MiR-27a-3p enhances the cisplatin sensitivity in hepatocellular carcinoma cells through inhibiting PI3K/Akt pathway

Abstract

MicroRNAs (miRNAs) play an important role in drug resistance, and it is reported that miR-27a-3p regulated the sensitivity of cisplatin in breast cancer, lung cancer and ovarian cancer. However, the relationship between miR-27a-3p and chemosensitivity of cisplatin in hepatocellular carcinoma (HCC) was unclear, especially the underlying mechanism was unknown. In the present study, we analyzed miR-27a-3p expression levels in 372 tumor tissues and 49 adjacent tissues in HCC samples from TCGA database, and found that the miR-27a-3p was down-regulated in HCC tissues. The level of miR-27a-3p was associated with metastasis, Child-Pugh grade and race. MiR-27a-3p was regarded as a favorable prognosis indicator for HCC patients. Then, miR-27a-3p was overexpressed in HepG2 cell, and was knocked down in PLC cell. Next, we conducted a series of in vitro assays, including MTT, apoptosis and cell cycle assays to observe the biological changes. Further, inhibitor rate and apoptosis rate were detected with pre- and post-cisplatin treatment in HCC. The results showed that overexpression of miR-27a-3p repressed the cell viability, promoted apoptosis and increased the percentage of cells in G0/G1 phase. Importantly, overexpression of miR-27a-3p significantly increased the inhibitor rate and apoptosis rate with cisplatin intervention. Besides, we found that miR-27a-3p added cisplatin sensitivity potentially through regulating PI3K/Akt signaling pathway. Taken together, miR-27a-3p acted as a tumor suppressor gene in HCC cells, and it could be useful for modulating cisplatin sensitivity in chemotherapy.

Keywords: Cisplatin; Hepatocellular cancer; PI3K/Akt pathway; Sensitivity; miR-27a-3p.

Figure 1. MiR-27a-3p was down-regulated in tumor tissue in HCC patients

(A) The scatter diagram represented the expression of miR-27a-3p in 49 paired HCC tissues and adjacent non-tumor tissues, which showed that miR-27a-3p was significantly low-expressed in tumor tissue. (B) The Kaplan–Meier curve revealed that high level of miR-27a-3p significantly correlated with better overall survival in HCC patients. (C–E) The correlation analysis reflected that miR-27a-3p expressions were associated with metastasis, Child–Pugh grade and race in HCC patients. (F) The miR-27a-3p expressions in different cell lines were measured by RT-qPCR. (G)The research flow chart of the article.

Figure 2. MiR-27a-3p acted as a tumor suppressor gene in HCC

HepG2 cells were chosen for miR-27a-3p overexpression and PLC cells were selected for miR-27a-3p knockdown. (A,B) MTT assays showed that high level of miR-27a-3p impaired the cell viability in HepG2, whereas its low level added viability in PLC. (C,D) Up-regulation of miR-27a-3p increased the apoptosis rate in HepG2. In contrast, knockdown of miR-27a-3p significantly reduced the apoptosis rate in PLC. (E,F) The cell cycle assays revealed that overexpression of miR-27a-3p led to an increase in G₀/G₁ phase and a decrease in S-phase in HepG2, while its knockdown resulted in a reverse trend in PLC. All experiments were performed in triplicate. *P<0.05.

Figure 3. MiR-27a-3p enhanced the cisplatin sensitivity of HCC cells

(A,B) MTT assays showed that overexpression of miR-27a-3p increased the inhibition rate in HepG2. On the contrary, knockdown of miR-27a-3p significantly decreased the inhibition rate in PLC. (C,D) MiR-27a+DDP group had higher apoptosis rate than that in miR-Con+DDP group in HepG2 (*P<0.05). MiR-inhibitor-27a+DDP group had lower apoptosis rate than that in miR-inhibitor-Con+DDP group in PLC (*P<0.05).

Figure 4. MiR-27a-3p added the cisplatin sensitivity potentially by inhibiting PI3K/Akt pathway

(A,B) MiR-27a group inhibited PI3K/p-Akt signaling and elevated C-caspase-3 in HepG2. MiR-inhibitor-27a group exhibited the opposite trend in PLC cells. And DDP group showed suppression of PI3K/p-Akt pathway and up-regulation of C-caspase-3. MiR-27a+DDP group showed more obvious trend than DDP group in HepG2. MiR-inhibitor-27a+DDP group revealed stronger expression in PI3K protein and weaker level of C-caspase-3, compared with DDP group in PLC. Silencing of miR-27a-3p attenuated the cisplatin effect. (C) The PI3K/Akt pathway inhibitors LY49002 was used, a significant suppression of PI3K/Akt signaling and an increased expression of C-caspase-3 was observed. When miR-27a-3p was knocked down, the above trend was attenuated in miR-inhibitor-27a group, compared with miR-inhibitor-Con group. β-actin was used as a loading control. All of the experiments were performed in triplicate. *P<0.05.