Sex and its mechanisms are not really understood.
為了改善醫療成效，提升口腔 癌患者的存活率，除了改善治療的方式外，發現並建立一個臨床上可以用來偵測以及監控病程的腫瘤標誌相信可以有效的減少口腔癌病人的死亡率，故本發明提供一種以微核酸-196a分子及微核酸-196b分子運用於偵測口腔癌之方法，係分析潛在病患之腫瘤組織、血漿、血清、口水或漱口水等生物檢體中miR-196a分子及miR-196b分子的表現量，其中該miR-196a分子具有SEQ ID NO：1所示之序列，該miR-196b分子具有SEQ ID NO：2所示之序列，若miR-196a分子或miR-196b分子的表現量異常高於正常值，即可推斷該潛在病患罹患口腔癌。To improve medical outcomes and improve oral cavity The survival rate of cancer patients, in addition to improving the way of treatment, discovering and establishing a tumor marker that can be used clinically to detect and monitor the course of the disease is believed to be effective in reducing the mortality of patients with oral cancer, so the present invention provides a micronucleic acid -196a molecular and micronucleic acid-196b molecule is used to detect oral cancer. It is a method for analyzing miR-196a and miR-196b molecules in biological tissues such as tumor tissues, plasma, serum, saliva or mouthwash of potential patients. The amount of the miR-196a molecule having the sequence of SEQ ID NO: 1, the miR-196b molecule having the sequence of SEQ ID NO: 2, if the miR-196a molecule or the miR-196b molecule is abnormally expressed Above normal, it can be inferred that the underlying patient is suffering from oral cancer.
又，本發明之分析潛在病患之生物檢體中miR-196a分子及miR-196b分子的表現量之分析步驟為：步驟一：首先取得血漿檢體，將血漿樣分裝出200μl以不會去除small RNA的試劑組來萃取出微核酸，第一步加入700μlQIAzol 使其均質化後再加入140μl氯仿分離萃取RNA，離心之後取上清液(約525μl)加入750μl純酒精沉澱核酸產物，並以試劑中所提供的離心管柱分離出沉澱物，並丟棄下層流出液，在潤洗管柱後，每個樣本以20μl無核酸水解酶(RNase-free)的水將核酸提取出來；步驟二：微核酸的定量，在微核酸的定量方式是以反轉錄定量聚合酶連鎖鍊反應試劑來進行，首先將所萃取的核酸取3μl進行反轉錄反應，加入4單位的反轉錄酶、10單位的核酸水解酶抑制劑及25mM dNTP於總量30μl反應體積內放在37℃培育30分鐘，接著以即時PCR檢測儀進行即時定量PCR，將8μl反轉錄後的產物與1μl特定微核酸探針混和，並加入6μl水和10μl定量PCR試劑後上機，結果以Ct值(threshold cycle)顯示，以相對表現量來呈現結果。Moreover, the analysis step of analyzing the expression amount of the miR-196a molecule and the miR-196b molecule in the biological sample of the potential patient of the present invention is as follows: Step 1: First, obtain a plasma sample, and put the plasma sample into 200 μl so as not to Remove the small RNA from the reagent set to extract the micronucleic acid. Add the first step to the 700 μl QIAzol. After homogenization, 140 μl of chloroform was added to separate the extracted RNA. After centrifugation, the supernatant (about 525 μl) was added to precipitate 750 μl of pure alcohol to precipitate the nucleic acid product, and the precipitate was separated by a centrifugal column provided in the reagent, and the lower layer was discarded. Liquid, after washing the column, each sample is extracted with 20 μl of RNase-free water; Step 2: Quantification of micronucleic acids, quantitative method of micronucleic acid is reverse transcription quantitative polymerization The enzyme chain reaction reagent is used to carry out the reverse transcription reaction by first taking 3 μl of the extracted nucleic acid, adding 4 units of reverse transcriptase, 10 units of nuclease inhibitor and 25 mM dNTP in a total volume of 30 μl of the reaction volume. Incubate for 30 minutes at °C, then perform real-time quantitative PCR with an instant PCR detector, mix 8 μl of the reverse transcribed product with 1 μl of specific micronucleic acid probe, and add 6 μl of water and 10 μl of quantitative PCR reagent to the machine, and the result is Ct ( Threshold cycle) shows the results in relative performance.
本發明透過實驗證明口腔癌病 人血漿中的miR-196a分子及miR-196b分子含量比起正常人有顯著上升(P<0.001,AUC>0.90)，且偵測敏感度達90%，專一性達85%，表示miR-196a分子及miR-196b分子具有良好的口腔癌偵測能力，因此，測定生物檢體中miR-196a分子及miR-196b分子的含量可以用於偵測口腔癌，以往並無適當之口腔癌腫瘤標誌，因此miR-196a分子及miR-196b分子應用於口腔癌的發現與偵測上為一醫學上的突破，且具有良好之功效。The invention proves oral cancer disease through experiments The content of miR-196a and miR-196b in human plasma was significantly higher than that in normal subjects (P<0.001, AUC>0.90), and the detection sensitivity was 90%, and the specificity was 85%, indicating miR-196a. Molecular and miR-196b molecules have good oral cancer detection ability. Therefore, the determination of miR-196a and miR-196b molecules in biopsies can be used to detect oral cancer. There is no suitable oral cancer tumor marker in the past. Therefore, miR-196a molecule and miR-196b molecule are used as a medical breakthrough in the discovery and detection of oral cancer, and have good effects.
有關本發明所採用之技術、手段及其功效，茲舉一較佳實施例並配合圖式詳細說明於后，相信本發明上述之目的、構造及特徵，當可由之得一深入而具體的瞭解。The above-mentioned objects, structures and features of the present invention will be described in detail with reference to the preferred embodiments of the present invention. .
本發明係提供一種以微核酸-196a分子及微核酸-196b分子運用於偵測口腔癌之方法，係分析潛在病患之血漿、血清、口水、漱口水或腫瘤組織等生物檢體中miR-196a分子及miR-196b分子的表現量，其中該miR-196a分子包含具有SEQ ID NO：1所示之序列，該miR-196b分子包含具有SEQ ID NO：2所示之序列，若該miR-196a分子或該miR-196b分子的表現量異常高於正常值，即可推斷該潛在病患罹患口腔癌。The present invention provides a method for detecting oral cancer by using micronucleic acid-196a molecule and micronucleic acid-196b molecule, and analyzing miR- in biological samples such as plasma, serum, saliva, mouthwash or tumor tissue of potential patients. The expression amount of the 196a molecule and the miR-196b molecule, wherein the miR-196a molecule comprises the sequence of SEQ ID NO: 1, and the miR-196b molecule comprises the sequence of SEQ ID NO: 2, if the miR- If the expression of the 196a molecule or the miR-196b molecule is abnormally higher than the normal value, it can be inferred that the potential patient suffers from oral cancer.
本發明首先係經由分析口腔癌細胞的微核酸，發現miR-196a分子及miR-196b分子在口腔癌細胞中扮演重要的致癌角色，而進一步偵測口腔癌病人以及正常人血漿中miR-196a分子及miR-196b分子的含量發現，在口腔癌病患的血漿中之miR-196a分子及miR-196b分子呈高度表現，因此認為miR-196a分子及miR-196b分子具有可以做為早期偵測口腔癌腫瘤標誌的潛力。故本發明利用微核酸微陣列(Agilent Technology，USA)的方式分析比對在口腔癌細胞與正常的口腔上皮細胞內的微核酸是否有差異，本發明總共分析6株口腔癌細胞以及5株的正常口腔上皮細胞內 470個微核酸的表現，結果如第二圖所示，第二圖係利用各微核酸在癌細胞即正常口腔上皮細胞的平均螢光表現含量作圖，其所示為偵測之470個微核酸的螢光強度圖，X軸為在5個正常口腔上皮細胞平均強度，Y軸為在6個口腔癌細胞中的平均表現強度。The invention firstly analyzes the micronucleic acid of oral cancer cells and finds that miR-196a molecule and miR-196b molecule play an important carcinogenic role in oral cancer cells, and further detect miR-196a molecules in oral cancer patients and normal human plasma. And miR-196b molecular content found that the miR-196a molecule and miR-196b molecule in the plasma of patients with oral cancer are highly expressed, so miR-196a molecule and miR-196b molecule can be considered as early detection of oral cavity. The potential of cancer tumor markers. Therefore, the present invention analyzes whether micronuclei in oral cancer cells and normal oral epithelial cells are different by using a micronucleic acid microarray (Agilent Technology, USA). The present invention analyzes a total of 6 oral cancer cells and 5 strains. Normal oral epithelial cells The performance of 470 micronucleic acids, the results are shown in the second figure, the second picture is the use of each micronucleic acid in the average fluorescence expression of cancer cells, ie normal oral epithelial cells, which shows 470 micro-detection The fluorescence intensity map of the nucleic acid, the X-axis is the average intensity in 5 normal oral epithelial cells, and the Y-axis is the average intensity of expression in 6 oral cancer cells.
表一係以微陣列方式篩選之有顯著差異的微小RNA，表列23個有顯著差異的微核酸在6個口腔癌細胞(C)與5個正常口腔上皮細胞(K)的平均表現量與相對倍數差及P值，P值低顯示在同群組中微核酸的表現趨勢相同，而在23個微核酸中，miR-196a及miR-196b在癌細胞顯著高表現(>10倍)，顯示出miR-196a及miR-196b在口腔癌形成時扮演一種重要角色。偵測470個人類微核酸，接著經過常態化(normalization)並去除微弱訊號後共190個微核酸進行群集分析(clustering analysis)，非預設性階層分級分析(The unsupervised hierarchical clustering analysis)結果顯示，這190個微核酸的表現數據圖譜可以將樣本分為癌細胞及正常口腔上皮細胞兩個群組，以統計方法(analysis of variance，ANOVA)計算，將條件設定為錯誤發現率(False discovery rate，FDR)<0.1，並同時有兩倍以上差異，得知有23個微小RNA的表現在口腔癌細胞與正常口腔上皮的樣本中有顯著差異，其中包含19個微核酸表現量上升，4個微核酸表現量下降於口腔癌細胞株(請參 考第一圖所示)。Table 1 shows the microRNA-screened microRNAs with significant differences, and the average performance of 23 slightly different micronuclei in 6 oral cancer cells (C) and 5 normal oral epithelial cells (K). Relative fold difference and P value, low P value showed the same trend of micronucleic acid in the same group, and miR-196a and miR-196b showed high performance (>10 times) in cancer cells in 23 micronucleic acids. It has been shown that miR-196a and miR-196b play an important role in the formation of oral cancer. Detection of 470 human micronuclei, followed by normalization and removal of weak signals, a total of 190 micronucleic acids for clustering analysis, the results of the unsupervised hierarchical clustering analysis showed that The performance data map of the 190 micronucleic acids can divide the sample into two groups of cancer cells and normal oral epithelial cells, and calculate the condition as the false discovery rate by the analysis of variance (ANOVA). FDR) <0.1, and at the same time more than twice the difference, it is known that there are 23 microRNAs in the oral cancer cells and normal oral epithelial samples are significantly different, including 19 micronuclear expression increased, 4 micro The amount of nucleic acid expression decreased in oral cancer cell lines (please refer to Test shown in the first figure).
此外，本發明利用活體外傷痕癒合實驗來分析miR-196a及miR-196b對於細胞移行能力的影響。將兩株口腔癌細胞(OECM1，SAS)處理miR-196a及miR-196b的抑制序列(antagomiR、anti-196a、anti-196b)來減低細胞內miR-196a及miR-196b的表現量，則細胞爬行至間隙的速度比控制組慢，如第三圖所示，第三圖係將miR-196a及miR-196b之抑制序列及控制序列(scramble control)處理口腔癌細胞OECM1及SAS後進行體外傷口癒合試驗， 其中控制序列SC是指序列中核酸基與抑制序列相同但順序為隨機打散的序列。In addition, the present invention utilizes an in vitro scar healing assay to analyze the effects of miR-196a and miR-196b on cell migration ability. Two oral cancer cells (OECM1, SAS) were treated with the inhibitory sequences of miR-196a and miR-196b (antagomiR, anti-196a, anti-196b) to reduce the expression of miR-196a and miR-196b in cells. The speed of crawling to the gap is slower than that of the control group. As shown in the third figure, the third image is the in vitro wound after treating the oral cancer cells OECM1 and SAS with the inhibitory sequence and scramble control of miR-196a and miR-196b. Healing test, Wherein the control sequence SC refers to a sequence in which the nucleic acid group in the sequence is identical to the inhibition sequence but the sequence is randomly dispersed.
兩株細胞都顯示出當控制組的細胞已經滿佈間隙的時候，不管是減低miR-196a或miR-196b的實驗組細胞仍未爬滿間隙。此外，為進一步證實miR-196a及miR-196b對細胞移行的影響，本發明亦設計可以高度表現miR-196a及miR-196b的質體，使細胞表現高量的miR-196a及miR-196b，如第四圖所示，在口腔癌細胞OECM1及SAS中以特定載體表現miR-196a、miR-196b後，以體外傷口癒合試驗測定細胞移行能力，並以空載體(pcDNA)處理的細胞當作控制組，顯示不管高度表現miR-196a或miR-196b都可以兩株口腔癌細胞的移行能力，高度表現miR-196的細胞在九小時後就可以爬滿間隙，顯著較控制組細胞(pcDNA)快。Both cells showed that the cells in the experimental group, which reduced miR-196a or miR-196b, did not fill the gap when the cells in the control group had been filled with gaps. In addition, in order to further confirm the effect of miR-196a and miR-196b on cell migration, the present invention also designed to highly express the plastids of miR-196a and miR-196b, so that cells express high amounts of miR-196a and miR-196b, As shown in the fourth panel, after miR-196a and miR-196b were expressed in specific cells in oral cancer cells OECM1 and SAS, cell migration ability was measured by an in vitro wound healing test, and cells treated with empty vector (pcDNA) were used as The control group showed that regardless of the high performance of miR-196a or miR-196b, the migration ability of two oral cancer cells could be observed. The cells with high expression of miR-196 could fill the gap after nine hours, significantly compared with the control group (pcDNA). fast.
本發明並利用基底膜穿透實驗(Matrigel invasion assay)來判斷miR-196a及miR-196b是否會改變細胞向外侵犯的能力，將miR-196a及miR-196b之抑制序列(antagomiR、anti-196a、anti-196b)及控制序列(scramble control)處理口腔癌細胞OECM1及SAS來減低細胞內miR-196a及miR-196b的表現量，其中控制序列SC是指序列中核酸基與抑制序列相同但順序為隨機打散的序列，結果如第五圖所示。在兩株口腔癌細胞中，以特 定的miR-196a及miR-196b抑制序列來減低細胞miR-196a及miR-196b的表現，則比控制組細胞出現較少穿越基底膜膠質的細胞數，與對照組細胞比較之後發現，抑制miR-196a的表現後，OECM1細胞的穿越數下降至49%，而SAS細胞更下降至31%；另外，抑制miR-196b則發現OECM1及SAS細胞穿越基底膜的數目減少到控制組的34%及47%。進一步高度表現miR-196a及miR-196b於細胞中，如第六圖所示，高度表現細胞中的miR-196a及miR-196b會促進口腔癌細胞的侵犯能力，在口腔癌細胞OECM1及SAS中以特定載體表現miR-196a及miR-196b後，以基底膜穿透試驗(Matrigel invasion assay)測定細胞侵犯能力，並以空載體(pcDNA)處理的細胞當作控制組，發現有miR-196a及miR-196b高度表現的細胞穿過基底膜到另一端的細胞數比控制組(pcDNA)多約2倍。The present invention also utilizes the Matrigel invasion assay to determine whether miR-196a and miR-196b alter the ability of cells to invade, and the inhibitory sequences of miR-196a and miR-196b (antagomiR, anti-196a) , anti-196b) and scramble control treatment of oral cancer cells OECM1 and SAS to reduce the expression of intracellular miR-196a and miR-196b, wherein the control sequence SC means that the nucleic acid sequence in the sequence is identical to the inhibition sequence but in sequence For a sequence that is randomly scattered, the result is shown in the fifth figure. In two oral cancer cells, The miR-196a and miR-196b inhibitory sequences were designed to reduce the expression of miR-196a and miR-196b, and the number of cells that passed through the basement membrane glial was smaller than that of the control group. After the performance of -196a, the number of OECM1 cells decreased to 49%, while SAS cells decreased to 31%. In addition, inhibition of miR-196b found that the number of OECM1 and SAS cells crossing the basement membrane was reduced to 34% of the control group. 47%. Further high expression of miR-196a and miR-196b in cells, as shown in Figure 6, high expression of miR-196a and miR-196b in cells can promote the invasion of oral cancer cells, in oral cancer cells OECM1 and SAS After miR-196a and miR-196b were expressed in a specific vector, cell invasion ability was measured by Matrigel invasion assay, and cells treated with empty vector (pcDNA) were used as control group, and miR-196a and The highly expressed cells of miR-196b crossed the basement membrane to the other end by about 2 times more cells than the control group (pcDNA).
除了miR-196a及miR-196b在細胞功能的鑑定，本發明也測定了臨床的組織檢體中miR-196a及miR-196b的表現，本發明總共操作了54個病人的腫瘤組織樣本(T)及其周圍正常組織樣本(N)，利用即時定量PCR方式檢測miR-196a及miR-196b的表現量，並以U6核酸作為標準化用的內部控制，以相對表現量來呈現結果，其中第七圖(A)表示在54組樣本中有52個(96.3%)腫瘤組織中miR-196a及miR-196b的表現量比其周圍正常組織中的表現量高出兩倍以上 (p<0.001)；第七圖(B)表示在54組樣本中有48個(88.6%)腫瘤檢體中miR-196b的表現量顯著比周圍正常組織中的表現量高過兩倍(p<0.001)。In addition to the identification of miR-196a and miR-196b in cell function, the present invention also measures the expression of miR-196a and miR-196b in clinical tissue samples. The present invention operates a total of 54 patient tumor tissue samples (T). And normal tissue samples (N), using real-time quantitative PCR to detect the expression of miR-196a and miR-196b, and U6 nucleic acid as the internal control for standardization, the results are presented in relative performance, the seventh figure (A) indicates that the expression of miR-196a and miR-196b in 52 (96.3%) of the 54 groups of samples is more than twice as high as that in the surrounding normal tissues. (p<0.001); Figure 7 (B) shows that the expression of miR-196b in 48 (88.6%) of the 54 samples was significantly higher than that in the surrounding normal tissues (p). <0.001).
為進一步確定miR-196a及miR-196b是否可以應用在臨床上，本發明另比較miR-196a及miR-196b在口腔癌病人與正常人的血漿中的表現量，本發明共偵測54個口腔癌病人與33個正常人的血漿，本發明所採用之測定血漿中miR-196a及miR-196b的方法為： 步驟一：首先取得血漿檢體，將血漿樣分裝出200μl以不會去除small RNA的試劑組來萃取出微核酸，在本發明之實驗係使用miRNeasy® mini kit(Qiagen Inc.，Valencia，CA)，第一步加入700μl QIAzol使其均質化後再加入140μl氯仿分離萃取RNA，離心之後取上清液(約525μl)加入750μl純酒精沉澱核酸產物，並以試劑中所提供的離心管柱分離出沉澱物，並丟棄下層流出液，在潤洗管柱後，每個樣本以20μl無核酸水解酶(RNase-free)的水將核酸提取出來；在微核酸的定量方式是以反轉錄定量聚合酶連鎖鍊反應試劑來進行，本發明之實驗係使用TaqMan® miRNA assays kit(ABI，Forest City，CA)作為反轉錄定量聚合酶連鎖鍊反應試劑，首先將所萃取的核酸取3μl進行反轉錄反應，加入4單位的反轉錄酶、10單位的核酸水解酶抑制劑及25mM脫氧核糖核苷三磷酸( deoxy-ribonucleoside triphosphate，dNTP)於總量30μl反應體積內放在37℃培育30分鐘，其中，本實驗係使用反轉錄酶AMV(HT Biotech Ltd，UK)及CalBiochem(CA，USA)的核酸水解酶抑制劑，接著，以即時PCR檢測儀(本發明係使用Bio-Rad之MiniOpticon)進行即時定量PCR，將8μl反轉錄後的產物與1μl特定微核酸探針混和，並加入6μl水和10μl定量PCR試劑後上機，本實驗所採用之定量PCR試劑為iQ supermix(Bio-Rad，Hercules，CA)，結果以Ct值(threshold cycle)顯示，以相對表現量來呈現結果。To further determine whether miR-196a and miR-196b can be used clinically, the present invention compares the expression levels of miR-196a and miR-196b in plasma of oral cancer patients and normal humans, and the present invention detects 54 oral cavityes. The plasma of cancer patients and 33 normal persons, the method for measuring miR-196a and miR-196b in plasma used in the present invention is as follows: Step 1: First, obtain a plasma sample, and pack the plasma sample into 200 μl so as not to remove small. The reagent set of RNA is used to extract the micronucleic acid. In the experimental system of the present invention, the miRNeasy ® mini kit (Qiagen Inc., Valencia, CA) is used, and the first step is to add 700 μl of QIAzol to homogenize it, and then add 140 μl of chloroform to separate and extract the RNA. After centrifugation, the supernatant (about 525 μl) was added to precipitate 750 μl of pure alcohol to precipitate the nucleic acid product, and the precipitate was separated by a centrifugal column provided in the reagent, and the lower effluent was discarded, and after washing the column, each sample was 20 μl of RNase-free water is used to extract the nucleic acid; the quantitative method of micronucleic acid is carried out by reverse transcription quantitative polymerase chain reaction reagent, and the experiment of the present invention uses TaqMan ® miRNA assays. Kit (ABI, Forest City, CA) as a reverse transcription quantitative polymerase chain reaction reagent, first take 3μl of the extracted nucleic acid for reverse transcription reaction, add 4 units of reverse transcriptase, 10 units of nuclease inhibitor and 25 mM deoxy-ribonucleoside triphosphate (dNTP) was incubated at 37 ° C for 30 minutes in a total volume of 30 μl. The experiment used reverse transcriptase AMV (HT Biotech Ltd, UK) and CalBiochem ( Nucleic acid hydrolase inhibitor of CA, USA), followed by real-time quantitative PCR using an instant PCR detector (this invention uses Bio-Rad's MiniOpticon), and mixing 8 μl of the reverse transcribed product with 1 μl of the specific micronucleic acid probe. After adding 6 μl of water and 10 μl of quantitative PCR reagent, the quantitative PCR reagent used in this experiment was iQ supermix (Bio-Rad, Hercules, CA). The results were shown by Ct value (threshold cycle) and presented in relative performance. result.
結果顯示，與正常人血漿中的miR-196a及miR-196b比較，癌症病人血漿中的miR-196a顯著增加，這表示miR-196a在口腔癌病人的血漿有過度表現的現象平均初出14.27(約為14)倍(P<0.0001)。請參閱第八圖(A)，係以散布的黑點表示出正常人血漿及口腔癌病人血漿群組中每一個樣本以RT-qPCR測定的miR-196a相對表現量，黑直線則為該群組的平均量。卡方測定(chi-squared)t-test計算後可得知兩群組間差異是否具有統計上的意義；第八圖(B)係以ROC曲線(receiver operational curve)分析，評估miR-196a在正常人與口腔癌病人檢體間的鑑別力，miR-196a可以得到曲線下面積(aera under curve，AUC)為0.938，顯示miR-196a具有高潛力用以將口腔癌的病人 從正常人中鑑別出來，邏輯回歸分析(logistic regression model)預測miR-196a用以區別口腔癌病人的敏感度達92.6%，專一性亦可達84.6%。The results showed that compared with miR-196a and miR-196b in normal human plasma, the plasma miR-196a was significantly increased in cancer patients, indicating that miR-196a has an overexpressed plasma in patients with oral cancer. It is 14) times (P < 0.0001). Please refer to the eighth figure (A), which shows the relative expression of miR-196a by RT-qPCR for each sample in the plasma group of normal human plasma and oral cancer patients with scattered black dots. The black line is the group. The average amount of the group. Chi-squared t-test calculation can be used to find out whether the difference between the two groups has statistical significance; the eighth figure (B) is analyzed by ROC curve (receiver operational curve) to evaluate miR-196a The discriminative power between normal and oral cancer patients, miR-196a can obtain an area under the curve (AUC) of 0.938, indicating that miR-196a has high potential for patients with oral cancer. From the normal person, logistic regression model predicts that miR-196a can distinguish 92.6% of patients with oral cancer, and the specificity can reach 84.6%.
又，miR-196b的偵測結果表示，miR-196在口腔癌病人血漿中同樣顯著增加，平均增加達10.03(約為10)倍(P<0.0001)，這表示miR-196b在口腔癌病人的血漿也有過度表現的現象，請參閱第九圖(A)，係以散布的黑點表示出正常人血漿及口腔癌病人血漿群組中每一個樣本以RT-qPCR測定的miR-196b相對表現量，黑直線則為該群組的平均量。卡方測定(chi-squared)t-test計算後可得知兩群組間差異是否具有統計上的意義。第九圖(B)係以ROC曲線(receiver operational curve)分析，評估miR-196b在正常人與口腔癌病人檢體間的鑑別力，miR-196b的線下面積為0.942，顯示miR-196b同樣有高潛力來從正常人血漿中鑑別出口腔癌病人血清，經邏輯回歸分析(logistic regression model)預測miR-196b偵測口腔病人的靈敏度可高達90.7%，專一性則達84.9%。Furthermore, the detection of miR-196b indicated that miR-196 also increased significantly in the plasma of patients with oral cancer, with an average increase of 10.03 (about 10) times (P < 0.0001), indicating that miR-196b is in patients with oral cancer. Plasma is also over-expressed. Please refer to Figure IX (A) for the relative expression of miR-196b by RT-qPCR for each sample in normal human plasma and oral cancer patients. The black line is the average amount of the group. Chi-squared t-test calculation can be used to find out whether the difference between the two groups is statistically significant. The ninth panel (B) is an analysis of the ROC curve (receiver operational curve) to evaluate the discrimination between miR-196b in normal human and oral cancer patients. The area under the line of miR-196b is 0.942, indicating that miR-196b is the same. There is high potential to identify sera from patients with oral cancer from normal human plasma. Logistic regression models predict that miR-196b can detect oral patients with sensitivity up to 90.7% and specificity of 84.9%.
綜上所述，本發明透過實驗發現表示miR-196a分子及miR-196b分子具有良好的口腔癌偵測能力，因此，測定生物檢體中miR-196a分子及miR-196b分子的含量可以利用於偵測口腔癌。In summary, the present invention shows that the miR-196a molecule and the miR-196b molecule have good oral cancer detection ability, and therefore, the content of the miR-196a molecule and the miR-196b molecule in the biological sample can be utilized. Detection of oral cancer.
前文係針對本發明之較佳實施例為本發明之技術特徵 進行具體之說明；惟，熟悉此項技術之人士當可在不脫離本發明之精神與原則下對本發明進行變更與修改，而該等變更與修改，皆應涵蓋於如下申請專利範圍所界定之範疇中。The foregoing is a technical feature of the present invention for a preferred embodiment of the present invention. The present invention is subject to change and modification without departing from the spirit and scope of the invention, and such modifications and modifications are intended to be included in the scope of the following claims. In the category.
第一圖：透過非預設性階層分級分析並以ANOVA計算，以FDR<0.1且具有兩倍以上差異的結果顯示有23個微小RNA的表現在口腔癌細胞與正常口腔上皮的樣本中有顯著差異。First panel: by non-predictive hierarchical analysis and calculated by ANOVA, the results of FDR<0.1 and more than twice the difference showed that the expression of 23 microRNAs was significant in the samples of oral cancer cells and normal oral epithelium. difference.
第二圖：係利用各微核酸在癌細胞即正常口腔上皮細胞的平均螢光表現含量作圖。Second panel: The average fluorescence expression level of each micronucleic acid in cancer cells, ie normal oral epithelial cells, was plotted.
第三圖：表示使用miR-196a及miR-196b的抑制核酸序列(antagomir)處理細胞會降低細胞移行能力。Figure 3: shows that treatment of cells with the antagomir of miR-196a and miR-196b reduces cell migration.
第四圖：表示高度表現miR-196a與miR-196b會促進細胞移行能力。Figure 4: High expression of miR-196a and miR-196b promotes cell migration.
第五圖：表示細胞向外侵犯能力會因為抑制miR-196a及miR-196b而下降。Figure 5: The ability of cells to invade outwards is reduced by inhibition of miR-196a and miR-196b.
第六圖：表示高度表現細胞中的miR-196a及miR-196b會促進口腔癌細胞的侵犯能力。Figure 6: shows that miR-196a and miR-196b in highly expressed cells promote the invasion of oral cancer cells.
第七圖(A)(B)：表示測定臨床組織檢體中口腔癌腫瘤樣本(T)與周圍正常組織樣本(N)內miR-196a與miR-196b的表現量。Fig. 7 (A) (B): shows the measurement of the expression levels of miR-196a and miR-196b in the oral cancer tumor sample (T) and the surrounding normal tissue sample (N) in the clinical tissue sample.
第八圖(A)(B)：表示miR-196a在口腔癌病人的血漿有過度表現的現象。Figure 8 (A) (B): shows the phenomenon that miR-196a is overexpressed in the plasma of patients with oral cancer.
第九圖(A)(B)：表示miR-196b在口腔癌病人的血漿有過度表現的現象。Fig. 9 (A) (B): shows the phenomenon that miR-196b is excessively expressed in the plasma of patients with oral cancer.
<110> 長庚大學<110> Chang Gung University
<120> 以微核酸-196(miR-196)分子運用於發現及診斷口腔癌之方法<120> Method for detecting and diagnosing oral cancer using micronucleic acid-196 (miR-196) molecule
<160> 2<160> 2
<210> SEQ ID NO 1<210> SEQ ID NO 1
<211> LENGTH：22<211> LENGTH: 22
<212> TYPE：RNA<212> TYPE:RNA
<213> ORGANISM：HOMO SAPIENS<213> ORGANISM: HOMO SAPIENS
<220> FEATURE： Two stem-loop,hsa-mir-196a-1(MI0000238)and hsa-mir-196a-2(MI0000279)<220> FEATURE: Two stem-loop, hsa-mir-196a-1 (MI0000238) and hsa-mir-196a-2 (MI0000279)
<222> GENE LOCATION： Has-mir-196a-1：17：46709852-46709921[-]； Has-mir-196a-2：12：54385522-54385631[+]<222> GENE LOCATION: Has-mir-196a-1:17:46709852-46709921[-]; Has-mir-196a-2:12:54385522-54385631[+]
<223> miRBase Accession MIMA0000226<223> miRBase Accession MIMA0000226
<400> 1 <400> 1
<210> SEQ ID NO 2<210> SEQ ID NO 2
<211> LENGTH：22<211> LENGTH: 22
<212> TYPE：RNA<212> TYPE:RNA
<213> ORGANISM：HOMO SAPIENS<213> ORGANISM: HOMO SAPIENS
<220> FEATURE： stem-loop,hsa-mir-196b MI0001150<220> FEATURE: stem-loop, hsa-mir-196b MI0001150
<222> GENE LOCATION： Has-mir-196b：7：27209099-27209182[-]<222> GENE LOCATION: Has-mir-196b: 7:27209099-27209182[-]
<223> miRBase Accession MIMA0001080<223> miRBase Accession MIMA0001080
<400> 2 <400> 2
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