A persistent infection with human papillomavirus (HPV) can induce precancerous lesions of the cervix that may ultimately develop into cancer. Cervical cancer development has been linked to altered ...microRNA (miRNA) expression, with miRNAs regulating anchorage‐independent growth being particularly important for the progression of precancerous lesions to cancer. In this study, we set out to identify and validate targets of miR‐129‐5p, a previously identified tumor suppressive miRNA involved in anchorage‐independent growth and HPV‐induced carcinogenesis. We predicted 26 potential miR‐129‐5p targets using online databases, followed by KEGG pathway enrichment analysis. RT‐qPCR and luciferase assays confirmed that 3'UTR regions of six genes (ACTN1, BMPR2, CAMK4, ELK4, EP300, and GNAQ) were targeted by miR‐129‐5p. Expressions of ACTN1, CAMK4, and ELK4 were inversely correlated to miR‐129‐5p expression in HPV‐transformed keratinocytes, and their silencing reduced anchorage‐independent growth. Concordantly, miR‐129‐5p overexpression decreased protein levels of ACTN1, BMPR2, CAMK4 and ELK4 in anchorage‐independent conditions. Additionally, c‐FOS, a downstream target of ELK4, was downregulated upon miR‐129‐5p overexpression, suggesting regulation through the ELK4/c‐FOS axis. ACTN1 and ELK4 expression was also upregulated in high‐grade precancerous lesions and cervical cancers, supporting their clinical relevance. In conclusion, we identified six targets of miR‐129‐5p involved in the regulation of anchorage‐independent growth, with ACTN1, BMPR2, ELK4, EP300, and GNAQ representing novel targets for miR‐129‐5p. For both ACTN1 and ELK4 functional and clinical relevance was confirmed, indicating that miR‐129‐5p‐regulated ACTN1 and ELK4 expression contributes to HPV‐induced carcinogenesis.
