Xu Mengfei, Huseinovic Angelina, Jaspers Annelieke, Yuan Lushun, Steenbergen Renske D M
Department of Pathology, Amsterdam UMC, Location Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, The Netherlands.
J Med Virol. 2023 Mar;95(3):e28589. doi: 10.1002/jmv.28589.
Cervical cancer is caused by a persistent infection with high-risk types of human papillomavirus (HPV) and an accumulation of (epi)genetic alterations in the host cell. Acquisition of anchorage-independent growth represents a critical hallmark during HPV-induced carcinogenesis, thereby yielding the most valuable biomarkers for early diagnosis and therapeutic targets. In a previous study, we found that miR-193a-3p and miR-193b-3p were involved in anchorage-independent growth. This study aimed to delineate the role of miR-193a/b-3p in HPV-induced carcinogenesis and to identify their target genes related to anchorage-independent growth. Cell viability and colony formation were assessed in SiHa cancer cells and HPV-16 and -18 immortalized keratinocytes upon miR-193a/b-3p overexpression. Both microRNAs reduced cell growth of all three cell lines in low-attachment conditions and showed a minor effect in adherent conditions. Online target-predicting programs and publicly available expression data were used to find candidate messenger RNA (mRNA) targets of miR-193a/b-3p. Seven targets showed reduced mRNA expression upon miR-193a/b-3p overexpression. For three targets, Western blot analysis was also performed, all showing a reduced protein expression. A direct interaction was confirmed using luciferase assays for six genes: LAMC1, PTK2, STMN1, KRAS, SOS2, and PPP2R5C, which are phosphatidylinositol 3-kinase/protein kinase B (PI3K-AKT) regulators. All six targets were overexpressed in cervical cancers and/or precursor lesions. Together with an observed downregulation of phosphorylated-AKT upon miR-193a/b-3p overexpression, this underlines the biological relevance of miR-193a/b-3p downregulation during HPV-induced cervical carcinogenesis. In conclusion, the downregulation of miR-193a-3p and miR-193b-3p is functionally involved in the acquisition of HPV-induced anchorage independence by targeting regulators of the PI3K-AKT pathway.
宫颈癌是由高危型人乳头瘤病毒（HPV）的持续感染以及宿主细胞中（表观）遗传改变的积累所引起的。获得不依赖贴壁生长是HPV诱导致癌过程中的一个关键标志，因此产生了用于早期诊断和治疗靶点的最有价值的生物标志物。在先前的一项研究中，我们发现miR-193a-3p和miR-193b-3p参与了不依赖贴壁生长。本研究旨在阐明miR-193a/b-3p在HPV诱导致癌过程中的作用，并鉴定其与不依赖贴壁生长相关的靶基因。在miR-193a/b-3p过表达后，对SiHa癌细胞以及HPV-16和-18永生化角质形成细胞的细胞活力和集落形成进行了评估。这两种微小RNA在低贴壁条件下均降低了所有三种细胞系的细胞生长，而在贴壁条件下显示出较小的影响。使用在线靶标预测程序和公开可用的表达数据来寻找miR-193a/b-3p的候选信使核糖核酸（mRNA）靶标。在miR-193a/b-3p过表达后，七个靶标的mRNA表达降低。对于三个靶标，还进行了蛋白质印迹分析，均显示蛋白质表达降低。使用荧光素酶测定法证实了六个基因的直接相互作用：LAMC1、PTK2、STMN1、KRAS、SOS2和PPP2R5C，它们是磷脂酰肌醇3激酶/蛋白激酶B（PI3K-AKT）调节剂。所有六个靶标在宫颈癌和/或癌前病变中均过表达。连同在miR-193a/b-3p过表达后观察到的磷酸化AKT的下调，这突出了miR-193a/b-3p下调在HPV诱导的宫颈癌发生过程中的生物学相关性。总之，miR-193a-3p和miR-193b-3p的下调通过靶向PI3K-AKT途径的调节剂在功能上参与了HPV诱导的不依赖贴壁生长的获得。
