MicroRNA是具有重要调控功能的非编码小RNA。虽然在癌症方面研究得很好,关于microRNAs在癌前病变中的作用知之甚少。完整的葡萄胎是妊娠滋养细胞疾病的良性形式,在多达20%的病例中发展为妊娠滋养细胞肿瘤;然而,目前还没有确定的生物标志物可以预测妊娠滋养细胞肿瘤的发展。
本研究旨在研究进展为妊娠滋养细胞肿瘤的完整痣和手术后消退的微RNA表达的可能差异。
从巴西子宫排空时收集的39个完整痣的新鲜冷冻组织中提取总RNA。在研究中,39例获得人绒毛膜促性腺激素正常化,无需进一步治疗,9例发生妊娠滋养细胞瘤,需要化疗。还从2个绒毛膜癌细胞系中提取总RNA,JEG-3和JAR,和永生化的正常胎盘细胞系,3A-subE.使用microRNA测序定量所有样品中的microRNA表达。使用基于定量探针的测定验证来自测序数据的命中。然后对显著改变的microRNA进行靶标预测和基因本体论分析以搜索关键信号传导途径的改变。通过定量实时聚合酶链反应和蛋白质印迹评估潜在微小RNA靶标的表达。最后,使用定量免疫组织化学方法,在一组独立的来自美国的福尔马林固定石蜡包埋患者样本(15个完全痣进展为妊娠滋养细胞肿瘤,12个自发消退)中验证了潜在的预后蛋白生物标志物.
总共,在所有样品中以〈1标签/百万的阈值鉴定462个微小RNA。MicroRNA测序揭示了与妊娠滋养细胞瘤形成相关的一组独特的microRNA。对大多数改变的转录本的基因本体论分析显示,前导通路与对缺血的反应有关(P<.001)。这里,前3个最显著改变的microRNAs中有2个是mir-181b-5p(1.65倍;调整后的P=0.014)和mir-181d-5p(1.85倍;调整后的P=0.014),两者均已显示调节BCL2的表达。通过定量实时聚合酶链反应,在进展为妊娠滋养细胞肿瘤的完整痣中,BCL2信使RNA表达显着低于消退的完整痣(-4.69倍;P=0.018)。通过蛋白质印迹证实组织样品中BCL2的表达降低。独立患者样本中的免疫组织化学显示,与那些消退的病例相比,注定要进展为妊娠滋养细胞瘤的绒毛滋养细胞中BCL2的细胞质表达显着降低,染色强度(光密度0.110±0.102vs0.212±0.036;P<.001)和阳性细胞百分比(16%±28%vs49.4%±28.05%;P=.003)。
发展为妊娠滋养细胞瘤的完整痣与不同的microRNA谱相关。miR-181家族成员和BCL2可能是预测妊娠滋养细胞瘤形成风险的预后生物标志物。
MicroRNAs are small noncoding RNAs with important regulatory functions. Although well-studied in cancer, little is known about the role of microRNAs in premalignant disease. Complete hydatidiform moles are benign forms of gestational trophoblastic disease that progress to gestational trophoblastic neoplasia in up to 20% of cases; however, there is no well-established biomarker that can predict the development of gestational trophoblastic neoplasia.
This study aimed to investigate possible differences in microRNA expression between complete moles progressing to gestational trophoblastic neoplasia and those regressing after surgical evacuation.
Total RNA was extracted from fresh frozen tissues from 39 complete moles collected at the time of uterine evacuation in Brazil. In the study, 39 cases achieved human chorionic gonadotropin normalization without further therapy, and 9 cases developed gestational trophoblastic neoplasia requiring chemotherapy. Total RNA was also extracted from 2 choriocarcinoma cell lines, JEG-3 and JAR, and an immortalized normal placenta cell line, 3A-subE. MicroRNA expression in all samples was quantified using microRNA sequencing. Hits from the sequencing data were validated using a quantitative probe-based assay. Significantly altered microRNAs were then subjected to target prediction and gene ontology analyses to search for alterations in key signaling pathways. Expression of potential microRNA targets was assessed by quantitative real-time polymerase chain reaction and western blot. Finally, potential prognostic protein biomarkers were validated in an independent set of formalin-fixed paraffin-embedded patient samples from the United States (15 complete moles progressing to gestational trophoblastic neoplasia and 12 that spontaneously regressed) using quantitative immunohistochemistry.
In total, 462 microRNAs were identified in all samples at a threshold of <1 tag per million. MicroRNA sequencing revealed a distinct set of microRNAs associated with gestational trophoblastic neoplasia. Gene ontology analysis of the most altered transcripts showed that the leading pathway was related to response to ischemia (P<.001). Here, 2 of the top 3 most significantly altered microRNAs were mir-181b-5p (1.65-fold; adjusted P=.014) and mir-181d-5p (1.85-fold; adjusted P=.014), both of which have been shown to regulate expression of BCL2. By quantitative real-time polymerase chain reaction, BCL2 messenger RNA expression was significantly lower in the complete moles progressing to gestational trophoblastic neoplasia than the regressing complete moles (-4.69-fold; P=.018). Reduced expression of BCL2 was confirmed in tissue samples by western blot. Immunohistochemistry in the independent patient samples revealed significantly lower cytoplasmic expression of BCL2 in the villous trophoblasts from cases destined for progression to gestational trophoblastic neoplasia compared with those that regressed, both with respect to staining intensity (optic density 0.110±0.102 vs 0.212±0.036; P<.001) and to the percentage of positive cells (16%±28% vs 49.4%±28.05%; P=.003).
Complete moles progressing to gestational trophoblastic neoplasia are associated with a distinct microRNA profile. miR-181 family members and BCL2 may be prognostic biomarkers for predicting gestational trophoblastic neoplasia risk.