本發(fā)明屬于腫瘤基因檢測技術(shù)領(lǐng)域，涉及一種用于肺腺癌早期診斷的circRNA標(biāo)志物、相關(guān)試劑盒及它們的用途。

背景技術(shù)：

肺癌是嚴(yán)重危害人類健康的第一大惡性腫瘤，其發(fā)病率(120萬/年)和死亡率(110萬/年)均居全球癌癥首位。2014年《中國腫瘤登記年報》顯示，在我國肺癌也是發(fā)病率和死亡率最高的惡性腫瘤(我國每年新發(fā)肺癌患者人數(shù)60萬以上)。肺癌5年生存率僅約15.6％，主要原因是多數(shù)患者在診斷時已屬晚期，因此肺癌的早期發(fā)現(xiàn)早期診斷至關(guān)重要。

環(huán)狀RNA是一類由特殊的選擇性剪切產(chǎn)生的首尾相接的非編碼RNA。起初它們被認(rèn)為是基因組轉(zhuǎn)錄的“噪音”，不具有生物學(xué)功能。然而，近年來的研究表明circRNA在人體中發(fā)揮著巨大的基因調(diào)節(jié)功能。與線性RNA分子相比，circRNA作為生物分子標(biāo)志物具有如下優(yōu)勢：(1)circRNA形成特殊的共價閉合環(huán)狀結(jié)構(gòu)，3’端沒有多聚腺苷酸尾，不會被核酸外切酶R所酶切，因此穩(wěn)定性高，便于樣品的保存；(2)circRNA可以通過外泌體而進(jìn)入血液、組織液及痰液中，便于取樣；(3)circRNA長度多在200-400堿基，使用qPCR即可定量檢測，可操作性強。目前已經(jīng)在食管癌、胃癌、肝癌等惡性腫瘤中相繼發(fā)現(xiàn)了具有早期診斷價值的circRNA，但是在肺癌領(lǐng)域中尚未見相關(guān)circRNA的報道。

技術(shù)實現(xiàn)要素：

解決的技術(shù)問題：本發(fā)明的目的在于提供一組用于肺腺癌早期診斷的circRNA標(biāo)志物及其應(yīng)用，為肺腺癌的早期診斷提供參考依據(jù)。本發(fā)明所提供的circRNA生物標(biāo)志物對于人肺腺癌具有很高的靈敏度和特異性，尤其對人早期肺腺癌具有很好的檢測效果，可以作為新型的生物標(biāo)志物用于肺腺癌的檢測。

技術(shù)方案：一組用于肺腺癌早期診斷的circRNA標(biāo)志物，標(biāo)志物包括：SEQ ID No:1所示的circRNA_100323、SEQ ID No:2所示的circRNA_69774、SEQ ID No:3所示的circRNA_403738、SEQ ID No:4所示的circRNA_404449四種circRNA。

上述肺腺癌為人早期肺腺癌。

上述circRNA標(biāo)志物的引物組合，所述circRNA引物的組合包括：針對circRNA_100323的特異性引物、針對circRNA_69774的特異性引物、針對circRNA_403738的特異性引物、針對circRNA_404449的特異性引物。

上述circRNA標(biāo)志物的引物組合，所述針對circRNA_100323的特異性引物包括如SEQ ID No：9所示的上游引物，如SEQ IDNo：10所示的下游引物；所述針對circRNA_69774的特異性引物包括如SEQ ID No：11所示的上游引物，如SEQ ID No：12所示的下游引物；所述針對circRNA_403738的特異性引物包括如SEQ ID No：13所示的上游引物，如SEQ ID No：14所示的下游引物；所述針對circRNA_404449的特異性引物包括如SEQ ID No：15所示的上游引物，如SEQ ID No：16所示的下游引物。

上述circRNA標(biāo)志物的引物組合，所述circRNA引物的組合還包括：針對SEQ ID No:5所示的circRNA_104855的特異性引物、針對SEQ ID No:6所示的circRNA_100983的特異性引物、針對SEQ ID No:7所示的circRNA_1011969的特異性引物、針對SEQ ID No:8所示的circRNA_006718的特異性引物。

上述circRNA標(biāo)志物的引物組合，所述針對circRNA_104855的特異性引物包括：如SEQ ID No：17所示的上游引物和如SEQ IDNo：18所示的下游引物；所述針對circRNA_100983的特異性引物包括：如SEQ ID No：19所示的上游引物和如SEQ IDNo：20所示的下游引物；所述針對circRNA_1011969的特異性引物包括：如SEQ ID No：21所示的上游引物和如SEQ IDNo：22所示的下游引物；所述針對circRNA_006718的特異性引物包括：如SEQ ID No：23所示的上游引物和如SEQ ID No：24所示的下游引物。

上述circRNA標(biāo)志物的引物組合在制備或篩選人肺腺癌的腫瘤診斷藥物中的用途。

一種人肺腺癌的腫瘤診斷試劑盒，包括所述的circRNA標(biāo)志物的引物組合。

一種檢測人肺腺癌的circRNA芯片，所述circRNA芯片包括固相載體以及固定于固相載體上的針對如上所述的circRNA生物標(biāo)志物的探針。

本發(fā)明所提供的人肺腺癌早期診斷用circRNA生物標(biāo)志物，可用于肺腺癌的早期診斷。其區(qū)分早期肺腺癌和配對正常肺組織的AUC可達(dá)0.841，顯著性高于這4個circRNAs單獨診斷早期肺腺癌的AUC(P<0.05)，其ROC曲線表明這4個circRNAs診斷早期肺腺癌的靈敏度和特異性分別為83.4％和73.5％，顯著性高于單個circRNA用于肺腺癌診斷的靈敏度和特異性(P<0.05)。

有益效果：(1)本方法通過定量PCR對一組circRNA的檢測，實現(xiàn)對肺腺癌的早期診斷，肺癌的早期發(fā)現(xiàn)和診斷提供參考價值。(2)本方法特異性強、敏感性高、結(jié)果穩(wěn)定等優(yōu)點，具有廣泛的臨床應(yīng)用前景和巨大的商業(yè)價值。

附圖說明

圖1.本發(fā)明8個circRNA聯(lián)合起來區(qū)分訓(xùn)練集(n＝30)肺腺癌及其配對正常肺組織的ROC曲線圖；

圖2.本發(fā)明優(yōu)化的4個circRNA聯(lián)合起來區(qū)分訓(xùn)練集(n＝30)肺腺癌及其配對正常肺組織的ROC曲線圖；

圖3.本發(fā)明優(yōu)化的4個circRNA聯(lián)合起來區(qū)分驗證集(n＝30)肺腺癌及其配對正常肺組織的ROC曲線圖。

具體實施方式

以下通過特定的具體實例說明本發(fā)明的實施方式。

實施例1

1臨床樣本收集：

收集東南大學(xué)附屬中大醫(yī)院2013-2014年期間存檔的早期(IA期)肺腺癌石蠟組織標(biāo)本及其配對正常肺組織60對。所有病人對參與科學(xué)研究均簽署知情同意書。

2 circRNA定量PCR實驗：

2.1 RNA抽提及定量PCR

利用Trizol從組織中抽提總RNA，用微體積分光光度計對RNA進(jìn)行定量。

2.2引物

本發(fā)明所提供的一組circRNA包括circRNA_100323、circRNA_69774、circRNA_403738、circRNA_404449、circRNA_104855、circRNA_100983、circRNA_1011969、circRNA_006718等8種circRNA。它們均是從早期肺腺癌患者circRNA芯片檢測的結(jié)果中挑選的，以GAPDH為內(nèi)參基因(SEQ ID No：27)，引物序列如表1所示：

表1定量PCR使用的引物序列

2.3逆轉(zhuǎn)錄反應(yīng)

使用TakaRa PrimeScript RT reagent Kit試劑盒進(jìn)行逆轉(zhuǎn)錄，RNA起始量為1μg，逆轉(zhuǎn)錄反應(yīng)組分的用量如表2所示，反應(yīng)條件：37℃15min，85℃5sec完成后低溫保存?zhèn)溆谩?/p>

表2逆轉(zhuǎn)錄反應(yīng)體系組成

2.4實時定量PCR反應(yīng)

實時定量PCR使用TaKaRa公司的SYBR Premix Ex Taq(Tli RnaseH Plus)試劑盒，定量儀器為BioRad公司CFX96型實時PCR儀。反應(yīng)體系如表3所示，反應(yīng)條件如表5所示步驟，每個實時定量PCR反應(yīng)分別做三個重復(fù)。

表3實時定量PCR反應(yīng)體系

反應(yīng)程序為：95℃2min；95℃30sec，56℃50sec，40個循環(huán)。

數(shù)據(jù)以平均值±標(biāo)準(zhǔn)差的形式表示，使用SPSS 18.0軟件對數(shù)據(jù)進(jìn)行統(tǒng)計分析。除非有特殊的說明。肺腺癌與其配對正常肺組織中circRNA的表達(dá)差異統(tǒng)計使用配對樣本t檢驗的方法，所有P值均為雙側(cè)，P<0.05被認(rèn)為有統(tǒng)計學(xué)意義。

2.5 8個circRNA區(qū)分肺腺癌及其配對正常肺組織的能力評估

我們利用ROC曲線來評估這8個區(qū)分肺腺癌及其配對正常肺組織的能力。單個circRNA用于肺腺癌診斷的AUC為0.53-0.75，靈敏度為54.4％-77.4％，特異性為49.1％-77.5％(表4)。我們將這8個circRNA聯(lián)合起來用于30對肺腺癌樣本的診斷，其AUC值可達(dá)到0.854(圖1)，在選擇合適閾值之后，靈敏度和特異性分別為80.1％和83.3％。

表4 8個circRNA在訓(xùn)練集(30對樣本)中的表達(dá)情況

2.6優(yōu)選的4個circRNA區(qū)分肺腺癌及其配對正常肺組織的能力評估

通過線性邏輯回歸分析，我們從8個circRNA中分析優(yōu)選得到了4個circRNA。我們利用ROC曲線來評估4個在訓(xùn)練集中區(qū)分肺腺癌及其配對正常肺組織的能力(所用30對樣本與2.5相同)。這4個circRNA結(jié)合起來區(qū)分早期肺腺癌和配對正常肺組織的AUC可達(dá)0.820(圖2所示)，接近8個circRNA聯(lián)合檢測水平，并顯著高于這4個circRNA單獨診斷早期肺腺癌的AUC(P<0.05)。在選擇合適的閾值之后，ROC曲線表明這4個circRNA診斷早期肺腺癌的靈敏度和特異性分別為83.3％和80.0％，顯著高于單個circRNA用于肺腺癌診斷的靈敏度和特異性(P<0.05)。

表5 4個circRNA在驗證集(30對樣本)中的診斷結(jié)果

我們在另外一組30例肺腺癌樣本及其配對正常肺組織(驗證集)中驗證從訓(xùn)練集篩選出的這4個circRNA的檢測特性。這4個circRNA在肺腺癌及其配對正常肺組織均中有顯著性差異表達(dá)(P<0.001)。單個circRNA用于檢測肺腺癌的AUC為0.73-0.82，靈敏度為65.6％-79.3％，特異性為67.9％-76.3％(表5)。這4個circRNA聯(lián)合檢測60對肺腺癌樣本的AUC可達(dá)0.841(圖3)，在選擇合適的閾值之后，ROC曲線表明其靈敏度為83.4％，特異性為73.5％，均顯著高于任何單個circRNA的檢測能力(P<0.05)。并且，這組circRNA在兩組獨立樣本(訓(xùn)練集和驗證集)中具有類似的檢測能力，說明這組circRNA可以作為肺腺癌早期檢測的生物標(biāo)志物。

以上所述，僅為本發(fā)明的較佳實施例，并非對本發(fā)明任何形式上和實質(zhì)上的限制，應(yīng)當(dāng)指出，對于本技術(shù)領(lǐng)域的普通技術(shù)人員，在不脫離本發(fā)明方法的前提下，還將可以做出若干改進(jìn)和補充，這些改進(jìn)和補充也應(yīng)視為本發(fā)明的保護(hù)范圍。凡熟悉本專業(yè)的技術(shù)人員，在不脫離本發(fā)明的精神和范圍的情況下，當(dāng)可利用以上所揭示的技術(shù)內(nèi)容而做出的些許更動、修飾與演變的等同變化，均為本發(fā)明的等效實施例；同時，凡依據(jù)本發(fā)明的實質(zhì)技術(shù)對上述實施例所作的任何等同變化的更動、修飾與演變，均仍屬于本發(fā)明的技術(shù)方案的范圍內(nèi)。

序列表

<110> 東南大學(xué)

<120> 一組用于肺腺癌早期診斷的circRNA標(biāo)志物及其應(yīng)用

<130>

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ucugcuggau auaaugugau uuuguaacca uuauuggauu gguguugguu uagacaguuu 3120

auuuucagcu gacuauauag uuaauuccgg gacucuuaua aaguuuuuuu uaaaaagaga 3180

aaaagagaag aucaaaagca cuggaacuca acgaguaaau aacccauggu aacagagaga 3240

uauaaugcag ugaugucaga gguguacaag aagugugaau cauuuaacau gugaagccag 3300

ggucuuuagu ccacuugggg agagcaaaga ggugaguggg cuucuccauc uuuuuccuuc 3360

ugccugcccu gcuaccccca gccauaugcu uaggaguggg guucccauuu acacugcccu 3420

gcuugucgcu ccuggcucag cugacccugc uugacauguc cacuucagua auugaacuga 3480

aacgcugauu gaugaacuua auucuuagug uucaucuuuu agcccuguca uuuaccccag 3540

uaucacauuu auuuuaaaac aauaacuauu uuggauuaag uuaauugugu gagugagaga 3600

uuaugaauau guaacuuugc agguauuuug uugaaguuuu agucauauug gaauuuuuaa 3660

gauagaaaug augguaggug uagagcuacu aauauccuuu uuaagaagcu aggaaaaauc 3720

uagccaugca gaaugccaca gugguagaaa caguguccag uaaacauauu aaauggucuu 3780

aaggauucuu gucuuuuaaa guuuuauuua auuuuuuuua uagagacaag gucuucuuau 3840

agcucauaac ucauugcagc cuccagcucc uggacucagg ccauccuccu gccucagccu 3900

ucugaguagc ugggaaggga uccuugucuu uaaaacuagg gguauggagg gaaggguggg 3960

uauaauauaa uaugauuuuu ccguuuaaua uuuucauaua uucuugguga uucauuuuag 4020

uuaggcauuu auuuauuauu guuuauucua auuuuguauu auaauagaau auaauugucu 4080

ucuuuucuuc cuuuuuuuuu uuuuuagaga cagggucuca cuauguugcu cagccugccc 4140

ucaaacuccu gggcucaagg gaucuucccc ucuuggccug ccaaaguggu gggauuacag 4200

gcaugagcca ucaugccugg ccuugucuug uuuuuauuuu auauauauaa uauauauaaa 4260

aaauaauaua uaaauauaua auuuauauuu auauuauaua uuaauauauu aauguaauua 4320

auauuauauu auauuaauau auaauauaua auauaauauu auauauuaua uauaauauua 4380

uauauaauau uaauacugua uuauauauaa uauaauauua auuauauuau auuaauauaa 4440

uuaauuauau uaauauaaua auaauaaaua auauuaauau aauuaauaua uuuauauuaa 4500

aauauaaaaa uauaauauau auaauauaua uauauuuuuu ugucucacuc ugucgcccag 4560

gcuggagugc aguggcguga ucuuggcuca cugcaagguc ugccuuccag gcucacgcca 4620

uucuccugcc ucagccucca gcauagcugg gacuacaggc gcccgccacc augcccgccu 4680

aauuuuuugu auuuuuagua gagacgaggu uuuaccgugu uagccaggag gucuccaucu 4740

ccugaccugg ugauccgccu gccucggccu cccaaagugc ugggauuaca ggagugagcc 4800

accacgccug gccuuaauua uauuuuuaug uaacuuuuug auacuacuca aguauuuuuu 4860

ccucuacggu acucacuaug cugaaagauu uuucuuucac auguuuucua aacuauguaa 4920

uauaaguguc auuggcuauu uuuguccacc ugauggaggu aagaaaugau caaaguacag 4980

uuuaacagca guauuagcaa gaguugagcc uugcugaaua uguaaguuuc aacggauuug 5040

cuuugcaacu acaugcagua gauguuuuaa gaggguaggu aggauauuua aauuuaaccu 5100

uuuugugcca cugugugaga gugggcuucu gaaaauggag gggggaagca ucucuccuug 5160

guuaguaggc auucaagugg aagauauguu auaaaaaccu aguuguaucu uauaggaugc 5220

uaaaagcaag gugacugaac uucuaacagg gcauucuaca uugaguaugu gcuuuucucu 5280

auaauuaggc uaaaaaaagg uugguaagua auaugaacuu aaaaucaaga uuaaccccca 5340

auuuacaaac ccaugauaaa aaguguuguu gauuacuugg auccuaucuu uucugcaguu 5400

uauguuucua gugacuuacu caauaguuuu uucuuacucu uucccuggac agaauaugua 5460

auauaagaaa cauaaguccu uugaguugag ggagaucaga uagauuuaau aaaagucauu 5520

uagacuuuau ugcauauucu ggaaguccaa agaagaauga cucugguugc auagcacuuc 5580

uaauaaagcc acaaaaucau uaccugacuc agugccuguu cuuguuauca uauuuuuacu 5640

uuauguuuug guauuucauu uauuuauuac guuucagcug ugagaguacc uucuacaaaa 5700

cauuuuaaac uuuuuucuau uuucauuaag uugcaguccc acuccaccca guccagccuc 5760

cuaauucuag ccuuuauaca gcuaagauug aggcaaucag uuauaaggac gccugcucau 5820

aauuaccuuc ucagaaggua auuauauuaa uacugaauuu auuuaguggu augcugucau 5880

caaaaaaauu cauuaaaaau aaaaucaaug aaggccgggc gcaguggcuc acgcuuguaa 5940

ucccuccacu uugggaggcc gaggugggug gaucaccuga ggucaagagu ucuagaccag 6000

ccuggccaac augaugaaac cccaucucua cugaaaauac aaaaaauuag cugggcgugg 6060

uggcacacac cuguaauccc agcuacucag gaggcugagg caggagaauc gcuugagccc 6120

gggaggugga gguugcagug aaccaagauc gugccacugc ccuccagccu gggcaacaag 6180

agcaaaacuc ugucuccaaa aaaaaaaaag aaacuaaaca cauuuaauaa auuucaagug 6240

cauaaauaca uuacaaaaca acauucauaa aaauaaacau ucauuaaaac aaaaaauaau 6300

uuuauuacaa aauauuacau auaaauucua auauaaauua acaaguaaca auaaaguauu 6360

uaaaaauagu gaaaccauuc acaaaaauac guaaugaaaa uuaaaauacu uuaauguuau 6420

cuuucgaaau cagguuuauc ugugguguca uuguuuaagu gcgugcgugu gugugugugu 6480

guauauauau auauauauau auauauauau auuuuuuuuu uuuuuuuuuu uuuuuuuuuu 6540

uuuuugagac agagucucac ugucacccag gcuggagugc aguggugaga ucuugguuca 6600

cuucaaccuc ugucuccggg guucaugcaa uucucaugcc ucagccucca gaguggcuga 6660

gacuacaggu gcaugccacc acgcccagcu gauuuuugua uuuuuaguag agaugggguu 6720

uugccacguu ggccaggcug gucucaagcu cccaaccucg agugauccac cuaccacgcc 6780

ucccaaguug cuaggauuac aggcaugagc caccaugccc ggccuaagug cauauauauu 6840

uuugggcaca uuuuuacaac uuuuaaaaaa agcuuuuuuu uuuuuuugag acggagucuu 6900

gcucugucgc ccaggcugga gugcaguggc gugaucucgg cucacugcaa gcucugccuc 6960

ccggguucac gccauucucc ugcgucagcc ucccaaguag cugggacuac aggugcccgc 7020

cacacgccca gcuaauuuuu uguauuuuua guagagaugg gguuucacca uguuagccag 7080

gauggucuca aucuccugac cucgugaucc gcccgccugg gccucccaaa gugcugggau 7140

uacaggcaug agccaccaca cccggccccu aaaaaaagcu uuuuugacuu aguagugccc 7200

aggagacagu ucuaagcaaa uuguaagcau uuucuucuga cuccuuuguc uucagauaau 7260

cucaccucuu guuugauaac uuauugagca acuuuguuuc uguuuuuuug cagggacauc 7320

aguuuuuuuc uugguagcaa gcuguagauu uuguuucaag gaaagcaucu ucaucuucuc 7380

uaguuucauc auguauagau ggagauuccg augcagguau cuguaucaau uucaauacag 7440

ucacauuugu guuuauuuga acucuagaac uagaaaagga auuacuuuuu ucuuugauuu 7500

ucuuuuucuu ccuucucuug aggaucauga aaguauaaaa gaauacuuuc uaaaugaggg 7560

ugccuuuuuc uuuuucuuuu uuuuuuuuuu acaauuuuua cauuuggaau uaaaauuuua 7620

acuagaaaag aguuuuuauu aagcaaguuu uuuuaaaaaa aucuuaaguu guuaauccug 7680

cucucaagau guaaguaagg uguguuucag acauuauuaa aauacugaaa uagugguauu 7740

uguaccaaaa cacuaguauc ucauuacuua ccaugauaua uaacacugua uaacugaaua 7800

guggaauacc aaaauugaaa uugcacacug caucuaaucc uguaugcauc caaguauauu 7860

cuucugugcc uguacugucu gccuuuuuca uguuuuggac aaaacuguca uccuuugugu 7920

accuccucuc uguguccacg uuccuauucu ccucccucag ugccuuuauu cucucccuuc 7980

ccauugcuuc cuuuguaucu gcuuacacau guucauaugu uucucuuauu uuggaaaaga 8040

aagaaaaagg ucauucauuu aaucuuaacu ugacuauuag uuucguuuuc uuuccuuccu 8100

uccuuccuuc cuucuuucuu uccuuuuuuu uuuuuuuuuu gacagugucu ugcucuguug 8160

cccagccugg agugcagugg gcacaaucuu ggcacacugc aaccucagcc uuccgagugg 8220

cuaggacuac aggugugcgc caccacgccu ggcuauuuuu uuuuuuugua cuuuuaguag 8280

agaugggguu ucaucauguu ugccaggcag cucuugaauu ccugaccuca gaucugucca 8340

ccucggccuc ccaaagugcu gggaugacag gcgugagcca ccaugcccag ccauauuagu 8400

uucuucaguu uuauaaacca aacuucuaac uaggggauuu gcugcuuuuu cacuuuacuu 8460

uaguauguug uuaucuggcu ucaguccuug ccuuuuccuu gaaauucuuu uuuuaauuuu 8520

aaagauaggg ucuugcucug ucgcccuggc uggaguucag uggcacaauc auagcuuacu 8580

gcagccugga acuagugggc ucaagugauc cucccaccuc agccucccaa guagcuggga 8640

cuacaggugc augccaccac accuggcuaa uuuuauuuau uuuauuuuuu uuuuuugugg 8700

agaugagguc ucacuauguu gcccaggcug gccucgaacu cuuggccuca agcaguccuc 8760

cugcaucggc cucccaaagu gcugggauua uaggcaugag ccaccacaaa uuccauuuuu 8820

ugaaagaauc uuguaaguua gcaaauugcu aaauccaggg aucuuuuuca guucucauuu 8880

gacacuguua auucucuacu guuuaagaau ccaucucagg uguaugaacu uggcagaaua 8940

augaagcccg uuugaccuca guuuuccugu auuaaaacuu aaaacagcuc cuaaguuuuu 9000

uugaguauca aaugcaacaa ugugucugaa aauaucucgc aucuaguuag uagcgcaccc 9060

caaccugacg gucugugaug cuuuaguugc ugccuggcuc gaaauaucua agauaguuuu 9120

uuuuccuauu uuuuuuccuc ccuccaccuu cuuugaauaa agacauucuc uaaggcgcau 9180

ucuuugaacu uuaucucuuu gccuuaucug uuggaaaucu caucuacuug cagagcuuua 9240

gcugauaaau uugggcugcc agcaaccaga uagaguacgu ccucacuuaa uguuauuaau 9300

agauucuugg aaauggucac uuuaagugaa augacaucaa ugaugucauu uuaauucagu 9360

guuguuucau uauaacauug augagaaaag aaaguugguu uucuuuuaca ucauuucacu 9420

uaaaguugca guuuccaaga gccuauugag gacacgaagg cugucaaccu ugaggcuaca 9480

uaccuugagg cuguccuagg cuaaacucug gugauccauu ucugauaacc ucuuucaaac 9540

cuucauuuga aaugcuuuuu aucaccccaa guggaaugua uaugaaacca aaccccuugu 9600

uuuuauuccc cuuuccgaca uaccuuuuaa gaguacuaaa uacauaauag cguuuuaaga 9660

guacuaauau uucugccaac cagaaaaccu caccaccauc uuugauuccc cccaucccgc 9720

cuaccuuuuu uuaaaggucu agugauuuuu cuuuccauug ugugcagaau uuuuuccuuc 9780

ucuuauuugu cuuaacacgg guuucaugca gcugaccucc ugauugaucc aguggcugau 9840

acuccccacu cugcccugau aauuggucac agugccuuga ugguguuuau guguuacaca 9900

aaaauaugua augacuuccc ucuugccucu gagcuugucc acaaacauaa agaauuucag 9960

gcccaguuua ucuuucucau cuugccccaa ccauucuucu gaugguuuac uucuuguucu 10020

cagaacaugc cuauauucuu ucacauuguc aguacagaaa agagguauau gcagaaaagg 10080

agugauguau uuucaaguua gccacuccau gagugacuag uacugaaucc cacugaaauc 10140

ccacugaaac uucccaggag gcuuaugaaa ugaaucuuag aaccagcuuc cauauuggua 10200

gcccuacagc uauuaacuuc ccauacuucc agguagugaa ugaguaaaua cagagcagug 10260

cuuuuugguu ggaccugccu gaagucaguc acagcuuaca agaaauugau ccuccauagc 10320

uggcauaaga uguuggguca ggaaguuuuu gaaaugguac auagcaugug ucuauaaugg 10380

ugugaaaguu aaaaauucca gaagauacag aagaauguag aaugagaagu uacgucucuu 10440

uuccuuuuuu guauucuaga aaaaguuugc caaccccugu ucuagucauu cugguuccuu 10500

uccuggaaac auucaucauu aaccuguuuc uugagacgug guguguauuu augauacuua 10560

caccauccac auguacacau acuuaguaua uacuuuuucu caacuucaaa auaucuuaau 10620

gauuuucuau auuaguguau agaacauucu uauuauucuu uuagcucuau aauauuccgu 10680

uauguggauc uaccauuuau uuaacuaguu uucuaucaau ggacauuuag uuguuuuuuu 10740

cuuucucuuu guuuugagac agggucucac ucuguugccc aggcuggagu gcagugaugu 10800

caucucugcu cacugcaacu uguaccuccu gggcucaagc gauccucuca ccucacagcc 10860

ucccaaauag cuggaaacua caggugugug ccaccacacc cagcuacuuu uguauuuuuu 10920

guagagacag ggucucacuu uguugcccag gcuggucuug aacuccuggg cucaagggcu 10980

ccacccaucu cccaaagugc ugggauuaca ggcaugagcc acugagccca gccgacauuu 11040

aguuauuuug ucucuuuauu acaaaugaua gcauuacauu uauacauauu uguaauccga 11100

aaaggucaau uccuugagug uuucugucuu gaacuuuuuu cuguaauagg ccucccauac 11160

ucauugccca caguuugcau ucaguuuuau uguagugggu caggaucauu aagagcugga 11220

guugcuggga uaggccuuuc ucaagagguu cuacuugagu uggaccuuga ggaaucugga 11280

ugaagagggg gauaacgaaa aggagguggg aagggaaaaa ggacugugug gcugaaggcu 11340

ugugguuagc aaaagccugu caugcuuucg agcugcacug cccagcacug uaaccaugag 11400

ccaugaguau guguggcugu gagcacuuga uauguggcua auccagaguu gaggugugcg 11460

gugaguaaau accuaccagg gucugaagau uuaauaugaa aaaaagaaug uuaaaauauc 11520

ucaguaguuu uaaaaauauu gcuuauaauu uaaauaguag cauuuuggau auauuggguu 11580

aaguaaaaug uuauuaaaau uuuuaccugg uuuuacuuuu aaauauaugg cuaccagaaa 11640

auguaaaauu guauuaugug gccuaccuuu guggcugcau gauuuuuugu uagacucugc 11700

ucuggcucau ccuucaggau ggagaagcag guucauuugg ggaaccuggu augugcuuca 11760

gugagaacug uuggauaguu accagaauca aguauuuggu augaaaagac cauuacguuu 11820

cuuaaaacag ugaaguaaca ugaaauuagu uucaggaaga uucaacuguu cauauuuugu 11880

ggcacuacaa aaacuaauug agcacuuacu guguacugag cacuguccua aagggcuuua 11940

uucauuuaau ccucacagca acccuuugag acauagguag cccuuauguu acagaugaac 12000

uugagucaca ggggggugua ccuauuggcc caaggucacg ccguaacuga uaaugccuug 12060

gcuaugaacu ggcagucuga uuccagagcc uauacacuua accacuacac ugugucccug 12120

ugaaggugaa uuagacaaag aagaugaucc uugcacauag cagugaagga augguaaugg 12180

aucacuuuuu aauccuguua auauuuuaau uaaaaaagau cuucuaucuu cacgguaccc 12240

cgauacuuuu cguuuuaagu ucuugucacc augauugguu guaugauuuu ggccacccag 12300

gaccagcuua guauguaaca agcuuuaugu uuauuuuucu uguguuuugu guguaggaag 12360

guuuuaagaa uauguagcau cgcuuaauga uggggaugug uucugagaaa cucauuauca 12420

ggcaauuuca uugugcagac guccuagagu guacucaaca caaaccuaga uguauaaccu 12480

gcuauacacc uaggcuaaau gguauagccu auggcuuuua ggcuacaaau cuguacagcg 12540

uguuacuaua uuaaauacug uaggcaguug uaacauuuua uauauauaaa aaaacauaga 12600

aaagguaaag uaaaaaauuu gguacuguaa ucuuauggga cuacugcugc auauucggcc 12660

caucauugau caaauuguca ucauguggug caugucugua cucuuaaagc aaauuuuauu 12720

uuaucccuac auuuuacaga ggaacagaga uuaaauaaug uggcugaagu uucauggcug 12780

ggaauuauaa aaccaugaga cuguguagcu uugaucacua uuguguuuga uuuaaaaagu 12840

aaacauuuug uguuugagcu uagagcuaag ugaucauaaa ugguguauau uuauuccuaa 12900

aauuuuucug ggauuucaga uccugaaauu uuauacaaac cuuuaaaaag cauuuucagg 12960

aggaaaaugu gucagagcca aguugaaauu ugaaaugccc uuuuuuuuuu uuuuaauaua 13020

aggauucuua auauuggaag agaaaggaag uuguuguuuu gagaaggggg cuugcuccau 13080

cacccaggcu ggagugcaau ggugccauua cagcucacug caggaagcuc guuuuuaaag 13140

gaccgucaac guagguggaa ccaaaauaau ggagagaaag caagaagccc uuaaaaugau 13200

acuugcuuaa uuaauuaaac ucaauucauu gagagccagc ugucaaggca acaaauacuu 13260

uccauuuugu uaaauuucuc caguauuuag cucagagguc uagucuuaau gguaaugccu 13320

aaauuuuucu gaucuuuacu guggaauuuu auaaucuuua uguuaugugu uaaggaauga 13380

caugcacacu aagaauuuau gauuuuauuc uuaccuguga cuagggccuu uguaauaaau 13440

cuguuuuuac gcuguccaaa auuacuaugu ucuuggguuu cuuuuucuuu aucucuuggg 13500

uuucuagaga uucauaagcu uaaaaaauua aaaauaaguu auuaggauug gaagagaccu 13560

uuggaagugg uuuaucuagc ccagggcguc ucagucuggg aacuguugac auuuuggaua 13620

uauaauucuu gguuguggaa gggcuguccu gugcauugua ggauguuuag cagcguuucu 13680

ggccucuacc cacuagaugc caguaguucc ccuuugauug uaacuaaaaa uguccgcaag 13740

cauugucaga uguccccugg gagacaaaau cgcccugguu gagaaccgcu ugucuagccc 13800

auuuccuggg aguggugguu cccuucucug gaacucagac guugagaaga gaaggguauu 13860

uaccaccucc ucuagcaguc uguuccaacu uugggugcgu cuuuggagac agaaaucucu 13920

ccucucuguu aagcuaaaac cugccaucuc uaacuuuuua guucugccuc uuaggaccgu 13980

auggauugga uagauucacu uaaaaacuuu cccaauuauu ggaacaugau accccguccu 14040

uccugguccc ucacgggauu guuuuagagc caagucagca aacuucuugu aguugccaug 14100

uauuugcagg cuaccaaucu cugaugcaua aucuuccuuu ccuucccucc cuccuccuug 14160

ccucccacca gccuucccuc cuuuucucuu ucuucuuauu ucuuguaacu ucuuaaaaau 14220

guaaaaaacc uuuuuuugcu cuuauaggcu ggguuuuggu ucaguaacuu gccaucccug 14280

guuuuagagu cugacaucug cuaaucuuca gagggcagug cagugcuuua gaacuguaua 14340

uagaacuuug gggaaggucu gagcagcaua gaauaacguu ggacuuguca uuaacuccuu 14400

caugcgaaga gcuuacugcu uuuuugaagc uuugucacau uuacagcgua augacagcca 14460

agacacuggu uugucuuuuu cauuaggcca cuuuaccuuc uuuuuuuuug agacagaguu 14520

ucacucauug cccauugugc aaugggcgca gucucagcuc acugcaaccu ccgccuccca 14580

gguugaagca auucucccgu cucagccucc caaguaacug ggauuauagg cuuccgccac 14640

caugccacuu uaccuucuaa gauauuuuua gacuucagcg uaggacgauc uguguuaaca 14700

gcuguauuaa acuugucuua gcuggaacca acucucuaua cccugcugag guuugugggu 14760

cuuacucugu ccuucagagu guuaaugaaa uuuuucuuag cuucccgucg uucaaauguu 14820

uuuuaaacau accugggucu uucaugcaag uuacuaauac aaauuugaau gagauguagc 14880

uaagaacuaa aucugaaauc acaaaugccu aacaaauaag guaauuuugg ccaagcgugg 14940

uggcucacgc cuguaaucuc agcacuuugg gaggcugagg uggguggauc acuugagguc 15000

aggaguuuga gaccagccug gccaacauga ugagaaaccc ugucuugacu aaaaauacaa 15060

aaauuagcug ggcuuggugg ugcauaccua uaaucccagc uacuugugag gcugaggcag 15120

gagaaucgcu ugaaccuggg agguggaggu ugcagugagc caagaucaca ccacugcacu 15180

ccagccugag ugacagagug agauuuuguc ucaaaaaaaa aaaaaaaaag ggaauuuuaa 15240

ugcauggaua uagcaggucu guacucuguu cuuaaaauug uuuuggagaa uuauauguaa 15300

aucugcauua uacauguuua guauccucaa ucuaaaaucc acagugcucc aaaaucugaa 15360

acuugagcac ugacauacau gaaaugcuca auggaacauu uuggauuucu ggauuagggg 15420

uacucagcua ugauauaaau auuccaaaau cuaaaaaaau ccaaaauucc aacuacuucu 15480

ggucccauac auuucaggua agggauacuc aaauuguagc aaccaucguc agagguuacc 15540

gucguuacug auggcuuuca gagugauuag acaguaaaua aguguaauga ccaugacaag 15600

aaaauuuuca aaaaaacuaa aucuuauuua cgugcaaaau aaaauaauuu ugaauaauga 15660

uuuuaagaag uaacugauuu uuuuuuucca gaaaauauuu acaccagcag cuccaguuca 15720

uaccaauaaa gaagauccug cuacccaaac uaauuuggga uuuauccaug cauuugucgc 15780

ugccauauca guuauuauug uaucugaauu gggugauaag acauuuuuua uagcagccau 15840

cauggcaaug cgcuauaacc gccugaccgu gcuggcuggu gcaaugcuug ccuugggacu 15900

aaugacaugc uugucag 15917

<210> 3

<211> 239

<212> RNA

<213> Homo sapiens

<400> 3

gaucagcauu gaggcccgca agucacccaa gcugccccgg gcugcucagg agcucucccg 60

guccccacgg uugccccugc gcaagcccuc ugugggcucg cccagccuga cucggagaga 120

guuuccuuuu gaagacauca cucagcacaa cuaucuugcu caggucacgu cuaauaucug 180

gggaaccaaa uuuaagauug ugggcuuggc ugcuuuccug ccaaccaacc ucggugcag 239

<210> 4

<211> 389

<212> RNA

<213> Homo sapiens

<400> 4

gcaauuuuua cccagaaaag caagccgggg ccugacccgu uggacacgag acgcuugcag 60

ggcuuucggc uggaggagua ucugauaggg caguccauug guaagggcug cagugcugcu 120

guguaugaag ccaccaugcc uacauugccc cagaaccugg aggugacaaa gagcaccggg 180

uugcuuccag ggagaggccc agguaccagu gcaccaggag aagggcagga gcgagcuccg 240

ggggccccug ccuuccccuu ggccaucaag augaugugga acaucucggc agguuccucc 300

agcgaagcca ucuugaacac aaugagccag gagcuggucc cagcgagccg aguggccuug 360

gcuggggagu auggagcagu cacuuacag 389

<210> 5

<211> 471

<212> RNA

<213> Homo sapiens

<400> 5

agggguguuu gaugcuuccc ucaaaauggc uggcuucuau ggauuguaua ccuggcugac 60

ucauacuaug uuuggcauca auauugucuu cauaccauca gcauuagcag caauccuugg 120

agcagugcca uuccugggga cauacugggc agcaguaccu gcaguucuug accuguggcu 180

gacacaaggg uuaggaugca aggccauuuu acuguugauu uuucaucucu ugccaacaua 240

cuuuguagau acugcaaucu acucugacau aucaggaggu ggccauccuu accugacagg 300

cuuggcagug gccgguggag cauacuaccu aggccuggaa ggagcaauca ucgguccuau 360

ucuucucugc auacuugugg uugcuuccaa uaucuauagu gccaugcuag ugagucccac 420

gaauucaguu cccacgccaa accagacccc auggccugcu cagccucagc g 471

<210> 6

<211> 359

<212> RNA

<213> Homo sapiens

<400> 6

guacuuuaau uaucggacua ccagguuccu ggcugaggag ggguuuuaua aauuccauaa 60

cugguuugau gaccgagccu gguacccuuu gggacgaauc auuggaggaa caauuuaccc 120

agguuuaaug aucaccucug cugcaaucua ccauguacuc cauuuuuucc acaucaccau 180

cgacauucgg aaugucugug uguuccuggc cccucucuuc uccuccuuca ccaccaucgu 240

cacguaccac cuuaccaaag agcucaagga ugcaggggcu gggcuucuug cugcugccau 300

gauugcugua guuccuggau auaucucccg aucuguggcu ggcuccuaug auaaugaag 359

<210> 7

<211> 146

<212> RNA

<213> Homo sapiens

<400> 7

uggcuggcgu ggcucaccga uuccacagua cuugcggcaa gaacgucacu cuagaggagg 60

auggcacgag ggcagugcgu gccgcuggcu augcucaugg ccuugucuuc aguaccaagg 120

agcugagggc ugaggaaguc uuugag 146

<210> 8

<211> 266

<212> RNA

<213> Homo sapiens

<400> 8

cuuggcuugu ggagauguug aaggaaaguu ugauauuuua uucaauagag uucaagcaau 60

ucagaagaaa aguggaaacu uugaucugcu guugugugua ggaaauuucu uuggcuccac 120

ccaagaugcu gaaugggagg aguauaagac uggcaucaag aaagcuccua uucagacaua 180

ugugcuuggu gcuaauaacc aggaaacagu aaaauauuuc caggaugcug auggauguga 240

auuagcugaa aacauuacuu aucugg 266

<210> 9

<211> 21

<212> DNA

<213> 人工序列

<400> 9

gtttgctggc tgggcttttc c 21

<210> 10

<211> 24

<212> DNA

<213> 人工序列

<400> 10

gggacctcta atagctgggg gttc 24

<210> 11

<211> 23

<212> DNA

<213> 人工序列

<400> 11

tgcttgcctt gggactaatg aca 23

<210> 12

<211> 20

<212> DNA

<213> 人工序列

<400> 12

cggcggctaa actggagtca 20

<210> 13

<211> 23

<212> DNA

<213> 人工序列

<400> 13

tttaagattg tgggcttggc tgc 23

<210> 14

<211> 17

<212> DNA

<213> 人工序列

<400> 14

gcgggcctca atgctga 17

<210> 15

<211> 20

<212> DNA

<213> 人工序列

<400> 15

gtggccttgg ctggggagta 20

<210> 16

<211> 23

<212> DNA

<213> 人工序列

<400> 16

ccggcttgct tttctgggta aaa 23

<210> 17

<211> 20

<212> DNA

<213> 人工序列

<400> 17

acgccaaacc agaccccatg 20

<210> 18

<211> 22

<212> DNA

<213> 人工序列

<400> 18

agccagccat tttgagggaa gc 22

<210> 19

<211> 24

<212> DNA

<213> 人工序列

<400> 19

tgtggctggc tcctatgata atga 24

<210> 20

<211> 25

<212> DNA

<213> 人工序列

<400> 20

atttataaaa cccctcctca gccag 25

<210> 21

<211> 24

<212> DNA

<213> 人工序列

<400> 21

ctgagggctg aggaagtctt tgag 24

<210> 22

<211> 20

<212> DNA

<213> 人工序列

<400> 22

agagtgacgt tcttgccgca 20

<210> 23

<211> 24

<212> DNA

<213> 人工序列

<400> 23

tgtggctggc tcctatgata atga 24

<210> 24

<211> 25

<212> DNA

<213> 人工序列

<400> 24

atttataaaa cccctcctca gccag 25

<210> 25

<211> 20

<212> DNA

<213> 人工序列

<400> 25

ttcttttgcg tcgccagccg 20

<210> 26

<211> 20

<212> DNA

<213> 人工序列

<400> 26

ggtgaccagg cgcccaatac 20

<210> 27

<211> 1455

<212> RNA

<213> Homo sapiens

<400> 27

ggcaccgcag gccccgggau gcuagugcgc agcgggugca ucccuguccg gaugcugcgc 60

cugcgguaga gcggccgcca uguugcaacc gggaaggaaa ugaaugggca gccguuagga 120

aagccugccg gugacuaacc cugcgcuccu gccucgaugg guggagucgc guguggcggg 180

gaagucaggu ggagcgaggc uagcuggccc gauuucuccu ccgggugaug cuuuuccuag 240

auuauucucu gauuuggucg uauugggcgc cuggucacca gggcugcuuu uaacucuggu 300

aaaguggaua uuguugccau caaugacccc uucauugacc ucaacuacau gguuuacaug 360

uuccaauaug auuccaccca uggcaaauuc cauggcaccg ucaaggcuga gaacgggaag 420

cuugucauca auggaaaucc caucaccauc uuccaggagc gagaucccuc caaaaucaag 480

uggggcgaug cuggcgcuga guacgucgug gaguccacug gcgucuucac caccauggag 540

aaggcugggg cucauuugca ggggggagcc aaaaggguca ucaucucugc ccccucugcu 600

gaugccccca uguucgucau gggugugaac caugagaagu augacaacag ccucaagauc 660

aucagcaaug ccuccugcac caccaacugc uuagcacccc uggccaaggu cauccaugac 720

aacuuuggua ucguggaagg acucaugacc acaguccaug ccaucacugc cacccagaag 780

acuguggaug gccccuccgg gaaacugugg cgugauggcc gcggggcucu ccagaacauc 840

aucccugccu cuacuggcgc ugccaaggcu gugggcaagg ucaucccuga gcugaacggg 900

aagcucacug gcauggccuu ccgugucccc acugccaacg ugucaguggu ggaccugacc 960

ugccgucuag aaaaaccugc caaauaugau gacaucaaga agguggugaa gcaggcgucg 1020

gagggccccc ucaagggcau ccugggcuac acugagcacc agguggucuc cucugacuuc 1080

aacagcgaca cccacuccuc caccuuugac gcuggggcug gcauugcccu caacgaccac 1140

uuugucaagc ucauuuccug guaugacaac gaauuuggcu acagcaacag ggugguggac 1200

cucauggccc acauggccuc caaggaguaa gaccccugga ccaccagccc cagcaagagc 1260

acaagaggaa gagagagacc cucacugcug gggagucccu gccacacuca gucccccacc 1320

acacugaauc uccccuccuc acaguugcca uguagacccc uugaagaggg gaggggccua 1380

gggagccgca ccuugucaug uaccaucaau aaaguacccu gugcucaacc aguuaaaaaa 1440

aaaaaaaaaa aaaaa 1455