MiR-375 is under-expressed in sorafenib-resistant HCC cells
The parental strains of HCC cells (HepG2 and Huh7) were continuously cultured in sorafenib of increasing concentrations to obtain sorafenib-resistant HCC cells (HepG2/so and Huh7/so). The morphology of HepG2 and Huh7 cells under the inverted microscope were changed from plump, pebble-like shape (epithelial phenotype) into spindle shape (mesenchymal phenotype) (Fig. 1A). CCK-8 assay detected that the IC50 of sorafenib in HepG2/so and Huh7/so cells was higher than in HepG2 and Huh7 cells (Fig. 1B, P < 0.05), suggesting that HepG2/so and Huh7/so cells had stronger tolerance to sorafenib.
MiR-375 was under-expressed in HepG2/so and Huh7/so cells compared to HepG2 and Huh7 cells (Fig. 1C, P < 0.05). To investigate the potential effect of miR-375 on sorafenib resistance, HepG2/so were transfected with miR-375 mimic or miR-375 inhibitor. MiR-375 was up-regulated in the miR-375 mimic group and down-regulated in the miR-375 inhibitor group (Fig. 1D, P < 0.05, vs the mimic NC and inhibitor NC group respectively), suggesting successful cell transfection. After the transfection, the cells were exposed to sorafenib for 24 hours. Sorafenib treatment impeded the survival of HepG2/so cells (P < 0.05). The survival of HepG2/so cells was further damaged by miR-375 mimic while improved by miR-375 inhibitor (Fig. 1E, P < 0.05). The results of the flow cytometry assessment of apoptosis showed that miR-375 mimic promoted sorafenib-induced apoptosis of HepG2/so cells while miR-375 inhibitor attenuated the apoptosis (Fig. 1F, P < 0.05).
Autophagy acts on miR-375-mediated drug resistance of HCC cells
The autophagy level was inhibited by 3-MA or activated by RAP in HepG2/so cells. CCK-8 assay detected that 3-MA treatment impaired the survivability of the cells with down-regulated miR-375 while RAP treatment enhanced the survivability of cells with over-expressed miR-375 (Fig. 2A, P < 0.05, vs the miR-375 inhibitor group and miR-375 mimic group respectively). The cell apoptosis was reduced in the miR-375 inhibitor group while enhanced in the miR-375 inhibitor + 3-MA group (Fig. 2B, P < 0.05, vs the inhibitor NC group and miR-375 inhibitor group respectively). The apoptosis promoted by miR-375 mimic was attenuated by RAP in the HepG2/so cells (Fig. 2B, P < 0.05).
Meanwhile, according to the Western blotting measurement of the expressions of p62, LC3I and LC3II, the autophagy was inhibited in the miR-375 inhibitor + 3-MA group while enhanced in the miR-375 mimic + RAP group (Fig. 2C, P < 0.05, vs the miR-375 inhibitor group and miR-375 mimic group respectively).
MiR-375 attenuates sorafenib resistance by mediating autophagy in HCC cells
According to the analysis of the cell survivability measured by CCK-8, HepG2/so cells were less susceptible to sorafenib than HepG2 cells (P < 0.05). The drug resistance of HepG2/so cells was enhanced by miR-375 inhibitor and reduced by miR-375 mimic (Fig. 3A, P < 0.05). The apoptosis rates of HepG2/so cells were significantly reduced compared to HepG2 cells (P < 0.05). The number of apoptotic HepG2/so cells was decreased after transfection of miR-375 inhibitor while increased after transfection of miR-375 mimic (Fig. 3B, P < 0.05).
Furthermore, the LC3II/LC3I ratio was increased and p62 was decreased in HepG2/so cells compared to HepG2 cells (P < 0.05). The expression trends of p62, LC3I and LC3II in HepG2/so cells were promoted by miR-375 inhibitor while perturbed by miR-375 mimic (Fig. 3C, P < 0.05).
SIRT5 is targeted and down-regulated by miR-375
SIRT5 was detected to be a downstream target of miR-375 based on the bioinformatic analysis of jefferson (Fig. 4A). Dual-luciferase reporter assay was designed to confirm the potential regulation between miR-375 and SIRT5. miR-375 mimic attenuated the relative luciferase activity of WT-SIRT5 (Fig. 4B, P < 0.01), but did not affect that of MUT-SIRT5. In cells, the expression of SIRT5 was negatively correlated with that of miR-375 (Fig. 4C-D, P < 0.01).
SIRT5 is over-expressed in sorafenib-resistant HCC cells and reverses the suppressive effect of miR-375 on sorafenib resistance
According to the results of qRT-PCR and Western blotting, HepG2/so and Huh7/so cells had higher expressions of SIRT5 compared to HepG2 and Huh7 cells (Fig. 5A-B, P < 0.05). To investigate the effect of SIRT5 on sorafenib resistance, SIRT5 was either knocked down or over-expressed in HepG2/so cells via transfection of sh-SIRT5 or pcDNA3.1-SIRT5 (Fig. 5C, P < 0.05). SIRT5 inhibition aggravated the damage to the survival of HepG2/so cells whereas SIRT5 overexpression improved the survival (Fig. 5D, P < 0.05). The suppressive or promotive effect of SIRT5 inhibition or overexpression on sorafenib resistance of HepG2/so cells was reversed by miR-375 inhibitor or miR-375 mimic (Fig. 5D, P < 0.05). SIRT5 inhibition exacerbated sorafenib-induced apoptosis of HepG2/so cells, which was later attenuated by miR-375 inhibitor (Fig. 5E, P < 0.05). SIRT5 overexpression ameliorated the cell death while the apoptosis rate was increased in the pcDNA3.1-SIRT5 + miR-375 mimic group (Fig. 5E, P < 0.05).
Meanwhile, the LC3II/LC3I ratio was reduced and p62 was up-regulated in the sh-SIRT5 group (vs the sh-NC group) and pcDNA3.1-SIRT5 + miR-375 mimic group (vs the pcDNA3.1-SIRT5 + mimic NC group); different expression patterns of LC3II, LC3I and p62 were found in the pcDNA3.1-SIRT5 group (vs the pcDNA3.1 group) and sh-SIRT5 + miR-375 inhibitor group (vs sh-SIRT5 + inhibitor NC group) (Fig. 5F, P < 0.05).
The above experiment data exhibited that SIRT5 knockdown inhibited autophagy to augment the susceptibility of HCC cells to sorafenib, and miR-375 down-regulation abolished the assistance of SIRT5 knockdown to sorafenib treatment. In addition, SIRT5 overexpression enhanced the resistance to sorafenib via autophagy activation in HCC cells, while miR-375 up-regulation made SIRT5 overexpressed HCC cells more sensitive to sorafenib. Taken together, miR-375 mediated the tolerance of HCC cells to sorafenib by regulating SIRT5.