Viral microRNA in HPV16-associated cervical cancer: expression, diagnostic potential, and biological functions

Abstract

BACKGROUND: Cervical cancer (CC) is the fourth most common cancer among women worldwide in terms of incidence and mortality. High-risk human papillomaviruses (HPVs) are etiologic factor of CC in more than 90% of cases, with type 16 HPV (HPV16) revealed in >50% of cancers. Dysregulation of expression of viral oncogenes E6 and E7 is the main cause of malignant transformation in infected cervical epithelial cells. The mechanisms of impaired expression of these genes are still underexplored. The dysfunction of viral microRNAs may be among the underlying factors.

AIM: To analyze the expression of HPV16-associated microRNA-H1 and microRNA-H2 in cervical cancer specimens, evaluate the correlation of their expression to viral load and overall patient survival, and analyze in silico their potential viral and cellular targets.

MATERIALS AND METHODS: The expression of HPV16 microRNA-H1 and HPV16 microRNA-H2 was evaluated in the real-time polymerase chain reaction. With this purpose, small RNAs were isolated from 36 specimens of HPV16-positive squamous cell carcinomas of the cervix. Further, the viral load was assessed after calculating the value of HPV16 DNA copies per cell. The association between microRNA expression and the viral load was evaluated using the nonparametric Spearman’s correlation coefficient. Kaplan-Meier curves were plotted to analyze the dependence of 5-year overall survival on the level of viral microRNA expression. The miRanda algorithm and online services mirDB, MR-microT and TargetScan Custom 5.2 were used for in silico search of theoretical microRNA targets.

RESULTS: MicroRNA-H1 expression was revealed in 33 of 38 specimens (86.8%), microRNA-H2 was detected in 37 of 38 specimens (97.4%) of HPV16-positive cervical cancer. There was a positive correlation between both microRNA-H1 (r=0.36, p=0.042) and microRNA-H2 (r=0.51, p=0.001) expression and HPV16 viral load. Higher level of expression of viral microRNA-H1 and microRNA-H2 tended to correlate with better overall patient survival. The theoretical microRNA-H1 (E7, E2, E5, L2 and URR) and microRNA-H2 (E1, E2, E5, L2, L1, URR) targets in the HPV16 genome were identified in silico, as well as theoretical cellular targets indicating possible regulation of cellular signaling pathways by means of viral microRNAs, both controlling normal viral cycle and promoting tumor transformation.

CONCLUSION: The results of this study demonstrate promising further investigation of the functions of viral microRNAs in relation with the infectious process and virus-induced malignant transformation, and their potential importance in the diagnosis of HPV16-associated cancers.

Keywords

HPV16 / epigenetics / viral microRNAs / HPV16-microRNA-H1 / HPV16-microRNA-H2 / cervical cancer

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Funding

Russian Scientific FoundationРоссийский научный фонд(23-15-00433)

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