The Interaction Between LIMD1 And AURKA Regulates Tumor Cell Proliferation

Background:With the increase of whole population and the aggravation of population aging,cancer has become a major threat to health and economic development nowadays.Therefore,how to find an effective treatment of cancer and improve the prognosis of patients have become key problems needed to be solved urgently.In previous studies,it has been found that many biological processes in the initiation and development of tumor cells are different from those in normal cells.One of the important biological characteristics is that during the mitotic process,the regulation of tumor cells is out of control,leading to a significant increase in their proliferation ability.The Aurora family(AURKA,AURKB and AURKC)plays key roles in the mitotic process of the cell cycle.As an oncogene,AURKA is found to be overexpressed in various types of tumor tissues,which has a strong correlation with the malignancy degree of tumor and the clinical prognosis of patients.And AURKA plays a crucial role in the initiation and development of tumor,and is regarded as a key target of tumor therapy.In the in vitro and in vivo experiments,significant achievements have been made in the research on AURKA inhibitors’ effect on tumor cell growth.Therefore,AURKA inhibitors are expected to be effective in tumor targeting therapy for clinical cancer patients.However,the results of clinical trial are not expected.Some patients have experienced adverse reactions in varying degrees after taking the drugs.In addition,it has been found that AURKA inhibitors have poor targeting specificity in clinical patients.Therefore,how to solve these problems is important for tumor treatment.Herein,we try to find a key protein that interacts with AURKA,and explore the regulatory mechanism between these two proteins We hope that the tumor growth will be inhibited by targeting the AURKA interacting protein.This research will provide a new idea for targeted cancer therapies.Objectives:Our project is based on the screening and analysis of interaction database,to find a key protein interacts with AURKA.Combining with biological experiments,we verify the interaction between these two proteins,and further explore the regulatory mechanism.This research will provide a new idea for targeted cancer therapies.Methods:(1)Co-immunoprecipitation(co-IP)and GST pull down assays were used to verify the interaction between LIMD1 and AURKA.(2)(1)si RNA assay: the expression of LIMD1 and AURKA was interfered with small interfering RNA in breast cancer cells,then the protein expression levels of LIMD1 and AURKA were detected by western blot.(2)Rescue assay: exogenous LIMD1-c DNA was transfected to MDA-MB-231 cells after the endogenous LIMD1 was interfered with si LIMD1,and the AURKA protein expression was detected by western blot.(3)Immunofluorescence(IF)assay was used to detect the intracellular localization of LIMD1 in MDA-MB-231 cells with AURKA knockdown or AURKA kinase inhibitor treatment.(4)CCK-8 kit was used to detect the proliferation ability of breast cancer cells(MDA-MB-231)after treatment LIMD1-overexpressed MDA-MB-231 cells with AURKA inhibitor(MLN8237).Results:(1)In the breast cancer cell line MDA-MB-231,AURKA interacts with LIMD1.(2)At the protein level,interfering with LIMD1 promotes AURKA expression in MDA-MB-231 cells. (3)AURKA knockdown could decrease LIMD1 localization in the nucleus of MDA-MB-231 cells.(4)After treating LIMD1-overexpressed MDA-MB-231 cells with AURKA inhibitor(MLN8237),the proliferation ability of breast cancer cell line MDA-MB-231 was significantly inhibited.Conclusions:(1)The interaction between LIMD1 and AURKA exists.(2)LIMD1 knockdown promotes AURKA protein expression.(3)The localization of LIMD1 in nucleus is regulated by AURKA.(4)The proliferation ability of breast cancer cells was significantly inhibited by treating LIMD1-overexpressed MDA-MB-231 cells with AURKA inhibitor.