Downregulation of miR‐193a/b‐3p during HPV‐induced cervical carcinogenesis contributes to anchorage‐independent growth through PI3K–AKT pathway regulators

Abstract

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.

miR‐129‐5p inhibits anchorage‐independent growth through silencing of ACTN1 and the ELK4/c‐FOS axis in HPV‐transformed keratinocytes

“…The 3'UTR regions were amplified from 50 ng of genomic DNA from FK18B cells using Phusion™ High‐Fidelity DNA Polymerase (Thermo Fisher Scientific) according to the manufacturers protocol. Subsequently, the amplified regions were cloned into pmirGLO Dual‐Luciferase miRNA Target Expression Vector plasmid (Promega), as described previously 21 . Primers used for cloning of luciferase reporter assays are listed in Supplementary File .…”

“…Subsequently, the amplified regions were cloned into pmirGLO Dual-Luciferase miRNA Target Expression Vector plasmid (Promega), as described previously. 21 Primers used for cloning of luciferase reporter assays are listed in Supplementary File 2. Mutagenesis of target sites was performed using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions.…”

Synaptotagmin-1 attenuated myocardial programmed necrosis and ischemia/reperfusion injury through the mitochondrial pathway

“…MicroRNAs, a class of single-stranded and highly-conserved small non-coding RNAs, suppress gene expression at post-transcriptional or translational levels, and is deeply implicated in cardiac development and disorders. Previous studies on the functions of miR-193b-3p were mainly focused on tumor [47,48], in ammation [49,50], and metabolism [36,51]. Recent advances have revealed that miR-193b-3p participates in the regulation of pulmonary hypertension in heart failure[36], cardiac hypertrophy [37] and the apoptotic pathway in myocardial ischemia/reperfusion injury [38].…”