Chiantore Maria Vincenza, Iuliano Marco, Mongiovì Roberta Maria, Dutta Sankhadeep, Tommasino Massimo, Di Bonito Paola, Accardi Luisa, Mangino Giorgio, Romeo Giovanna
Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy.
Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome - Polo Pontino, Latina, Italy.
Infect Agent Cancer. 2022 Jun 15;17(1):29. doi: 10.1186/s13027-022-00445-z.
The β3 human papillomavirus (HPV)49 induces immortalization of primary keratinocytes through the action of E6 and E7 oncoproteins with an efficiency similar to alpha high risk (HR)-HPV16. Since HR-HPV oncoproteins are known to alter microRNA (miRNA) expression and extracellular vesicle (EV) production, we investigated the impact of HPV49 E6 and E7 proteins on miRNA profile and EV expression, and their involvement in the control of cell proliferation.
The miRNA expression was evaluated by a miRNA array and validated by RT-qPCR in primary human keratinocytes immortalized by β3 HPV49 (K49) or α9 HR-HPV16 (K16), and in EVs from K49 and K16. The modulation of miRNA target proteins was investigated by immunoblotting analyses.
By comparing miRNA expression in K49 and K16 and the derived EVs, six miRNAs involved in HPV tumorigenesis were selected and validated. MiR-19a and -99a were found to be upregulated and miR-34a downregulated in both cell lines; miR-17 and -590-5p were upregulated in K49 and downmodulated in K16; miR-21 was downregulated only in K16. As for EV-carried miRNAs, the expression of miR-17, -19a, -21 and -99a was decreased and miR-34a was increased in K49 EVs. In K16 EVs, we revealed the same modulation of miR-19a, -34a, and -99a observed in producing cells, while miR-21 was upregulated. Cyclin D1, a common target of the selected miRNAs, was downmodulated in both cell lines, whereas cyclin-dependent kinase 4 was down-modulated in K49 but upregulated in K16.
These data suggest that E6 and E7 proteins of β3 HPV49 and α9 HR-HPV16 affect key factors of cell cycle control by indirect mechanisms based on miRNA modulation.
β3人乳头瘤病毒（HPV）49通过E6和E7癌蛋白的作用诱导原代角质形成细胞永生化，其效率与α高危（HR）-HPV16相似。由于已知HR-HPV癌蛋白会改变微小RNA（miRNA）表达和细胞外囊泡（EV）产生，我们研究了HPV49 E6和E7蛋白对miRNA谱和EV表达的影响，以及它们在细胞增殖控制中的作用。
通过miRNA阵列评估miRNA表达，并在由β3 HPV49（K49）或α9 HR-HPV16（K16）永生化的原代人角质形成细胞以及来自K49和K16的EV中通过RT-qPCR进行验证。通过免疫印迹分析研究miRNA靶蛋白的调节。
通过比较K49和K16以及衍生的EV中的miRNA表达，选择并验证了六种参与HPV肿瘤发生的miRNA。发现miR-19a和-99a在两种细胞系中均上调，而miR-34a下调；miR-17和-590-5p在K49中上调而在K16中下调；miR-21仅在K16中下调。至于EV携带的miRNA，K49 EV中miR-17、-19a、-21和-99a的表达降低，而miR-34a增加。在K16 EV中，我们发现miR-19a、-34a和-99a在产生细胞中观察到相同的调节，而miR-21上调。所选miRNA的共同靶标细胞周期蛋白D1在两种细胞系中均下调，而细胞周期蛋白依赖性激酶4在K49中下调但在K16中上调。
这些数据表明，β3 HPV49和α9 HR-HPV16的E6和E7蛋白通过基于miRNA调节的间接机制影响细胞周期控制的关键因素。
