MicroRNA-9-5p downregulates Klf4 and influences the progression of hepatocellular carcinoma via the AKT signaling pathway

Kruppel-like factor 4 (Klf4) is a transcriptional factor involved in the progression of hepatocellular carcinoma (HCC). However, the underlying regulatory mechanisms associated with the Klf4 gene as a tumor suppressor in HCC remain unclear. microRNAs (miRNAs or miRs) are a series of small non-coding RNAs that serve a vital role in regulating gene expression via their influence on protein translation and the associated degradation of mRNA. The mRNA expression levels of the miRNA, miR-9-5p, and Klf4 were measured using reverse transcription-quantitative polymerase chain reaction. The protein expression levels of Klf4, protein kinase B (AKT), phosphorylated (p-)AKT, mechanistic target of rapamycin (mTOR), p-mTOR, B cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) were determined by western blot analysis. Dual luciferase reporter assay was used to confirm a direct interaction between miR-9-5p and the 3-untranslated region (3-UTR) sequence of Klf4. Cell counting kit-8 assay, wound healing assay, Transwell migration assay and flow cytometric analysis were performed to evaluate the proliferative, migratory and apoptotic capabilities of the HCC cells. In the present study, miR-9-5p was revealed to be overexpressed in HCC as a novel upstream gene of Klf4. miR-9-5p expression was inversely associated with Klf4 expression in clinical samples. Additionally, Kaplan-Meier analysis revealed a markedly poor prognosis of HCC in the miR-9-5p high-expression group. Bioinformatics analysis revealed that miR-9-5p bound directly to the 3-UTR of Klf4, which reduced the expression levels of Klf4. The miR-9-5p/Klf4 axis promoted HCC proliferation and migration, and inhibited HCC apoptosis. Furthermore, miR-9-5p upregulated the Bcl-2/Bax protein ratio and activated AKT/mTOR signaling. Taken together, these data demonstrated that the miR-9-5p/Klf4 axis was able to promote HCC progression, which may occur via regulation of the AKT signaling pathway, highlighting a potential novel target in HCC treatment.