The Role And Mechanism Of Aurora Kinase AURKC In The Development Of Lung Adenocarcinoma

BackgroundLung cancer is ranking the first around the world with high incidence and mortality in malignant tumors,in which non-small cell lung cancer accounts for more than 80%,and abundant studies showed that the incidence of lung adenocarcinoma in non-small cell lung cancer is increasing year by year,leading to terrible effects on people's life and health.Generally,it is intractable to treat with patients who have been diagnosed in the middle and late stages of lung cancer.Therefore,it is of great significance to explore the mechanism of lung cancer occurrence and development for the diagnosis and treatment of lung cancer.Currently,many studies have shown that malignant progression of lung cancer is associated with biological behaviors such as cell proliferation,migration,invasion,apoptosis and cell cycle.The formation of tumor cells results from the disorder of the cell cycle,which then causes the cells to undergo aneuploid expansion,since proteins involved in the cell cycle are crucial.Aurora kinase C,as one of the members of the Aurora kinase family that regulates the cell cycle process,is mainly involved in the division of cells to ensure the cell cycle proceeds normally,however,abnormality of this kinase will lead to tumor cell formation.There are three members of the Aurora kinase family,namely Aurora kinase A(AURKA),Aurora kinase B(AURKB),and Aurora kinase C(AURKC),all of which belong to serine or threonine protein kinases at different stages of the cell cycle different positioning,using its kinase activity to coordinate with other cell cycle proteins to maintain cell cycle progression.AURKA and AURKB are usually expressed in mammalian somatic cells.Most studies have shown that AURKA and AURKB can affect the biological behavior of tumor cells through various signaling pathways.AURKC is less expressed in somatic cells but specifically expressed in testicular tissues,which plays an irreplaceable role in the development of sperm and eggs,and other studies also exhibited that it is also involved in the occurrence and development of malignant diseases such as breast cancer,colon cancer,and malignant gliomas.AURKC,as a protein kinase that regulates the cell cycle,may affect the occurrence and development of lung adenocarcinoma,however,the mechanism of action in lung adenocarcinoma is still unclear.In this study,the effect of knocking out AURKC on the function of lung adenocarcinoma A549 cells was tested by in vitro experiments.The results demonstrated that knock-out of AURKC lead to the tumor suppressive effect by up-regulating the expression of p53 protein,and the regulatory mechanism between AURKC and p53 was preliminary discussed.ObjectiveAURKC CRISPR/Cas9 knockout plasmid was constructed,through infecting human lung adenocarcinoma cells A549 cells,and the stable knockout cell lines abtained with Puro screening,were used for in vitro experiments to clarify the effects of AURKC on cell growth,migration,invasion,cell cycle and apoptosis.Moreover,the mechanism of AURKC in the occurrence and development of lung adenocarcinoma will provide theoretical basis and effective targets for diagnosis and treatment of clinical lung adenocarcinoma in future.MethodsFirst,the protein expression level of AURKC in various of human lung adenocarcinoma cell lines was evaluated,and A549 cells with obvious high-expression level of AURKC was selected for experiment The AURKC CRISPR/Cas9 knockout plasmid was constructed by the lentiviral packaging method,the knockout plasmid was then transfected into 293T cells for virus packaging,subsequently,the packaged virus was used to infect A549 cells.To verify the AURKC protein knockout effect,Western Blot was conducted after screening with Puro and picking monoclonal cells strains.Meanwhile,genome-level knockout effect and stable control cell line(A549-AURKC-ctrl)and stable knockout AURKC cell lines(A549-AURKC-sgRNAl,A549-AURKC-sgRNA2)were determined by PCR amplification and sequencing with extraction of genomic DNA.Subsequently,the stable cell lines were used for in vitro cell function experiments.The abilities of cell cloning formation and lateral migration after AURKC knockout were detected by the plate cloning and scratch healing experiment,respectively.In addition,the abilities of longitudinal migration and invasion of AURKC knockout cells were also evaluated with Transwell chamber.The effect of apoptosis was determined by flow cytometry with the help of the PE-Annexin V and 7-AAD staining and PI staining.Finally,Western Blot and Co-IP experiments were used to detect the level of major protein molecules in related signaling pathways and protein-protein interactions.Results(1)Obtaining A549 monoclonal cell line stably knocking out AURKC:PCR amplification and sequencing results of bacterial liquid PCR proved that the AURKC CRISPR/Cas9 knockout plasmid was successfully constructed;genome sequencing and Western Blot results proved that A549 stable cell line knocked out AURKC was successfully constructed.(2)The roles of AURKC in lung cancer was confirmed in vitro experiments:after knockout AURKC,the cloning ability of A549 cells was significantly reduced proved with plate cloning experiments;the wound healing ability and the lateral mobility were slightly enhanced in comparison with the control group.However,the longitudinal mobility of the A549 cells was significantly reduced after knocking out AURKC(P<0.001).Additionally,Matrigel matrix gel test proved that the ability of A549 cells to digest matrix glue after knocking out AURKC was significantly reduced,and the number of A549 cells passing through the chamber was significantly lower than that of the control group(P<0.001).Apoptosis experiments showed that there was no significant change in cell apoptosis and cell cycle experiment results showed that most cells had G2/M phase arrest after AURKC knocked out.(3)AURKC and p53 synergetically regulate the occurrence and development of lung adenocarinoma:Western Blot experiments indicated that the cell cycle-related protein CDK1,Cyclin B1 and p53 are significantly up-regulated after knockout of AURKC.The Co-IP results showed that there was an interaction between AURKC and p53,and p53 inhibited the expression of AURKC protein,which was independent of increased protein degradation.Conclusion(1)Knockout of AURKC lead to significant changes in cell morphology,cell proliferation,migration and invasion ability decreased,and at the same time lead to cell G2/M phase arrest;(2)It was found that AURKC could affect the growth of A549 cells by interacting with p53.