miR-424-5p/Rictor/mTORC信号轴诱导肝癌细胞自噬性死亡的机制

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Abstract

Objective To study the mechanism of miR-424-5p/Rictor/mammalian target of rapamycin(mTORC)signaling axis inducing autophagic death of hepatoma cells.Methods Hep-3B was selected as liver cancer cell,the control group was treated with PBS,and the overexpression miR-424-5p group,interference miR-424-5p expression group,overexpression Rictor group and interference Rictor expression group were respectively transfected with miR-424-5p mimics,miR-424-5p inhibitor,Rictor overexpression plasmid,Rictor siRNA.Cell viability,apoptosis and cycle were detected,the shape of autophagosomes in slices was observed,Lc3-Ⅱ,Lc3-Ⅰ,Beclin1,P62 mRNA and protein were detected in cells,and the mRNA expression of miR-424-5p/Rictor/mTORC2/Akt/mTORC1 was determined by RT-PCR,Western blot method was used to detect related proteins,and to verify the targeting relationship between miR-424-5p and Rictor.Results The expression of miR-424-5p in Hep-3B cells was low(0.45±0.05).After transfection,the G0+G1 phase cells in the overexpression miR-424-5p group were higher than those in the control group,and the cell viability was lower than that in the control group,the differences were statistically significant(P<0.05).The proportion of cells in G0+G1 phase in the interference miR-424-5p expression group and overexpression Rictor group were lower than those in the overexpression miR-424-5p group,and the cell viability was higher than that in the overexpression miR-424-5p group,interference Rictor expression group,the differences were statistically significant(P<0.05).The autophagosomes and autophagolysosomes in the overexpression miR-424-5p group at excitation wavelengths of 488 nm and 587 nm were higher than those in the interference miR-424-5p expression group and overexpression Rictor group,the differences were statistically significant(P<0.05).The expression levels of Rictor,mTORC2,Akt and mTORC1 in the overexpression miR-424-5p group were lower than those in the interference miR-424-5p expression group and the overexpression Rictor group,but higher than those in the interference Rictor expression group,the differences were statistically significant(P<0.05).The expression levels of Lc3-II,Lc3-I and Beclin1 in the overexpression miR-424-5p group were lower than those of the other four groups,and the expression level of P62 was higher than that of the other four groups,the differences were statistically significant(P<0.05).Dual luciferase experiments confirmed that miR-424-5p could directly inhibit Rictor expression,and Western blot showed that miR-424-5p could inhibit Rictor expression in Hep-3B cells.Conclusion MiR-424-5p can affect autophagy by regulating the expression of Rictor/mTORC2/Akt/mTORC1,thereby inhibiting the proliferation of liver cancer cells and promoting their apoptosis.

Key words： MiR-424-5p Rictor Mammalian target of rapamycin Immunofluorescence

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