The Study Of Effect Of AURKA Protein Kinase On Hepatocellular Carcinoma And Its Mechanism

Objective:First,HCC cell line HepG2 was transfected with shAURKA and AURKA overexpression vectors to obtained stable cell lines.The aim of the present study was to determine whether AURKA mediates DNA damage pathway in vitro and in vivo.Then,to investigate the effect and mechanism of Aurora kinase A(AURKA)inhibitor MLN8237 on cell proliferation of HCC cells in vitro.Method:(1)HCC cell line HepG2 was transfected with shAURKA and AURKA overexpression vectors to obtained stable cell lines.The cells were then analyzed by CCK8 and cell clone formation to explore the effects of AURKA on the proliferation of HCC cells;wound healing assay was used to explore the effects of AURKA on the migration and invasion ability;Western blotting was performed to detect the mechanism of AURKA involved in the development of HCC cells;and the effect of AURKA on mitosis of HCC cells was detected by immunofluorescence assay.(2)Mice xenograft models of human liver cancer was established using HCC stably transfected cell lines.In vivo immunofluorescence assay and HE and TUNEL staining were performed to explore the effects of AURKA on the growth and apoptosis of mice xenograft models of human liver cancer.(3)HepG2 cells were divided into two groups,including the control group and the observation groups with MLN8237 in different dose.The cell proliferation inhibitory rate was examined by CCK8 assay;the clonality was investigated by plate clone formation assay;cellular invasion and migration ability was determined by wound healing assay;apoptosis was determined using the PI and Annexin V-FITC staining method;the protein expression level of cell signal pathway was detected by Western blotting;after different concentrations of MLN8237 acted on HepG2 cells and received different doses of β-ray irradiation,CCK8 assay was used to detect cell survival rate to explore the radiosensitizing effect of MLN8237 on HCC cells.Results:(1)The transient transfection method was used to successfully construct two groups of stably transfected HCC cell lines.(2)Through a series of in vitro experiments on HCC stably transfected cell lines,the experimental results indicate that silencing AURKA expression can inhibit the proliferation activity of HCC cells,inhibit the ability of HCC cells to migrate and invade.Then,α-tubulin immunofluorescence staining of HCC stably transfected cell lines was performed.Observation under a confocal microscope showed that silencing AURKA expression can inhibit mitosis and induce apoptosis in HCC cells.(3)Western blot results indicate that the mechanism of AURKA involved in the development of HCC may be through the DNA damage repair pathway.AURKA can directly change the DNA damage repair network and DNA damage response protein mediated by ATM / Chk2 and ATR / Chk1.(4)Mice xenograft models of human liver cancer was established by using the obtained stable transfected cell lines,observe the growth of subcutaneous tumor formation of human liver cancer,and perform HE and TUNEL staining on tissue sections of experimental tumor specimens of human liver cancer.The results showed that AURKA promoted the growth of subcutaneous tumors in human liver cancer nude mice;AURKA could promote the proliferation of liver cancer cells and inhibit the apoptosis and necrosis of liver cancer cells,thus promoting the growth of tumor tissues.(5)Aurora kinase A inhibitor MLN8237 inhibits HepG2 cell proliferation and inhibits cell migration,and the higher the concentration of MLN8237,the more obvious the inhibitory effect.At the same time,MLN8237 promoted the apoptosis of HepG2 cells,as the concentration of MLN8237 increased,the promotion effect became more obvious.The results of Western blot showed that MLN8237 could promote DNA damage repair response in cells.(6)Apply different concentrations of MLN8237 to HepG2 cells and receive different doses of β-ray irradiation.CCK8 test was used to detect HepG2 cell survival rate.The results show that MLN8237 can effectively increase the sensitivity of HCC cells to radiation therapy and increase the lethality to HCC cells.Conclusion:The results of this study suggest that AURKA-mediated DNA damage repair pathway promotes the occurrence and development of HCC.Inhibition of AURKA can achieve antitumor activity by inhibiting cell proliferation and pro-apoptotic effects,etc.,and provide experimental basis for clinical treatment and diagnosis of liver cancer.Become a new target for the treatment of HCC.At the same time,the results of this study show that MLN8237,an Aurora kinase A inhibitor,can reduce the malignant degree of HepG2 cells,and by down-regulating the expression of AURKA protein in cells,it can promote apoptosis and DNA damage repair response,prevent malignant transformation of cells,and is expected to become a treatment for HCC and specific molecular targeted drug.