本文選題：miRNA + 宮頸上皮內(nèi)瘤變　； 參考：《浙江大學(xué)》2014年博士論文

【摘要】：官頸癌是目前世界上第三高發(fā)的女性惡性腫瘤,其發(fā)生一般需經(jīng)歷從正常宮頸上皮到不同級別癌前病變的過程,平均需5-20年。根據(jù)宮頸癌發(fā)病率較高和發(fā)病過程較緩慢的特點,結(jié)合官頸易于被暴露和取材的特點,宮頸癌是目前唯一被世界衛(wèi)生組織確定為可以通過篩查降低浸潤癌發(fā)生率的惡性腫瘤。 高危型人乳頭瘤病毒(HPV)檢測和宮頸細(xì)胞學(xué)檢查是目前最主要的兩種宮頸癌篩查方法。HPV檢測具有敏感性高,陰性預(yù)測值高,結(jié)果客觀可靠等優(yōu)點。歐洲生殖道感染和腫瘤研究組織(European Research Organization on Genital Infection and Neoplasia,EUROGIN)已經(jīng)推薦HPV檢測作為宮頸癌篩查的初篩手段。在許多歐洲國家和一些細(xì)胞病理學(xué)醫(yī)生相對較為缺乏的發(fā)展中國家,HPV檢測已被廣泛用于宮頸癌的初篩。HPV檢測的最大局限性是陽性預(yù)測值低,在HPV檢測結(jié)果為陽性的大量人群中,實際發(fā)生或?qū)l(fā)生宮頸上皮內(nèi)瘤變或?qū)m頸癌的患者很少。因此,HPV檢測可能導(dǎo)致HPV陽性婦女產(chǎn)生不必要的心理焦慮,甚至過度診療。宮頸細(xì)胞學(xué)檢查具有特異性高和陽性預(yù)測值高的優(yōu)點,已被EUROGIN推薦用于HPV初篩后的分層篩查。但宮頸細(xì)胞學(xué)檢查的敏感性較低,僅38%～65%,而且,結(jié)果判斷有較大的主觀性,依賴于經(jīng)過專業(yè)培訓(xùn)的細(xì)胞病理學(xué)醫(yī)生,而這在我國許多欠發(fā)達(dá)地區(qū)仍是嚴(yán)重缺乏的。因此,在HPV初篩陽性婦女中,探索一種新的分層篩查方法意義重大。 MicroRNA (miRNA)是一類基因轉(zhuǎn)錄后調(diào)節(jié)因子,參與細(xì)胞凋亡,細(xì)胞增殖,和細(xì)胞分化等許多重要生物學(xué)過程。在許多人類惡性腫瘤中均已發(fā)現(xiàn)存在miRNA表達(dá)異常。近來,一些研究評估了miRNA作為生物標(biāo)記物在腫瘤診斷中的價值,并取得了較好的結(jié)果。宮頸癌及其癌前病變中也存在miRNA表達(dá)異常。研究表明miR-218和miR-34a在官頸癌的發(fā)生中具有抑癌基因的作用。在我們先前的研究中,首次在宮頸癌及其癌前病變組織中發(fā)現(xiàn)了14個表達(dá)下調(diào)的miRNA(包括miR-375和miR-424等)和17個表達(dá)上調(diào)的miRNA(包括miR-93和miR-92a等)。我們進(jìn)一步研究發(fā)現(xiàn)miR-375,miR-424,和miR-93分別通過作用于Spl,Chk1,和RAB11FIP1參與官頸癌的發(fā)生和發(fā)展。其他研究也發(fā)現(xiàn)miR-34a參與HPV E6-p53降解途徑,miR-218通過靶基因LAMB3參與宮頸癌的發(fā)生、發(fā)展。這些miRNA在宮頸癌及其癌前病變中的異常表達(dá)提示它們可作為宮頸癌的生物標(biāo)記物,在宮頸癌篩查中具有潛在的臨床應(yīng)用價值。 根據(jù)一些miRNA在官頸癌及其癌前病變組織中異常表達(dá)的情況,以及它們在宮頸癌發(fā)生、發(fā)展中的作用機制,我們在本研究中選擇了6個miRNA(miR-424, miR-375, miR-218, miR-34a,miR-92a和miR-93)作為宮頸癌篩查的候選生物標(biāo)記物。首先研究了miRNA在正常宮頸、宮頸上皮內(nèi)瘤變及浸潤性宮頸癌的宮頸脫落細(xì)胞中的表達(dá)情況；在此基礎(chǔ)上,進(jìn)一步評估比較了宮頸脫落細(xì)胞miRNA檢測和宮頸細(xì)胞學(xué)檢查在HPV初篩陽性婦女中的分層篩查價值。 第一部分miRNA在正常宮頸、宮頸上皮內(nèi)瘤變及浸潤性官頸癌的宮頸脫落細(xì)胞中的表達(dá) 目的 比較并證實在正常宮頸、宮頸上皮內(nèi)瘤變及浸潤性宮頸癌的宮頸脫落細(xì)胞中候選miRNA的表達(dá)水平存在顯著差異。 方法 研究對象入組標(biāo)準(zhǔn)：年齡30-65歲；宮頸完整；否認(rèn)既往宮頸上皮內(nèi)瘤變(Cervical intraepithelial neoplasia, CIN),或?qū)m頸癌,或其他惡性腫瘤病史；非孕婦。收集宮頸脫落細(xì)胞,用Stem loop RT-qPCR方法在宮頸脫落細(xì)胞中檢測候選miRNA (miR-424,miR-375, miR-34a,miR-218,miR-93,miR-92a)的相對表達(dá)水平(以U6為內(nèi)參)。以宮頸組織學(xué)診斷作為分組依據(jù),比較CINl-(≤CIN1)組和CIN2+(≥CIN2)組,CIN2-組和CIN3+組,CIN3-組和ICC(Invasive cervical cancer)組之間各候選miRNA的相對表達(dá)水平。用SPSS17.0軟件統(tǒng)計分析,具體統(tǒng)計方法為Student t檢驗和非參數(shù)Mann-Whitney U檢驗。所有統(tǒng)計分析都采用雙側(cè)檢驗,P0.05時差異具有統(tǒng)計學(xué)顯著性。 結(jié)果 1.共收集有效病例735例,其中：240例正常宮頸,100例CIN1,111例CIN2,117例CIN3,167例ICC。 2.CIN1-組與CIN2+組、CIN2-組與CIN3+組、CIN3-組與ICC組之間的年齡分布均無顯著性統(tǒng)計學(xué)差異(P0.05)。 3.CIN1-組、CIN2-組和CIN3-組的宮頸脫落細(xì)胞中miR-424、miR-375、 miR-34a和miR-218的相對表達(dá)水平分別顯著低于它們在CIN2+組、CIN3+組和ICC組中的相對表達(dá)水平(P0.001)。miR-92a和miR-93的相對表達(dá)水平在上述各組之間無顯著性統(tǒng)計學(xué)差異(P0.05)。 結(jié)論： 1.在宮頸脫落細(xì)胞中檢測miRNA簡便可行。 2. miR-424、miR-375、miR-34a和miR-218在高級別宮頸上皮內(nèi)瘤變(CIN2-3)和浸潤性宮頸癌的宮頸脫落細(xì)胞中的相對表達(dá)水平顯著低于它們在低級別宮頸上皮內(nèi)瘤變(CIN1)和正常官頸中的表達(dá)。miR-92a和miR-93在不同宮頸組織病理狀態(tài)的宮頸脫落細(xì)胞中的表達(dá)沒有顯著差異。 3. miR-424、miR-375、miR-34a和miR-218在官頸癌篩查中具有潛在的臨床應(yīng)用價值。 第二部分比較官頸脫落細(xì)胞miRNA檢測和官頸細(xì)胞學(xué)檢查在HPV陽性婦女中的分層篩查作用 目的： 比較宮頸脫落細(xì)胞miRNA檢測和宮頸細(xì)胞學(xué)檢查在HPV陽性婦女中的分層篩查作用。 方法 研究對象入組標(biāo)準(zhǔn)：年齡30-65歲；宮頸HPV初篩陽性；宮頸完整；否認(rèn)既往CIN,或?qū)m頸癌,或其他惡性腫瘤病史；非孕婦。所有研究對象均行宮頸細(xì)胞學(xué)檢查和宮頸脫落細(xì)胞miRNA檢測(miR-424,miR-375,miR-34a, miR-218, miR-93,miR-92a),并接受陰道鏡檢查。宮頸細(xì)胞學(xué)檢查和陰道鏡檢查均陰性者不做宮頸活檢術(shù),視作組織學(xué)正常；宮頸細(xì)胞學(xué)檢查陰性,但陰道鏡檢查發(fā)現(xiàn)異常者行宮頸活檢術(shù)；宮頸細(xì)胞學(xué)檢查異常者,無論陰道鏡檢查是否發(fā)現(xiàn)異常,均行宮頸活檢術(shù)。宮頸細(xì)胞學(xué)檢查采用ThinPrep液基細(xì)胞學(xué)方法；高危型HPV檢測采用雜交捕獲-2(Hybrid Capture-2,HC2)的方法；miRNA檢測采用Stem loop RT-qPCR方法(以U6為內(nèi)參)。所有統(tǒng)計學(xué)分析均采用SPSS17.0軟件。用非參數(shù)Mann-Whitney U檢驗比較不同宮頸組織學(xué)分組之間的宮頸脫落細(xì)胞miRNA表達(dá)水平；用ROC曲線,Pearson卡方檢驗和Logistic回歸分析等比較miRNA檢測與宮頸細(xì)胞學(xué)檢查在診斷高級別宮頸上皮內(nèi)瘤變中的作用。所有統(tǒng)計學(xué)分析均采用雙側(cè)檢驗,P0.05時差異具有統(tǒng)計學(xué)顯著性。 結(jié)果 1.共收集有效病例1021例,這些病例最終被診斷為：579例正常宮頸,83例CIN1,144例CIN2,208例C1N3,和7例ICC。 2.CIN1-組與CIN2+組、CIN2-組與CIN3+組之間的年齡分布無顯著性統(tǒng)計學(xué)差異(P0.05). miR-424, miR-375, miR-34a, miR-218在CIN2+組和CIN3+組中的相對表達(dá)水平分別顯著低于它們在CIN1-組和CIN2-組中的相對表達(dá)水平(P0.001)。 3.在HPV陽性婦女中分層篩查CIN2+時,與官頸細(xì)胞學(xué)檢查相比：miR-424和miR-375檢測的敏感性(76.4%和74.9%vs.63.8%;P0.05),陽性預(yù)測值(65.3%和66.3%vs.58.4%； P0.05)和陰性預(yù)測值(85.7%和85.4%vs.79.3%；P0.05)均顯著高于宮頸細(xì)胞學(xué)檢查；特異性無顯著性統(tǒng)計學(xué)差異。在HPV陽性婦女中分層篩查CIN3+時,與宮頸細(xì)胞學(xué)檢查相比：miR-424和miR-375檢測的敏感性(82.3%和80.9%vs.69.8%；P0.05)和陰性預(yù)測值(93.8%和93.4%vs.89.7%；P0.05)均顯著高于宮頸細(xì)胞學(xué)檢查；特異性和陽性預(yù)測值均無顯著性統(tǒng)計學(xué)差異。 4.通過Logistic回歸分析,得到兩個分別用于分層篩查CIN2+和CIN3+的聯(lián)合檢測模型：miR-424/375/218和miR-424/375。這兩個聯(lián)合檢測模型在篩查高級別宮頸上皮內(nèi)瘤變時的敏感性、特異性、陽性預(yù)測值、陰性預(yù)測值均顯著高于宮頸細(xì)胞學(xué)檢查；與單一miR-424或miR-375檢測相比,兩種聯(lián)合檢測的特異性有顯著提高,敏感性、陽性預(yù)測值、陰性預(yù)測值均無顯著差異。 結(jié)論 1.在HPV陽性婦女中分層篩查高級別宮頸上皮內(nèi)瘤變時,與宮頸細(xì)胞學(xué)檢查相比,miR-424和miR-375檢測在保持特異性和陽性預(yù)測值不下降的同時,敏感性和陰性預(yù)測值均顯著優(yōu)于宮頸細(xì)胞學(xué)檢查。 2.兩種聯(lián)合檢測(miR-424/375/218和miR-424/375)均優(yōu)于單一miR-424或miR-375檢測,或?qū)m頸細(xì)胞學(xué)檢查。 3.在HPV陽性婦女中,官頸脫落細(xì)胞miRNA檢測可能成為一種優(yōu)于宮頸細(xì)胞學(xué)檢查的分層篩查方法。
[Abstract]:Official neck cancer is the third high - onset female malignant tumor in the world . It takes about 5 - 20 years . According to the characteristics of higher incidence and slow onset of cervical cancer , the cervical cancer is the only malignant tumor which is currently identified by the World Health Organization as the only malignant tumor that can reduce the incidence of invasive cancer .

HPV detection and cervical cytology are the most important methods for cervical cancer screening . HPV testing has the advantages of high sensitivity , high negative predictive value and objective and reliable results . HPV testing has been widely used in cervical cancer screening .

MicroRNA ( miRNA ) is a class of important biological processes such as regulation factor , cell apoptosis , cell proliferation , and cell differentiation . In recent studies , there are 14 downregulated miRNA ( including miR - 375 and miR - 424 , etc . ) and 17 up - regulated miRNA ( including miR - 93 and miR - 92a , etc . ) in cervical cancer and precancerous lesions .

In this study , six miRNA ( miR - 424 , miR - 375 , miR - 218 , miR - 34a , miR - 92a and miR - 93 ) were selected as candidate biomarkers for cervical cancer screening .
On the basis of this , we further evaluated the value of hierarchical screening in HPV primary screen positive women compared with the detection of cervical exfoliated cells and cervical cytology .

Expression of the first miRNA in the cervical exfoliated cells of normal cervix , endocervical neoplasia and invasive cervical cancer

Purpose

There was a significant difference in the level of expression of the candidate miRNA in the cervical exfoliated cells of normal cervix , endocervical neoplasia and invasive cervical cancer .

method

Inclusion criteria for study subjects : 30 - 65 years of age ;
Cervical integrity ;
To deny the history of prior cervical neoplasia ( CIN ) , or cervical cancer , or other malignant tumors ;
The relative expression level of candidate miRNA ( miR - 424 , miR - 375 , miR - 34a , miR - 218 , miR - 93 , miR - 92a ) was detected by stem loop RT - qPCR in cervical exfoliated cells .

Results

1 . 735 cases of effective cases were collected , including 240 normal cervix , 100 CIN1 , 111 CIN2 , 117 CIN3 and 167 ICC .

2 . There was no statistical difference between CIN1 - group and CIN2 + group , CIN2 - group and CIN3 + group , CIN3 - group and ICC group ( P0.05 ) .

The relative expression levels of miR - 424 , miR - 375 , miR - 34a and miR - 218 in CIN1 - group , CIN2 - group and CIN3 - group were significantly lower than those in CIN2 + , CIN3 + and ICC ( P0.05 ) .

Conclusion :

1 . Detection of miRNA in cervical exfoliated cells is simple and feasible .

2 . The relative expression levels of miR - 424 , miR - 375 , miR - 34a and miR - 218 in the cervical exfoliated cells of the high - level cervical epithelial neoplasia ( CIN2 - 3 ) and invasive cervical cancer were significantly lower than their expression in the low - grade cervical epithelial neoplasia ( CIN1 ) and the normal official neck . The expression of miR - 92a and miR - 93 in the cervical exfoliated cells of different cervical tissues was not significantly different .

3 . miR - 424 , miR - 375 , miR - 34a and miR - 218 have potential clinical application values in cervical cancer screening .

The second part compares the role of miRNA detection and cervical cytology in the detection of cervical cytology in HPV - positive women .

Purpose :

To compare the effect of cervical cytology and cervical cytology on the screening of HPV positive women .

method

Inclusion criteria for study subjects : 30 - 65 years of age ;
Positive cervical HPV screen ;
Cervical integrity ;
denying the history of previous CIN , or cervical cancer , or other malignancy ;
Non - pregnant women . All study subjects were examined for cervical cytology and cervical exfoliated cell miRNA ( miR - 424 , miR - 375 , miR - 34a , miR - 218 , miR - 93 , miR - 92a ) , and underwent vaginal speculum examination .
The cervical cytology test was negative , but the vaginoscopy revealed abnormal cervical biopsy ;
Abnormal cervical cytology was performed , and cervical biopsy was performed regardless of whether it was found abnormal or not . Cytological examination of cervical cytology was performed by ThinPrep liquid - based cytology .
high - risk HPV detection adopt hybrid capture - 2 ( hybrid Capture - 2 , HC2 ) method ;
Stem loop RT - qPCR was used to detect miRNA expression . SPSS 17.0 software was used in all statistical analyses . Non - parametric Mann - Whitney U test was used to compare the level of miRNA expression between different cervical histological subgroups .
ROC curves , Pearson chi - square test and Logistic regression analysis were used to examine the role of miRNA detection and cervical cytology in the diagnosis of high - grade cervical neoplasia . All statistical analyses showed statistically significant difference between the two - sided test ( P0.05 ) .

Results

1 . There were 1021 effective cases , which were eventually diagnosed as : 579 normal cervix , 83 cases CIN1 , 144 CIN2 , 208 cases C1N3 , and 7 ICC .

The relative expression levels of miR - 424 , miR - 375 , miR - 34a and miR - 218 in CIN2 + and CIN3 + groups were significantly lower than those in CIN2 + and CIN3 + groups ( P0.001 ) .

3 . The sensitivity of miR - 424 and miR - 375 detection ( 76.4 % and 74.9 % vs . 63.8 % ; P0.05 ) , positive predictive value ( 65.3 % and 66.3 % vs . 58.4 % ) were compared with cervical cytology .
P0.05 ) and negative predictive value ( 85.7 % and 85.4 % vs . 79.3 % ;
P0 . 05 ) was significantly higher than that of cervical cytology ;
There was no significant difference in specificity . In HPV - positive women , the sensitivity of miR - 424 and miR - 375 was 82.3 % and 80.9 % vs . 69.8 % compared with cervical cytology .
P0.05 ) and negative predictive value ( 93.8 % and 93.4 % vs . 89.7 % ;
P0 . 05 ) was significantly higher than that of cervical cytology ;
There was no significant statistical difference between specificity and positive predictive value .

4 . Two joint detection models for screening CIN2 + and CIN3 + were obtained by Logistic regression analysis : miR - 424 / 375 / 218 and miR - 424 / 375 . The sensitivity , specificity , positive predictive value and negative predictive value of the two joint detection models in screening of high - grade cervical epithelial neoplasia were significantly higher than that of cervical cytology ;
Compared with single miR - 424 or miR - 375 detection , the specificity of the two joint detection is obviously improved , the sensitivity , the positive predictive value and the negative predictive value are not significantly different .

Conclusion

1 . The sensitivity and negative predictive values of miR - 424 and miR - 375 were significantly better than cervical cytology compared with cervical cytology when the high - level cervical neoplasia was stratified by stratified screening in HPV - positive women .

2 . Both joint assays ( miR - 424 / 375 / 218 and miR - 424 / 375 ) are superior to single miR - 424 or miR - 375 detection , or cervical cytology .

3 . In HPV - positive women , miRNA detection of cervical exfoliated cells can be a hierarchical screening method superior to cervical cytology .

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.33

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Wanqing Chen;Rongshou Zheng;Siwei Zhang;Ping Zhao;Guanglin Li;Lingyou Wu;Jie He;;The incidences and mortalities of major cancers in China, 2009[J];Chinese Journal of Cancer;2013年03期

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本文編號：1782046