宫颈脱落细胞microRNA检测在宫颈癌筛查中的作用

发布时间：2018-04-21 10:30

本文选题：miRNA + 宫颈上皮内瘤变　；参考：《浙江大学》2014年博士论文

【摘要】：官颈癌是目前世界上第三高发的女性恶性肿瘤,其发生一般需经历从正常宫颈上皮到不同级别癌前病变的过程,平均需5-20年。根据宫颈癌发病率较高和发病过程较缓慢的特点,结合官颈易于被暴露和取材的特点,宫颈癌是目前唯一被世界卫生组织确定为可以通过筛查降低浸润癌发生率的恶性肿瘤。高危型人乳头瘤病毒(HPV)检测和宫颈细胞学检查是目前最主要的两种宫颈癌筛查方法。HPV检测具有敏感性高,阴性预测值高,结果客观可靠等优点。欧洲生殖道感染和肿瘤研究组织(European Research Organization on Genital Infection and Neoplasia,EUROGIN)已经推荐HPV检测作为宫颈癌筛查的初筛手段。在许多欧洲国家和一些细胞病理学医生相对较为缺乏的发展中国家,HPV检测已被广泛用于宫颈癌的初筛。HPV检测的最大局限性是阳性预测值低,在HPV检测结果为阳性的大量人群中,实际发生或将发生宫颈上皮内瘤变或宫颈癌的患者很少。因此,HPV检测可能导致HPV阳性妇女产生不必要的心理焦虑,甚至过度诊疗。宫颈细胞学检查具有特异性高和阳性预测值高的优点,已被EUROGIN推荐用于HPV初筛后的分层筛查。但宫颈细胞学检查的敏感性较低,仅38%～65%,而且,结果判断有较大的主观性,依赖于经过专业培训的细胞病理学医生,而这在我国许多欠发达地区仍是严重缺乏的。因此,在HPV初筛阳性妇女中,探索一种新的分层筛查方法意义重大。 MicroRNA (miRNA)是一类基因转录后调节因子,参与细胞凋亡,细胞增殖,和细胞分化等许多重要生物学过程。在许多人类恶性肿瘤中均已发现存在miRNA表达异常。近来,一些研究评估了miRNA作为生物标记物在肿瘤诊断中的价值,并取得了较好的结果。宫颈癌及其癌前病变中也存在miRNA表达异常。研究表明miR-218和miR-34a在官颈癌的发生中具有抑癌基因的作用。在我们先前的研究中,首次在宫颈癌及其癌前病变组织中发现了14个表达下调的miRNA(包括miR-375和miR-424等)和17个表达上调的miRNA(包括miR-93和miR-92a等)。我们进一步研究发现miR-375,miR-424,和miR-93分别通过作用于Spl,Chk1,和RAB11FIP1参与官颈癌的发生和发展。其他研究也发现miR-34a参与HPV E6-p53降解途径,miR-218通过靶基因LAMB3参与宫颈癌的发生、发展。这些miRNA在宫颈癌及其癌前病变中的异常表达提示它们可作为宫颈癌的生物标记物,在宫颈癌筛查中具有潜在的临床应用价值。根据一些miRNA在官颈癌及其癌前病变组织中异常表达的情况,以及它们在宫颈癌发生、发展中的作用机制,我们在本研究中选择了6个miRNA(miR-424, miR-375, miR-218, miR-34a,miR-92a和miR-93)作为宫颈癌筛查的候选生物标记物。首先研究了miRNA在正常宫颈、宫颈上皮内瘤变及浸润性宫颈癌的宫颈脱落细胞中的表达情况；在此基础上,进一步评估比较了宫颈脱落细胞miRNA检测和宫颈细胞学检查在HPV初筛阳性妇女中的分层筛查价值。第一部分miRNA在正常宫颈、宫颈上皮内瘤变及浸润性官颈癌的宫颈脱落细胞中的表达目的比较并证实在正常宫颈、宫颈上皮内瘤变及浸润性宫颈癌的宫颈脱落细胞中候选miRNA的表达水平存在显著差异。方法研究对象入组标准：年龄30-65岁；宫颈完整；否认既往宫颈上皮内瘤变(Cervical intraepithelial neoplasia, CIN),或宫颈癌,或其他恶性肿瘤病史；非孕妇。收集宫颈脱落细胞,用Stem loop RT-qPCR方法在宫颈脱落细胞中检测候选miRNA (miR-424,miR-375, miR-34a,miR-218,miR-93,miR-92a)的相对表达水平(以U6为内参)。以宫颈组织学诊断作为分组依据,比较CINl-(≤CIN1)组和CIN2+(≥CIN2)组,CIN2-组和CIN3+组,CIN3-组和ICC(Invasive cervical cancer)组之间各候选miRNA的相对表达水平。用SPSS17.0软件统计分析,具体统计方法为Student t检验和非参数Mann-Whitney U检验。所有统计分析都采用双侧检验,P0.05时差异具有统计学显著性。结果 1.共收集有效病例735例,其中：240例正常宫颈,100例CIN1,111例CIN2,117例CIN3,167例ICC。 2.CIN1-组与CIN2+组、CIN2-组与CIN3+组、CIN3-组与ICC组之间的年龄分布均无显著性统计学差异(P0.05)。 3.CIN1-组、CIN2-组和CIN3-组的宫颈脱落细胞中miR-424、miR-375、 miR-34a和miR-218的相对表达水平分别显著低于它们在CIN2+组、CIN3+组和ICC组中的相对表达水平(P0.001)。miR-92a和miR-93的相对表达水平在上述各组之间无显著性统计学差异(P0.05)。结论： 1.在宫颈脱落细胞中检测miRNA简便可行。 2. miR-424、miR-375、miR-34a和miR-218在高级别宫颈上皮内瘤变(CIN2-3)和浸润性宫颈癌的宫颈脱落细胞中的相对表达水平显著低于它们在低级别宫颈上皮内瘤变(CIN1)和正常官颈中的表达。miR-92a和miR-93在不同宫颈组织病理状态的宫颈脱落细胞中的表达没有显著差异。 3. miR-424、miR-375、miR-34a和miR-218在官颈癌筛查中具有潜在的临床应用价值。第二部分比较官颈脱落细胞miRNA检测和官颈细胞学检查在HPV阳性妇女中的分层筛查作用目的：比较宫颈脱落细胞miRNA检测和宫颈细胞学检查在HPV阳性妇女中的分层筛查作用。方法研究对象入组标准：年龄30-65岁；宫颈HPV初筛阳性；宫颈完整；否认既往CIN,或宫颈癌,或其他恶性肿瘤病史；非孕妇。所有研究对象均行宫颈细胞学检查和宫颈脱落细胞miRNA检测(miR-424,miR-375,miR-34a, miR-218, miR-93,miR-92a),并接受阴道镜检查。宫颈细胞学检查和阴道镜检查均阴性者不做宫颈活检术,视作组织学正常；宫颈细胞学检查阴性,但阴道镜检查发现异常者行宫颈活检术；宫颈细胞学检查异常者,无论阴道镜检查是否发现异常,均行宫颈活检术。宫颈细胞学检查采用ThinPrep液基细胞学方法；高危型HPV检测采用杂交捕获-2(Hybrid Capture-2,HC2)的方法；miRNA检测采用Stem loop RT-qPCR方法(以U6为内参)。所有统计学分析均采用SPSS17.0软件。用非参数Mann-Whitney U检验比较不同宫颈组织学分组之间的宫颈脱落细胞miRNA表达水平；用ROC曲线,Pearson卡方检验和Logistic回归分析等比较miRNA检测与宫颈细胞学检查在诊断高级别宫颈上皮内瘤变中的作用。所有统计学分析均采用双侧检验,P0.05时差异具有统计学显著性。结果 1.共收集有效病例1021例,这些病例最终被诊断为：579例正常宫颈,83例CIN1,144例CIN2,208例C1N3,和7例ICC。 2.CIN1-组与CIN2+组、CIN2-组与CIN3+组之间的年龄分布无显著性统计学差异(P0.05). miR-424, miR-375, miR-34a, miR-218在CIN2+组和CIN3+组中的相对表达水平分别显著低于它们在CIN1-组和CIN2-组中的相对表达水平(P0.001)。 3.在HPV阳性妇女中分层筛查CIN2+时,与官颈细胞学检查相比：miR-424和miR-375检测的敏感性(76.4%和74.9%vs.63.8%;P0.05),阳性预测值(65.3%和66.3%vs.58.4%； P0.05)和阴性预测值(85.7%和85.4%vs.79.3%；P0.05)均显著高于宫颈细胞学检查；特异性无显著性统计学差异。在HPV阳性妇女中分层筛查CIN3+时,与宫颈细胞学检查相比：miR-424和miR-375检测的敏感性(82.3%和80.9%vs.69.8%；P0.05)和阴性预测值(93.8%和93.4%vs.89.7%；P0.05)均显著高于宫颈细胞学检查；特异性和阳性预测值均无显著性统计学差异。 4.通过Logistic回归分析,得到两个分别用于分层筛查CIN2+和CIN3+的联合检测模型：miR-424/375/218和miR-424/375。这两个联合检测模型在筛查高级别宫颈上皮内瘤变时的敏感性、特异性、阳性预测值、阴性预测值均显著高于宫颈细胞学检查；与单一miR-424或miR-375检测相比,两种联合检测的特异性有显著提高,敏感性、阳性预测值、阴性预测值均无显著差异。结论 1.在HPV阳性妇女中分层筛查高级别宫颈上皮内瘤变时,与宫颈细胞学检查相比,miR-424和miR-375检测在保持特异性和阳性预测值不下降的同时,敏感性和阴性预测值均显著优于宫颈细胞学检查。 2.两种联合检测(miR-424/375/218和miR-424/375)均优于单一miR-424或miR-375检测,或宫颈细胞学检查。 3.在HPV阳性妇女中,官颈脱落细胞miRNA检测可能成为一种优于宫颈细胞学检查的分层筛查方法。
[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 .

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33

【参考文献】