Protein Kinase Aurora-A Promotes Liver Fibrosis And Its Mechanism Investigation

BackgroundHepatic fibrosis is the result of accumulation of extracellular matrix proteins due to chronic liver injury,causing by a variety of pathogenic factors.Activated hepatic stellate cells(HSCs)are the main source of extracellular matrix protein production and deposition.Protein kinase Aurora-A(AURKA)is a serine,threonine protein kinase which participate in the regulation of mitosis during centrosome maturation,spindle assembly and its positioning and other events.As an oncogene,AURKA has been reported to promote the progression of hepatocellular carcinoma and Primary myelofibrosis.However,the effect of AURKA on hepatic fibrosis has not been reported.Here,we aimed to investigate whether AURKA affects liver fibrosis via promoting the activation of HSCs and its potential mechanism,providing a new target and therapeutic strategy for clinical intervention of liver fibrosis.Methods1.The expression level and cellular localization of AURKA in normal liver and different degrees of hepatic fibrosis were examined by bioinformatics analysis and immunohistochemical method.The expression levels of AURKA and HSCs activation marker gene(PDGFRβ)in four different mouse primary liver cells were examined by Western Blot.2.The changes of AURKA and HSCs activation marker gene(a-SMA)expression were determined by immunofluorescences technique during activation of primary HSCs in vitro.AURKA expression in LX-2 cells was inhabited by siRNA,and the relationship between AURKA and HSCs activation markers and liver fibrosis related genes were further analyzed by real-time PCR and Western Blot.3.The effects of AURKA expression on LX-2 cell proliferation,cell cycle and apoptosis were examined by using cell counting kit-8(CCK8),Edu cell proliferation assay and flow cytometry.4.The effect of AURKA inhibitor on HSCs activation was investigated by using real-time PCR and Western Blot in LX-2 cells.A mouse model of CCl4-induced hepatic fibrosis was established to evaluate the effect of AURKA inhibitors on the progression of hepatic fibrosis in vivo.5.The possible mechanism of AURKA in promoting liver fibrosis were further explored by RNA sequencing and a variety of bioinformatics analysis in LX-2 cells that treats with AURKA siRNA and control cells.Results1.AURKA expression was upregulated in hepatic fibrosis and highly expressed in activated HSCs in the septal region.Compared with hepatocyte,kupffer cells and liver sinusoidal endothelial cell,AURKA was highly expressed in HSCs.2.AURKA and a-SMA were significantly upregulated during culture activation of mouse primary HSCs in vitro.The transcription lever of a-SMA,PDGFR and profibrogenic genes,such as TIMP1,COL1A1 was significantly down-regulated and expression of a-SMA,PDGFR protein were significantly decreased in AURKA siRNA treated LX-2 cells.3.Interference of AURKA expression with siRNA in LX-2 cells significantly inhibited cell proliferation,led to G2/M phase arrest and promoted cell apoptosis.4.AURKA inhibitors(MLN8237)treatment significantly suppressed the proliferation of LX-2 cells in a concentration-dependent manner in vitro,with an IC50 of 23.58 μM.It also blocked the activation of LX-2,causing cell G2/M phase arrest and promoting cell apoptosis in a concentration dependent manner.5.Pharmacology inhibition of AURKA phosphorylation by MLN8237 significantly alleviated liver fibrosis and improved liver function,inhibiting the expression of α-SMA and have no obvious toxic and side effects on normal mice in vivo.6.RNA sequencing analysis found that the Wnt/β-catenin signaling pathway is the downstream signaling pathway of AURKA.AURKA can promote the activation of HSCs by activating the Wnt/β-catenin signaling pathway.ConclusionOur results suggest that AURKA protein promote HSCs activation by activating the Wnt/β-catenin signaling pathway,further aggravate liver fibrosis,Thus,AURKA may serve as a new potential therapeutic target for liver fibrosis.