Mig-6 induced the apoptosis and reduced the autophagy of HCC cell lines.
To validate the role of Mig-6 in HCC cell lines,we examined the impact exerted by increasing Mig-6 on HCC cell apoptosis and autophagy with Mig-6 plasmid.HepG-2 and HLE cells were transfected with Mig-6 plasmid, respectively. As expected, two cell lines transfected with Mig-6 showed the enhanced percentage of apoptosis(Fig. 1a) .In the meanwhile western blotting were using for evaluated the expression levels of autophagy-associated protein (Fig. 1b). The expression levels of p62, which is mainly degraded by autophagy pathway, is greatly induced in the Mig-6 plasmid groups compared with those of the PC groups. But microtubule associated protein 1 light chain 3β (LC3b), following transfection with Mig-6 plasmid were significantly decreased. Autophagic flux experiment showed that in two cell lines, Mig-6 plasmid weakened the flux of autophagy, with a less number of autophagosomes (yellow) and autolysosomes (red) in two celllines(Fig. 1c).
Identification of Mig-6-regulated miRNAs
Several underlying mechanisms of Mig-6 were hypothesized; the regulation of miRNAs by Mig-6 was an area that had never been studied. Mig-6-regulated miRNAs were identified by transducing 293T with lentiviruses encoding control virus or lentivirus-mediated Mig-6 (Gene Chem, Shanghai, China). After transduction for 48 h, the cells were treated with puromycin for 48 h to remove the nontransduced cells. Successful transduction was examined by fluorescence microscopy of GFP-expressing cells (Fig. 2a), which was further confirmed by western blot analysis (Fig. 2b). The GFP-positive cell population was used immediately for analysis using the GeneChip assay. The results showed that a total of 44 Mig-6-regulated miRNAs were identified, of which 11 were upregulated and 33 downregulated (Fig. 2c).Among them the expression of 4 and 16 miRNAs in Mig-6 overexpressed cells was increased or inhibited by more than 2.5-fold, respectively (Table 1). Among all the miRNAs, miR-193a-3p is the most potent, with downregulated expression reported in many types of cancers. The HepG-2 and HLE cells were transfected with Mig-6 plasmid and siRNA to further verify the relationship between Mig-6 and miR-193a-3p. The transfection efficiency was measured using Western blot analysis (Fig. 3a). When the expression of Mig-6 was promoted, the miR-193a-3p level correspondingly increased in each cell line, and when the expression of Mig-6 was suppressed, the decrease in the miR-193a-3p was observed (Fig. 3b).
Table 1
Expression changes of Mig6-regulated miRNAs
miRNA | Regulation | Control virus or LV-Mig-6 Fold change |
hsa-miR-1269a | up | 3.663214041 |
hsa-miR-193a-3p | up | 2.960149418 |
hsa-miR-1244 | up | 2.512778203 |
hsa-miR-371b-5p | up | 2.503430956 |
hsa-miR-5093 | down | -5.363445158 |
hsa-miR-297 | down | -4.571560354 |
hsa-miR-4793-3p | down | -4.556744037 |
hsa-miR-610 | down | -4.539741026 |
hsa-miR-4440 | down | -4.535851264 |
hsa-miR-195-3p | down | -4.458473988 |
hsa-miR-6877-3p | down | -4.173369522 |
hsa-miR-3148 | down | -3.956889197 |
hsa-miR-8071 | down | -3.647623099 |
hsa-miR-185-3p | down | -3.48629987 |
hsa-miR-3064-5p | down | -3.347459653 |
hsa-miR-595 | down | -3.344313283 |
hsa-miR-3907 | down | -3.319532324 |
hsa-miR-7844-5p | down | -2.856073948 |
hsa-miR-4455 | down | -2.736743073 |
hsa-mir-297 | down | -2.606974853 |
MiR-193a-3p induced the apoptosis and reduced the autophagy of HCC cell lines.
MiR-193a-3p is reported to relate to various pathological processes of liver cancer, including proliferation, migration, invasion, apoptosis, radioresistance, and chemotherapeutic tolerance. However, the relationship between miR-193a-3p and autophagy has not been reported to date. This study attempted to clarify the impact exerted by miR-193a-3p. First, the HepG-2 and HLE cells were transfected with miR-193a-3p mimics or miR-193a-3p inhibitors, and the transfection efficiency was measured by reverse transcription-polymerase chain reaction (Fig. 4a). Then, apoptosis was inspected in the HepG-2 and HLE cells with flow cytometric analysis. A larger percentage of apoptotic cells were presented in the cells transfected with miR-193a-3p mimics; however, miR-193a-3p inhibitors decreased the apoptotic rate (Fig. 4b ,c). Finally, the effect of miR-193a-3p on the autophagy capacity of HepG-2 and HLE cells was verified using western blot analysis and autophagic flux experiment. As anticipated, miR-193a-3p mimics increased the expression of p62 but reduced the expression of LC3b, as well as weakened the flux of autophagy. The reverse consequence was also observed in the miR-193a-3p inhibitor group (Fig. 4d,e).
MiR-193a-3p affected the apoptosis and autophagy of HCC cells by regulating the expression of TGF-β2.
The miR-193a-3p target predicted by all three target gene prediction software was regarded with high confidence (Table S1). Thus, 62 nominated target genes were hit by miR-193a-3p. Among them, the TGF-β2 gene gained attention. Previous studies reported that TGF-β2 was regulated by multiple miRNAs and associated with apoptosis and autophagy in certain cell lines. In this study, the expression of TGF-β2 protein in both HepG-2 and HLE cells decreased after the transfection of miR-193a-3p mimics, whereas treatment with miR-193a-3p inhibitors increased the expression of TGF-β2 (Fig. 5a). TGF-β2 mRNA was also quantified after transfecting miR-193a-3p mimics or inhibitors into HCC cells, but no statistically significance difference was found at the mRNA level (Fig. 5b). Next, the apoptosis and autophagy were applied to assess the impact of miR-193a-3p and TGF-β2 on HCC. The HepG-2 and HLE cells transfected with miR-193a-3p mimics showed an increased percentage of apoptotic cells and weakened flux of autophagy. However, TGF-β2 plasmid could not only inhibit apoptosis and enhance the flux of autophagy but also weaken the effects exerted by miR-193a-3p mimics (Fig. 5c,d,e). These results substantiated that miR-193a-3p affected the apoptosis and autophagy of HCC cells by controlling the expression of TGF-β2.
Mig-6 modulated apoptosis and autophagy of HCC cells through the miR-193a-3p/TGF-β2 pathway.
The relationship between Mig-6 and TGF-β2 has not been reported to date. Therefore, the correlation between Mig-6 and TGF-β2 protein level was analyzed in five HCC cell lines. An inverse relationship was observed between the expression of Mig-6 and TGF-β2. High expression of Mig-6 was found in Huh7 and PLC/PRF/5 cells; both of them showed low levels of TGF-β2. On the contrary, the HepB3 and HLE cells with the low level expression of Mig-6 had high expression of TGF-β2 (Fig. 6a). A series of function experiments were performed in HepG-2 and HLE cells to assess further impacts of overexpression of Mig-6. As indicated by western blot analysis, the level of TGF-β2 protein decreased after HCC cells were transfected with Mig-6 plasmid (Fig. 6b,c), without any mRNA alteration (data not show). Meanwhile, miR-193a-3p reversed the decrease in TGF-β2 induced by the Mig-6 plasmid (Fig. 6b,c). As presented in Fig. 1A, a larger percentage of apoptotic cells were present in the cells transfected with Mig-6 plasmid compared with the control group, whereas they were rescued by miR-193a-3p inhibitors (Fig. 6d). The autophagy of HCC cells was tested by western blot analysis and autophagic flux assay. Weakened autophagy due to the overexpression of Mig-6 was reversed by miR-193a-3p inhibitors (Fig. 6b,c,e).