Natalia Martinez-Acuna, Alejandro Gonzalez-Torres, Virginia Tapia-Vieyra Juana, Alvarez-Salas Luis Marat
Laboratorio de Terapia Genica, Departamento de Genetica y Biologia Molecular, Centro de Investigacion y de Estudios Avanzados del I.P.N., Ciudad de Mexico, Mexico.
Microrna. 2018;7(1):54-61. doi: 10.2174/2211536606666171024160244.
Aberrant miRNA expression is associated with the development of several diseases including cervical cancer. Dysregulation of miR-125a-5p is present in a plethora of tumors, but its role in cervical cancer is not well understood.
The aim was to analyze the expression profile of miR-125a-5p in tumor and immortal cell lines with further target prediction, validation and function analysis.
MiR-125a-5p expression was determined by real-time RT-PCR from nine cervical cell lines. In silico tools were used to find target transcripts with an miR-125-5p complementary site within the 3'UTR region. Further target selection was based on gene ontology annotation and ΔG analysis. Target validation was performed by transfection of synthetic miR-125a-5p mimics and luciferase assays. Functional evaluation of miR-125a-5p on migration was performed by transwell migration assays.
Differential miR-125a-5p expression was observed between immortal and tumor cells regardless of the human papillomavirus (HPV) content. Thermodynamic and ontological analyses showed Microtubule-Affinity-Regulating Kinase1 (MARK1) as a putative target for miR-125a-5p. An inverse correlation was observed among miR-125a-5p expression and MARK1 protein levels in tumor but not in immortal cells. Luciferase assays showed direct miR-125a-5p regulation over MARK1 through recognition of a predicted target site within the 3'-UTR. HeLa and C-33A cervical tumor cells enhanced migration after transfection with miR-125a-5p mimics and stimulation of cell migration was reproduced by siRNA-mediated inhibition of MARK1.
The results showed MARK1 as a novel functional target for miR-125a-5p with implications on cell migration of tumor cervical cancer cells.
异常的微小RNA（miRNA）表达与包括宫颈癌在内的多种疾病的发生发展相关。miR-125a-5p的失调存在于大量肿瘤中，但其在宫颈癌中的作用尚不清楚。
旨在分析miR-125a-5p在肿瘤细胞系和永生化细胞系中的表达谱，并进行进一步的靶标预测、验证及功能分析。
通过实时逆转录聚合酶链反应（RT-PCR）检测9种宫颈细胞系中miR-125a-5p的表达。利用生物信息学工具在3'非翻译区（UTR）内寻找具有miR-125-5p互补位点的靶转录本。进一步的靶标选择基于基因本体注释和ΔG分析。通过转染合成的miR-125a-5p模拟物和荧光素酶测定进行靶标验证。通过Transwell迁移试验对miR-125a-5p对迁移的功能进行评估。
无论人乳头瘤病毒（HPV）含量如何，在永生化细胞和肿瘤细胞之间均观察到miR-125a-5p的差异表达。热力学和本体分析显示微管亲和力调节激酶1（MARK1）是miR-125a-5p的潜在靶标。在肿瘤细胞而非永生化细胞中，观察到miR-125a-5p表达与MARK1蛋白水平呈负相关。荧光素酶测定显示miR-125a-5p通过识别3'-UTR内的预测靶位点直接调控MARK1。HeLa和C-33A宫颈肿瘤细胞在转染miR-125a-5p模拟物后迁移增强，并且通过小干扰RNA（siRNA）介导的MARK1抑制可重现细胞迁移的刺激。
结果表明MARK1是miR-125a-5p的一个新的功能靶标，对宫颈癌细胞的细胞迁移有影响。
