| Purpose:Aurora kinase A(AURKA),which belongs to the serine/threonine protein kinase family,has been identified as a key driver of the genesis and progression of diverse tumors.The aim of this study was to determine the clinical significance of AURKA in patients with hepatoblastoma(HB)and the effect of inhibiting AURKA in the HuH-6 human HB cells.Methods:1.Thirty-three paraffin-embedded HB tissue sections were obtained from the Children’s Hospital of Chongqing Medical University.These sections contained 33 cases of HB tissues and 14 cases of adjacent liver tissues.Immunohistochemistry(IHC)was used to detect the expression of AURKA in hepatoblastoma tissue and normal liver tissue adjacent to the tumor.Then,the relationship between the expression of AURKA and the clinicopathological characteristics of HB was statistically analyzed.2.After HuH-6 cells were transfected with NC-siRNA orAURKA-siRNA,Western blot was used to check the knockdown efficiency and CCK-8 was performed to detected the effect of AURKA knockdown on cell viability in HuH-6 cells.3.CCK-8,EdU,flow cytometry and Western blot were used to detect the effects of ALS on cell cycle,proliferation and the expression of cycle-related protein Cyclin-D1 in HuH-6 cells.4.Flow cytometry,TEM and Western blot were used to detect the effects of ALS on apoptosis,autophagy,apoptosis-related and autophagy-related proteins Bcl-2,Bcl-xL,cleaved caspase-3 and LC3-II in HuH-6 cells.5.CCK-8,flow cytometry and Western blot were used to examine the effect of autophagy inhibitor 3-MA on the viability,apoptosis,and LC3 II expression in HuH-6 cells induced by ALS.6.Western blot was used to detect the effects of ALS on the expression of p-AURKA,p38 MAPK and p-p38 MAPK in HuH-6 cells.Results:1.AURKA was highly expressed in 21 of the 33 HB tissues and in 1of 14 adjacent normal tissues.The expression of AURKA in HB tissues was significantly higher than that in adjacent normal tissues(63.34% vs 7.14%,p <0.001).High AURKA expression was significantly correlated with tumor metastasis and COG stage.2.Western blot results showed that AURKA expression wassignificantly down-regulated in the cells of AURKA-siRNA group compared with NC-siRNA group,while there was no significant difference in AURKA expression between Control group and NC-siRNA group.CCK-8 results showed that compared with NC-siRNA group,the HuH-6cell viability was decreased significantly in AURKA-siRNA group at 48 and 72 hours after transfection.3.The results of EdU and CCK-8 showed that ALS significantly inhibited the proliferation of HuH-6 cells.Flow cytometry results showed that ALS induced G1 phase arrest of HuH-6 cell.Western blot results showed that ALS significantly down-regulated the expression of Cyclin-D1 in HuH-6 cells in a concentration-dependent manner.4.Flow cytometry results showed that ALS significantly promoted HuH-6 cell apoptosis.TEM results showed that the number of autophagosomes of HuH-6 cells was increased in the ALS group compared with Control group.Western blot results showed that ALS significantly down-regulated the expression of Bcl-2 and Bcl-xL and up-regulated the expression of cleaved caspase-3 and LC3 II.5.Western blot results showed that the expression of LC3 II was significantly down-regulated in 3-MA + ALS group compared with ALS group,while there was no statistical difference in the expression of LC3 II between 3-MA group and Control group.The CCK-8 results showed that compared with the ALS group,the viability of HuH-6 cells was decreasedsignificantly in 3-MA + ALS group,while there is no statistical difference in cell viability between 3-MA group and Control group.Flow cytometry results showed that compared with the ALS group,the apoptosis rate was increased significantly in 3-MA + ALS group.6.Western blot results showed that ALS significantly inhibited p-AURKA expression and p38 MAPK phosphorylation in HuH-6 cells.Conclusion:The expression of AURKA in human HB tissues was significantly higher than that in adjacent normal tissues,and high expression of AURKA was positive related to COG stage and the tumor metastasis.In vitro,knockdown of AURKA significantly inhibited HuH-6 cell viability.These results suggested that AURKA may be a potential prognostic biomarker and therapeutic target of HB.AURKA-specific inhibitor ALS could inhibit HuH-6 cell proliferation and promote apoptosis and autophagy.Meanwhile,after supplying 3-MA to inhibit ALS-induced cell autophagy,the apoptosis rate was further increased.Furthermore,ALS could inhibit p-38 MAPK phosphorylation.These results indicated that ALS may be a potential targeting drug for HB,and its mechanism may be related to inhibiting p38 MAPK signaling pathway. |
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