血清miRNA-9-5p、miRNA-21-5p和miRNA-3923对子宫颈癌的诊断价值

Diagnostic value of serum miRNA-9-5p, miRNA-21-5p and miRNA-3923 in cervical cancer

目的探讨血清miRNA-9-5p(miR-9-5p)、miRNA-21-5p(miR-21-5p)和miRNA-3923(miR-3923)对子宫颈癌的诊断价值。方法收集2016年7月至2018年6月山西省肿瘤医院收治的100例子宫颈癌患者(子宫颈癌组)和同期100名健康体检者(健康对照组)资料。采用实时荧光定量聚合酶链反应(qRT-PCR)结合探针杂交检测子宫颈癌患者石蜡包埋组织和健康对照者子宫颈脱落细胞中人乳头瘤病毒(HPV)E6/E7 mRNA的表达水平, 以Ct值≤40个循环为HPV E6/E7阳性。取3例子宫颈癌患者与3名健康对照者血清样本, 采用微RNA(miRNA)Array检测384个miRNA基因表达量, 并进行差异miRNA表达筛选及聚类分析, 采用qRT-PCR对筛选的miRNA进行验证。绘制最终筛选的各miRNA及HPV E6/E7单独或联合诊断子宫颈癌的受试者工作特征(ROC)曲线, 比较诊断子宫颈癌效能。结果 100例子宫颈癌患者石蜡包埋组织中, HPV E6/E7阳性65例(65%);100名健康对照者子宫颈脱落细胞HPV E6/E7均为阴性。3例子宫颈癌患者与3名健康对照者血清样本中发现248个差异表达的miRNA, 经聚类分析鉴定出前16个调控异常miRNA。经qRT-PCR验证, 确认在健康对照组和子宫颈癌组中miR-9-5p、miR-21-5p和miR-3923表达水平差异均有统计学意义(均P<0.01), 选取三者用于后续子宫颈癌的诊断。与健康对照组比较, HPV E6/E7阳性子宫颈癌组miR-9-5p、miR-21-5p和miR-3923表达水平均增高(均P<0.05), HPV E6/E7阴性子宫颈癌组miR-21-5p和miR-3923表达水平均增高(P=0.008和P=0.038);HPV E6/E7阳性组3个miRNA表达水平均高于HPV E6/E7阴性组(均P<0.05)。经ROC曲线分析, miR-9-5p、miR-21-5p和miR-3923单独诊断子宫颈癌时, miR-3923的曲线下面积(AUC)最大(0.843), 特异度也最高(82%, 截断值为2.88);miR-21-5p的灵敏度最高(85%, 截断值为4.08);3个miRNA联合诊断子宫颈癌的AUC(0.924)和灵敏度、特异度(85%、94%, 截断值为4.04)均高于单一指标。HPV E6/E7单独诊断的AUC为0.766, miR-9-5p、miR-21-5p、miR-3923分别联合HPV E6/E7的诊断效能进一步增高, AUC分别为0.914、0.848和0.932;四者联合的诊断效能最高, AUC为0.942。结论 miR-9-5p、miR-21-5p和miR-3923可能有助于子宫颈癌诊断。

Objective To investigate the diagnostic value of serum miRNA-9-5p(miR-9-5p),miRNA-21-5p(miR-21-5p)and miRNA-3923(miR-3923)in patients with cervical cancer.Methods The data of 100 cervical cancer patients in Shanxi Provincial Cancer Hospital from July 2016 to June 2018(the experimental group)and 100 healthy subjects(the healthy control group)during the same period were collected.The real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)combined with probe hybridization was used to detect the expression levels of human papillomavirus(HPV)E6/E7 mRNA in paraffin-embedded tissues of patients with cervical cancer and in cervical exfoliated cells of the healthy control people.Ct value≤40 cycles was considered as HPV E6/E7 positive.Serum samples from 3 patients with cervical cancer and 3 healthy people were taken out;microRNA(miRNA)Array was used to detect the expression level of 384 miRNA;the differential expression of miRNA was screened out and cluster analysis was performed,and then the screened miRNAs was verified by using qRT-PCR.Finally,the receiver operation characteristics(ROC)curves of screened miRNAs alone and HPV E6/E7 alone or the combination of three miRNAs and HPV E6/E7 in the diagnosis of cervical cancer were drawn to make comparison of diagnostic efficacy.Results Among the paraffin-embedded tissues from 100 patients with cervical cancer,there were 65 cases(65%)HPV E6/E7 positive;in the healthy control group,HPV E6/E7 in cervical exfoliated cells of 100 people was negative.A total of 248 differentially expressed miRNAs were detected from the serum samples in 3 patients with cervical cancer and 3 healthy people.The cluster analysis finally identified 16 abnormally regulated miRNAs.qRT-PCR verification confirmed that differences in the expression levels of miR-9-5p,miR-21-5p and miR-3923 in the healthy control group and cervical cancer group were statistically significant(all P<0.01),and then the three were selected to make diagnosis of cervical cancer.The expression levels of miR-9-5p,miR-21-5p and miR-3923 in HPV E6/E7 positive cervical cancer group were higher than those in the healthy control group(all P<0.05),expression levels of miR-21-5p and miR-3923 in HPV E6/E7 negative cervical cancer group were increased(P=0.008,P=0.038);expression levels of the three miRNAs in HPV E6/E7 positive group were higher than those in HPV E6/E7 negative group(all P<0.05).ROC curve analysis showed that the area under the curve(AUC)of miR-3923 was the biggest(0.843),the specificity was the highest(82%,the cut-off value was 2.88)and the sensitivity of miR-21-5p was the highest(85%,the cut-off value was 4.08)when miR-9-5p,miR-21-5p and miR-3923 were respectively applied to diagnose the cervical cancer;AUC(0.924),the sensitivity and the specificity(85%,94%;the cut-off value was 4.04)of the combination of the three indicators were higher than those of the single indicator in the diagnosis of cervical cancer.AUC was 0.766 when HPV E6/E7 was kused alone to diagnose.The diagnostic efficacy of HPV E6/E7 combined with miR-9-5p,miR-21-5p,miR-3923 respectively was further improved,the corresponding AUC was 0.914,0.848,0.932,respectively;the diagnostic efficacy of the combination of the four indicators was the highest(AUC was 0.942).Conclusion miR-9-5p,miR-21-5p and miR-3923 may be helpful in the diagnosis of cervical cancer.