Altered microRNA expression associated with chromosomal and epigenetic changes contributes to cervical carcinogenesis. Homo sapiens

Little is known about the alterations in microRNA (miRNA) expression patterns during the consecutive stages of cervical cancer development and their association with chromosomal instability and epigenetic changes. We integrated miRNA expression profiles in normal cervical squamous epithelium, high-grade precancerous lesions (CIN2-3), squamous cell carcinomas (SCC) and adenocarcinomas (AdCAs) with previously generated chromosomal profiles of the same samples. In addition, the DNA methylation status of downregulated miRNAs located in a CpG island was determined. Significantly differential expression during the consecutive stages of cervical SCC development was observed for 106 miRNAs. Altered expression of 5 significantly differentially expressed miRNAs, hsa-miR-9 (1q23.2), hsa-miR-15b (3q25.32), hsa-miR-28-5p (3q27.3), hsa-miR-100 and hsa-miR-125b (both 11q24.1) was directly linked to frequent chromosomal alterations. Another 9 significantly downregulated miRNAs were located within a CpG island. Three of these 9 miRNAs, hsa-miR-203, hsa-miR-572, and hsa-miR-638, showed increased methylation in cervical cancer cell lines and HPV-immortalised cells compared to primary keratinocytes. Functional analyses were performed for hsa-miR-9, representing a potential oncogene with increased expression linked to a chromosomal gain, and hsa-miR-203, representing a potential tumour suppressor gene silenced by DNA methylation. Hsa-miR-9 and hsa-miR-203 were found to alter cell viability and anchorage independent growth in vitro, respectively, supporting their oncogenic and tumour suppressive function in cervical cancer. In conclusion, differential expression of 106 miRNAs, partly associated with chromosomal alterations and epigenetic changes, was observed during cervical SCC development. Altered expression of hsa-miR-9 and hsa-miR-203 was shown to be functionally relevant, underlining the importance of deregulated miRNA expression in cervical carcinogenesis. Overall design: 10 squamous cell carcinomas of the cervix, 9 adenocarcinomas of the cervix, 18 high-grade cervical intraepithelial neoplasias (CIN2-3), and 10 cervical squamous epithelial samples with normal histology were analysed using single channel (Cy3) miRNA microarrays from Agilent (G4471A-016436).

目前对于宫颈癌发生连续阶段中微小RNA（microRNA, miRNA）表达模式的改变，及其与染色体不稳定、表观遗传改变的关联尚不清楚。
我们整合了正常宫颈鳞状上皮、高级别癌前病变（CIN2-3）、鳞状细胞癌（squamous cell carcinomas, SCC）以及腺癌（adenocarcinomas, AdCAs）的miRNA表达谱，与此前获取的相同样本的染色体谱进行联合分析。
此外，我们还对位于CpG岛中的下调miRNA的DNA甲基化状态进行了检测。
在宫颈鳞状细胞癌发生的连续阶段中，共有106个miRNA呈现出显著差异表达。
5个显著差异表达的miRNA的表达改变——hsa-miR-9（1q23.2）、hsa-miR-15b（3q25.32）、hsa-miR-28-5p（3q27.3）、hsa-miR-100以及hsa-miR-125b（均位于11q24.1）——与频繁发生的染色体改变直接相关。
另有9个显著下调的miRNA位于CpG岛区域。在这9个miRNA中，hsa-miR-203、hsa-miR-572和hsa-miR-638在宫颈癌细胞系及HPV永生化细胞中的甲基化水平相较于原代角质形成细胞有所升高。
我们针对hsa-miR-9和hsa-miR-203开展了功能分析：前者作为潜在致癌基因，其表达升高与染色体拷贝数增加相关；后者则是被DNA甲基化沉默的潜在抑癌基因。研究发现，hsa-miR-9与hsa-miR-203可分别在体外改变细胞活力及非锚定依赖性生长，证实了二者在宫颈癌发生中的致癌与抑癌功能。
综上，在宫颈鳞状细胞癌发生过程中，共有106个miRNA呈现差异表达，其中部分与染色体改变及表观遗传改变相关。hsa-miR-9与hsa-miR-203的表达改变具有功能相关性，凸显了miRNA表达失调在宫颈癌变过程中的重要作用。
整体实验设计：本研究采用安捷伦（Agilent）G4471A-016436型号单通道（Cy3）miRNA微阵列，对10例宫颈鳞状细胞癌、9例宫颈腺癌、18例高级别宫颈上皮内瘤变（CIN2-3）以及10例组织学正常的宫颈鳞状上皮样本进行了检测分析。