miR‑576‑3p/M‑phase phosphoprotein 8 axis regulates the malignant progression of hepatocellular carcinoma cells via the PI3K/Akt signaling pathway

Abstract

Hepatocellular carcinoma (HCC) is a fatal digestive system cancer with unclear pathogenesis. M-phase phosphoprotein 8 (MPP8) has been shown to play a vital role in several cancer types, such as non-small cell lung cancer, gastric cancer and melanoma; however, there have been no studies into its role in HCC. The present study aimed to evaluate the role of MPP8 in regulating malignant phenotypes of liver cancer cells, and to further investigate the underlying mechanism. Bioinformatics analysis was performed to analyze related data from a public database, and to predict the potential microRNAs (miRNAs) that might target MPP8 mRNA; reverse transcription-quantitative PCR was used to measure the levels of mRNA and miRNA; western blotting was employed to detect protein levels; Cell Counting Kit-8 (CCK-8) and plate colony formation assays, wound healing assay and Transwell invasion assay were performed to evaluate the ability of cell proliferation, migration and invasion, respectively; dual-luciferase reporter gene assay was performed to identify the target association. The results showed that MPP8 was a risk factor for the survival of patients with HCC, and was up-regulated in HCC tissue samples and cell lines; MPP8 knockdown inhibited the proliferation, migration and invasion of liver cancer cells; MPP8 knockdown suppressed the PI3K/Akt pathway, and activation of this pathway reversed the inhibited liver cancer cell phenotypes by down-regulating MPP8; miR-576-3p, which was low in liver cancer cells, negatively regulated MPP8 expression by directly targeting its mRNA; up-regulating MPP8 expression reversed the inhibited signaling pathway and malignant phenotypes of liver cancer cells by miR-576-3p overexpression. In conclusion, the miR-576-3p/MPP8 axis regulates the proliferation, migration, and invasion of liver cancer cells through the PI3K/Akt signaling pathway. These findings lead novel insights into HCC progression, and propose MPP8 as a potential therapeutic target for HCC.

Keywords: M-phase phosphoprotein 8; PI3K/Akt pathway; hepatocellular carcinoma; malignant phenotype; miR-576-3p.

Figure 1.

Expression and prognostic value of MPP8 in HCC. (A) Expression levels of MPP8 in the samples of HCC tissues (n=371) and normal tissues (n=50) were analyzed using UALCAN. (B) Survival analysis of 365 patients with HCC was performed using UALCAN. (C) Levels of MPP8 mRNA in normal liver cells (THLE-2) and liver cancer cells (HepG2, Huh-7 and MHCC97-H) were detected by RT-qPCR. (D) Levels of MPP8 protein in normal liver cells and liver cancer cells were detected by western blotting. **P<0.01 vs. corresponding control. MPP8, M-phase phosphoprotein 8; HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription-quantitative PCR; UALCAN, The University of Alabama at Birmingham Cancer data analysis Portal.

Figure 2.

Role of MPP8 in the proliferation, migration and invasion of HCC cells. The effect of siRNA transfection on MPP8 expression in HCC cells was detected by (A) RT-qPCR for mRNA expression and (B) western blotting for protein expression. The effect of MPP8 knockdown on the proliferation of HCC cells was measured by (C) CCK-8 assay and (D) colony formation assay. (E) Effect of MPP8 knockdown on the migration of HCC cells was measured by wound-healing assay (scale bar, 200 µm). (F) Effect of MPP8 knockdown on the invasion of HCC cells was measured by Transwell invasion assay (scale bar, 50 µm). **P<0.01 vs. corresponding control. MPP8, M-phase phosphoprotein 8; HCC, hepatocellular carcinoma; siRNA, small interfering RNA; RT-qPCR, reverse transcription-quantitative PCR; CCK-8, Cell Counting Kit-8; OD, optical density.

Figure 3.

Role of the PI3K/Akt signaling pathway in MPP8-mediated regulatory effects on the malignant phenotypes of HCC cells. (A) Western blotting was used to detect the effect of MPP8 knockdown on the PI3K/Akt pathway, and to confirm the validity of IGF-1 as the agonist of this pathway. Rescue experiments with activation of the PI3K/Akt pathway were performed to determine whether this pathway was involved in MPP8-mediated regulation of the proliferation assessed by (B) CCK-8 and (C) colony formation assay, (D) migration was assessed by wound-healing assay (scale bar, 200 µm), and (E) invasion was assessed by Transwell invasion assay (scale bar, 50 µm) in HCC cells. *P<0.05 and **P<0.01 vs. corresponding control. MPP8, M-phase phosphoprotein 8; HCC, hepatocellular carcinoma; siRNA, small interfering RNA; p-, phosphorylated; t-, total; CCK-8, Cell Counting Kit-8; OD, optical density; IGF-1, insulin-like growth factor-1.

Figure 4.

Targeted regulatory relationship between miR-576-3p and MPP8 in HCC cells. (A) Sequence alignment of miR-576-3p with MPP8 3′UTR-WT and SPRR3 3′UTR-MUT. (B) Levels of miR-576-3p in normal liver cells (THLE-2) and HCC cells (Huh-7) were detected by RT-qPCR. (C) Effect of miR-mimic transfection on miR-576-3p expression in HCC cells was detected by RT-qPCR. The effect of miR-576-3p overexpression on MPP8 expression in HCC cells was detected by (D) RT-qPCR for mRNA expression and (E) western blotting for protein expression. (F) Target relationship between miR-576-3p and MPP8 was verified by dual-luciferase reporter gene assay. **P<0.01 vs. corresponding control. MPP8, M-phase phosphoprotein 8; HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription-quantitative PCR; WT, wild-type; -MUT, mutant.

Figure 5.

Role of miR-576-3p/MPP8 axis in HCC cells. The validity of MPP8 overexpression plasmid was confirmed by (A) RT-qPCR for mRNA expression and (B) western blotting for protein expression. (C) Western blotting used to detect the effect of up-regulating miR-576-3p on the PI3K/Akt pathway, and to detect the effect of MPP8 overexpression on miR-576-3p-mediated regulation of the PI3K/Akt pathway. Rescue experiments with MPP8 overexpression were performed to determine whether MPP8 was involved in miR-576-3p-mediated regulation of the proliferation assessed by (D) CCK-8 and (E) colony formation assay, (F) migration assessed by wound-healing assay (scale bar, 200 µm) and (G) invasion assessed by Transwell invasion assay (scale bar, 50 µm) in HCC cells. *P<0.05 and **P<0.01 vs. corresponding control. MPP8, M-phase phosphoprotein 8; HCC, hepatocellular carcinoma; p-, phosphorylated; t-, total; CCK-8, Cell Counting Kit-8; OD, optical density; RT-qPCR, reverse transcription-quantitative PCR.

Figure 6.

Schematic illustration of the regulatory mechanism of miR-576-3p/MPP8 axis via the PI3K/Akt pathway in HCC cells, created with BioRender.com. MPP8, M-phase phosphoprotein 8; HCC, hepatocellular carcinoma; P, phosphorylated.