Data from: Loss of miR-143 and miR-145 in condyloma acuminatum promotes cellular proliferation and inhibits apoptosis by targeting NRAS

The expression profile of miRNAs and their function in condyloma acuminatum (CA) remains unknown. In this study, we aimed to detect the effects of miR-143 and miR-145, the most downregulated in CA samples using high-throughput sequencing, on cell proliferation and apoptosis, to determine a novel therapeutic target for CA recurrence. RT-qPCR was used to validate the lower expression of miR-143 and miR-145 in a larger size of CA samples, and the expression of NRAS in CA samples was significantly higher than self-controls as determined western blotting assay. Luciferase assay was performed to confirm that miR-143 or miR-145 targeted NRAS directly. Transduction of LV-pre-miR-143 or LV-pre-miR-145 to human papilloma virus (HPV)-infected SiHa cells led to reduced proliferation, greater apoptosis, and inhibition of expression of NRAS, PI3Kp110α, and pAKT. However, knockout of miR-143 or miR-145 in human epidermal keratinocytes (HEKs) by delivery of CRISPR/CAS9-gRNA for target miRNAs protected cells from apoptosis and upregulated expression of target genes as described above. MiR-143 and miR-145 sensitized cells to nutlin-3a, a p53 activator and MDM2 antagonist, while their loss protected cells from the stress of nutlin-3a. Furthermore, siRNA targeting NRAS showed similar effects on proliferation and apoptosis as miR-143 or miR-145. Taken together, our results suggest that loss of miR-143 or miR-145 in CA protects HPV-infected cells from apoptosis induced by environmental stress, in addition to promoting cellular proliferation and inhibiting apoptosis by targeting NRAS/PI3K/ATK. Restoration of miR-143 or miR-145 might provide an applicable and novel approach to block the recurrence and progression of CA.

尖锐湿疣（condyloma acuminatum，CA）中微小RNA（microRNAs，miRNAs）的表达谱及其功能目前仍不明确。本研究旨在通过高通量测序筛选出尖锐湿疣样本中表达下调最显著的miR-143与miR-145，探究其对细胞增殖与凋亡的影响，以期为尖锐湿疣复发寻找全新治疗靶点。本研究采用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）在更大样本量的尖锐湿疣组织中验证了miR-143与miR-145的低表达水平；同时通过蛋白质印迹法（Western blotting）检测发现，尖锐湿疣样本中NRAS的表达水平显著高于自身对照组织。通过荧光素酶报告基因实验证实，miR-143与miR-145可直接靶向结合NRAS基因。将携带pre-miR-143或pre-miR-145的慢病毒载体（lentiviral vector，LV）转染至人乳头瘤病毒（human papilloma virus，HPV）感染的SiHa细胞后，可显著抑制细胞增殖、促进细胞凋亡，并下调NRAS、PI3Kp110α以及pAKT的蛋白表达水平。而通过靶向miRNAs的成簇规律间隔短回文重复序列相关蛋白9-向导RNA（clustered regularly interspaced short palindromic repeats-associated protein 9-guide RNA，CRISPR/Cas9-gRNA）系统敲除人表皮角质形成细胞（human epidermal keratinocytes，HEKs）中的miR-143或miR-145后，则可抑制细胞凋亡，并上调上述靶基因的表达水平。miR-143与miR-145可增强细胞对nutlin-3a（一种p53激活剂与MDM2拮抗剂）的敏感性，而敲除这两种miRNA则可使细胞免受nutlin-3a诱导的应激损伤。此外，靶向NRAS的小干扰RNA（small interfering RNA，siRNA）可产生与miR-143或miR-145相似的调控细胞增殖与凋亡的效果。综上，本研究结果表明，尖锐湿疣组织中miR-143与miR-145的缺失，可通过靶向NRAS/PI3K/AKT通路促进细胞增殖、抑制细胞凋亡，同时还可使人乳头瘤病毒感染的细胞免受环境应激诱导的细胞凋亡。恢复miR-143与miR-145的表达，或许可为阻断尖锐湿疣的复发与进展提供一种可行的全新治疗策略。