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Objective:To explore the effect of miR-223-3p regulating FOXO3a-mediated autophagy in hepatic injury-reperfusion injury (LIRI).Methods:The model of hepatic ischemia-reperfusion injury (IR) was established in C57BL6 mice. According to different reperfusion timepoints, mice were randomly divided into 2 h, 6 h, 12 h and 24 h group. For sham group, there was no intraoperative clamping of hepatic pedicle. Murine hepatic AML12 cells were treated with miR-223-3p mimics, miR-223-3p inhibitor and FOXO3a interfering RNA. A hypoxic 1 h reoxygenation 6 h model was established. And miRNA-NC, miR-223-3p mimics, miR-223-3p inhibitor and siRNA-NC and FOXO3a siRNA groups were assigned. Hepatic injury and apoptosis were detected by hematoxylin eosin or TdT-mediated nick end labeling (HE/TUNEL) at different timepoints. The changes of proliferating cell nuclear antigen (PCNA) and Caspase-3 in hepatocytes were detected by immunohistochemistry. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were employed for detecting the expressions of miR-223-3p, FOXO3a, LC3, p62 and Caspase-3 in hepatocytes.Results:The results of HE/TUNEL indicated that reperfusion injury and apoptosis of hepatic tissue were most severe in 12 h group. In hepatic ischemia-reperfusion model, RT-PCR results showed that the expressions of miR-223-3p and FOXO3a were higher in IR group than those in sham group (1.00±0), the expression level of miR-223-3p mRNA peaked at 12 h (9.13±2.12) after reperfusion and FOXO3a was the highest at 6 h (5.23±0.90, P<0.05). Western blot showed that the expression of FOXO3a peaked at 6 h post-reperfusion and the expressions of LC3 and caspase-3 were the highest at 12 h. ( P<0.05). In the model of cell hypoxia and reoxygenation, RT-PCR indicated that the expression of FOXO3a mRNA decreased in miR-223-3p mimics group (0.45±0.21) as compared with miRNA-NC group (1.00±0). In contrast, miR-223-3p inhibitor group increased (2.73±0.53, P<0.05). Western blot indicated that FOXO3a protein expression was highest in miR-223-3p inhibitor and miR-223-3p mimics groups whereas LC3 and Caspase-3 were the highest in miR-223-3p mimics group ( P<0.05). The expression of FOXO3a was higher in siRNA-NC group than that in FOXO3a siRNA group while the expressions of Caspase-3 and LC3 were the higher in FOXO3a siRNA group. Conclusions:FOXO3a has protective effect on hepatic ischemia-reperfusion injury. It may be related to its inhibition of autophagy and apoptosis and miR-223-3p promotes injury through a down-regulation of FOXO3a-mediated autophagy. It suggests that miR-223-3p and FOXO3a are negatively correlated and may be potential gene therapeutic targets for hepatic injury.
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Objective To investigate the effect of miRNA-30a-3p on the proliferation,invasion and metastasis of liver cancer cells by targeting Atg3-mediated autophagy pathway.Methods The immunohistochemical staining was used to detect content of miRNA-30a-3p and Atg3 and their correlation in human hepatocellular carcinoma.Liver cancer cells were cultured in vitro and hunger environment was used to induce autophagy.RFP-GFP-LC3 double-labeled adenovirus infected hepatoma cells were used to detect autophagosomes in hepatoma cells.The expressions of autophagy-related proteins (autophagocytosis associated protein (Atg3),polyubiquitin-binding protein p62,autophagy microtubule-associated protein light chain 3 (LC3)) and EMT-related proteins (N-cadherin,vimentin,snail,ZO-1) were detected by Western blot.Platelet cloning assay and transwell assay were carried out to detect the proliferation,invasion and metastasis of carcinoma cell.CCK-8 kit was used to detect hepatocarcinoma cells' viability.Results The expression of miRNA-30a-3p was down-regulated.The expression of Atg3,E-cadherin and N-cadherin in miRNA-30a-3p high-expressed hepatocellular carcinoma was lower than that in miRNA-30a-3p low-expressed hepatocellular carcinoma.Increasing the expression of miRNA-30a-3p in hepatocellular carcinoma cells can decrease the expression of Atg3 and LC3,increase the expression of p62 and inhibit the formation of autophagosomes;otherwise,Atg3 and LC3 were increased,p62 was decreased and the formation of autophagosomes was promoted.Inhibition of Atg3 expression could decrease the expression of EMT-related proteins.When miRNA-30a-3p was inhibited,the cell viability of HCC was increased at each time point (F1 =10.314,P ＜0.05).When miRNA-30a-3p and Atg3 were inhibitor together,the cell viability of HCC was decreased at each time point(F2 =6.599,P ＜ 0.05).Conclusion miRNA-30a-3p can inhibit Atg3-mediated autophagy pathway and reduce cell autophagy activity,thus inhibiting the proliferation,invasion and metastasis of hepatocarcinoma cells.
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Objective To explore the role of miR-137 in the proliferation and migration of hepatocellular carcinoma (HCC) cells by regulating Notch1 and mediating autophagy.Methods The human SMMC7721 hepatoma cell line was transfected with miR-137 mimics,miR-137 inhibitor and Notch1 interfering RNA (siRNA),and divided into normal control group (NC group),miR-137 mimics group,miR-137 inhibitor group,Notch1 siRNA group.The expression levels of miR-137 and Notch1 mRNA after the transfection were detected by RT-PCR in SMMC7721 cells.Transwell experiments were performed to analyze the effect of miR-137 and Notch1 on the migration and invasion of SMMC7721 cells.The expression levels of β-catenin and vimentin in SMMC7721 cells were detected by immunohistochemistry.The number of autophagosomes was detected by double labeled adenovirus.Western blot was utilized to detect the expression of Notch1,E-Cadherin,N-Cadherin,vimentin,P62,and LC3.Results The results of RT-PCR showed that the relative expression level of Notch1 in miR-137 inhibitor group (5.71 ± 0.45) was significantly higher than that in miR-137 mimics group (0.21 ± 0.06) with statistical significance (P ＜ 0.05).The Transwell experiments showed that there were fewer invasive metastatic hepatoma cells in miR-137 mimics group (66.00 ± 4.55) and Notch1 siRNA group (88.00 ± 6.78) than that in the miR-137 inhibitor group (515.00 ±35.12) (P ＜0.05).The expression levels of β-catenin in miR-137 mimics group and Notch1 siRNA group were significantly increased and the expression level of vimentin was decreased (P ＜ 0.05).The results of autophagy double labeled adenovirus test showed that the number of autophagosomes in miR-137 mimics group (5.50 ± 3.70) was significantly fewer than that in miR-137 inhibitor group (32.75 ± 4.11),and the difference was statistically significant (P ＜ 0.05).The expression levels of Notch1,N-cadherin,vimentin,and LC3 protein in miR-137 mimics group were much lower than that in miR-137 inhibitor group and NC group,and the expression levels of E-Cadherin and P62 protein were greatly increased.The expression level of Notch1,N-cadherin,and LC3 protein in Notch1 siRNA group were significantly lower than that in NC group,and the expression levels of E-cadherin and P62 protein were much higher than that in NC group.Conclusion MiR-137 can inhibit the proliferation,migration and invasion of HCC cells by inhibiting the expression of Notch1 and autophagy,which may become a new target for the treatment of HCC.
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Objective To investigate the role of miRNA-30a-3p on inhibiting the proliferation,invasion and metastasis of HCC cells by targeting Caspase 1 involved in pyroptosis.Methods The qRT-PCR and immunohistochemical staining were used to detect the expressions of miRNA-30a-3p and Caspase 1 in HCC cells.SMMC-7721 cells were transfected with miR-30a-3p agonists,inhibitors and Caspase 1-specific inhibitors.Western blot was obtained to detect the expression of EMT-related proteins (N-cadherin,vimentin,snail,MMP-2) and Caspase 1,IL-18 and IL-1β.Plate clone assay,CCK-8 kit and Transwell were carried out to detect the proliferation and immigration of HCC cells.Results Caspase 1 was highly expressed (t =17.54,P ＜ 0.05) in HCC tissues.Overexpression of miR-30a-3p inhibited HCC cells proliferation and metastasis,while miR-30a-3p inhibition increased the proliferation and metastasis of HCC cells.Overexpression of miR-30a-3p decreased the expression of Caspase 1 (t =12.73,P ＜ 0.05) and inhibited the induction of pyroptosis,inhibiting the expression of IL-18 (t =7.32,P ＜ 0.05) and IL-1 β (t =7.32,P ＜0.05).When miRNA-30a-3p was inhibited,the cell viability of HCC was increased (F1 =9.57,P ＜0.05).When miRNA-30a-3p and Caspase 1 were inhibited together,the cell viability of HCC decreased (F2 =10.66,P ＜ 0.05).Conclusion MiRNA-30a-3p regulate cell pyroptosis through Caspase 1 pathway,inhibiting the proliferation,invasion and metastasis of HCC cells.