Roles Of TP53INP1 In Ir-induced Autophagy Of Breast Cancer MCF-7 Cells

Breast Cancer(BC)is the most common cancer among women around the world,and it is estimated that about 1.3 million women suffer from BC each year.BC ranks second in the female cancer mortality rate,resulting in the death of about 350,000 women a year in both developed and developing countries.Many factors are involved in the pathogenesis and progression of BC,including genetic?biological and environmental factors,as well as lifestyle.Radiation therapy(RT)is one of the methods of breast cancer treatment,the treatment effect depends on the radiation target tumor target and the optimal irradiation program(single dose,cumulative dose,segmentation program,etc.),and ultimately to give priority to kill tumor cells less damage to the role of normal cells.Ionizing radiation can cause different types of death of tumor cells,including: apoptosis? autophagy?necrosis?mitosis(MC)and so on.The mechanism of autophagy-induced autophagy in tumor cells is more complex,especially in breast cancer with autophagy gene Beclin-1 single allele deletion.TP53INP(tumor protein 53-induced nucleoprotein)gene family has two members,namely TP53INP1 and TP53INP2.In the late 1990 s,three different independent laboratories found that in 2002,the human genome gene naming committee finally identified the gene TP53INP1,TP53INP1 gene located on the chromosome 8q22 in the human gene.It has been found that TP53INP1 is a tumor suppressor gene that is low in mosttumor cells.TP53INP1 is an important target gene of p53.The expression of TP53INP1 can be regulated by transcription factors such as p53?p73and E2F1.It has been confirmed that TP53INP1 interacts with MAPLC3?GABARAP and GABARAP-like protein 2,and the amino-terminal fraction of TP53INP1 contains LC3 interacting region(LIR)interacting with MAPLC3,and TP53INP1 promotes autophagic cell death.Our research group confirmed that the expression of TP53INP1 was increased by ionizing radiation in the breast cancer MCF-7 cell,the role of TP53INP1 in autophagy of breast cancer cells caused by ionizing radiation is worthy of further study.Objective:This paper is intended to elaborate the role and mechanism of TP53INP1 in autophagy of breast cancer cells induced by ionizing radiation and found that a new regulatory pathway of autophagy induced by ionizing radiation,which provides a new target for radiotherapy and improves the effect of radiotherapy.Methods:1.The human breast cancer cell line MCF-7 was selected to construct a stable TP53INP1 gene silencing cell model(TP53INP1Ri)and control empty vector(p SUPER)cells.2.Cells were irradiated with deep X-ray irradiator at 0? 2?4?6?8Gy,respectively,the irradiation dose rate of 1.02 Gy / min and target skin distance of 70 cm.3.The cell death rate after irradiation was measured by trypan blue staining and analyzed by flow cytometry.4.Genetic engineering construction and transfection of RFP-LC3 into the cells were carried out.The autophagy of the cells was observed by fluorescence microscopy.5.Cell viability was measured by colony forming method after ionizing radiation.6.The autophagy and apoptosis were detected by Fluorescence staining flowcytometry.7.Protein expression was detected by Western blot.8.Immunofluorescence staining was used to detect the localization of different proteins in cells.9.Immunoprecipitation method to detect the interaction between different proteins.10.Chromatin immunoprecipitation method to detect the interaction between DNA and protein.Results:1 ionizing radiation induced breast cancer MCF-7 cell autophagyMDC staining and flow cytometry were used to detect the changes of autophagy after irradiation with different doses,that is,0?2?4?6?8 Gy.The results showed that the autophagic vacuoles in the irradiated cells were significantly increased and the incidence of autophagy showed a dose-dependent increase.Microtubule associated protein 1 light chain 3(MAPLC3)is a homologue of yeast autophagy related gene atg8 in mammals,which is an autophagy initiation factor.LC3-? was transformed into LC3-? and LC3-II was accumulated on the autophagic membrane during autophagy.The RFP-LC3 plasmid(RFP-LC3)was transfected into MCF-7 cells,and the cells transfected with RFP-LC3 were irradiated with 4Gy for 24 hours.The expression of RFP-LC3 in the cells was observed by fluorescence microscopy,and the spotted aggregation of MAPLC3 was significantly increased by microscopic images.The expression of MAPLC3 and Beclin1 in MCF-7 cells was detected by Western blot 24 hours after 4Gy irradiation.The results showed that MAPLC3 and Beclin1 expression were increased after irradiation,suggesting that ionization induced MCF-7 autophagy.2 ionizing radiation induced MCF-7 cell autophagic deathThe mortality of MCF-7 cells at 1?2?4?12? 24? 48 and 72 hoursafter 8Gy irradiation with the highest autophagy rate was detected by trypan blue staining.The results showed that MCF-7 cell mortality showed a time-dependent increase.3-methyladenosine(3MA)is an autophagic inhibitor that inhibits the formation of autophagy by inhibiting the formation of LC3-I in LC3-? by inhibiting the formation of Beclin1-Ptd Ins3KC3 complex.MCF-7 cells were added with 3MA,then given 0?2?4?6?8 Gy irradiation,the results showed that after irradiation3 MA group compared with no 3MA group significantly increased cell survival by using MDC staining flow cytometry.The changes of autophagy and apoptosis were observed by flow cytometry after 3MA acting and non-acting MCF-7 cells.The results showed that 3MA inhibited the autophagy induced by ionizing radiation while the inhibitory effect on apoptosis was not significant.Silencing MCF-7 cells autophagy-related genes Beclin1 and ATG5,4Gy irradiation after trypan blue staining to detect cell mortality and found that radiation-induced cell death reversal,suggesting that ionizing radiation induced autophagic cell death.3 TP53INP1 participates in autophagy caused by ionizing radiationFirst,it was found that ionizing radiation induced upregulation of TP53INP1 in breast cancer MCF-7 cells by high-throughput gene chip analysis,the expression of TP53INP1 was further confirmed by Western blot.To investigate whether TP53INP1 is involved in autophagy caused by ionizing radiation,we constructed TP53INP1 Ri and the control empty vector p SUPER cell model.Five dose points of 0?2?4?6?8 Gy were selected to irradiate the cell model,the cell viability fraction of TP53INP1 Ri and p SUPER was significantly increased by 6 Gy and 8Gy irradiation after cell colony formation assay.TP53INP1 Ri and p SUPERcells in the logarithmic growth phase were implanted into the 96-well plate and irradiated with 8 Gy.Autophagic inhibitors(1 mmol / L)3MA and autophagic inducers(100nmol / L)rapamycin were added before irradiation,the viability of the cells was measured by CCK-8 method 48 hours after irradiation.The results showed that compared with p SUPER,TP53INP1 Ri cells had significant effect on cell proliferation after 3MA,and there was no significant difference in the effect of rapamycin.The changes of autophagy and apoptosis were analyzed by flow cytometry 24 hours after 8Gy irradiation.The results showed that the incidence of autophagy after TP53INP1 Ri cells was significantly lower than that of p SUPER,but the change of apoptosis was not significant.It has been confirmed that the amino-terminal portion of TP53INP1 contains a part of the LC3-interacting region(LIR)that can interact with LC3,allowing TP53INP1 and LC3 to interact,TP53INP1 participates in autophagy and promotes autophagic cell death.Whether or not this mechanism of action occurs after ionizing radiation.We found that there was co-localization of TP53INP1 and autophagy protein LC3 in MCF-7cells at 24 hours after 8Gy irradiation by immunofluorescence.The expression of MAPLC3 in TP53INP1 Ri cells was significantly lower than that in p SUPER cells 24 hours after 8Gy irradiation,suggesting that TP53INP1 was involved in autophagy caused by ionizing radiation.4 TP53INP1 interacts with p53TP53INP1 is the target gene of p53,our laboratory has confirmed that increased expression of p53 after ionizing radiation and regulation of DRAM to promote breast cancer MCF-7 cells autophagy.Then whether p53 also interacts with TP53INP1,whether there is a link between the two gene by ionization radiation.The First,immunofluorescence was usedto confirm the co-localization of TP53INP1 and p53 in MCF-7 cells after ionizing radiation.The interaction between TP53INP1 and p53 was confirmed by IP(immunoprecipitation method),but compared with non-irradiated groups there was no increase in the interaction with TP53INP1 and p53.The results of Q-PCR and WB after silencing TP53INP1 cells by 8Gy showed that TP53INP1 did not affect the expression of p53 protein after ionizing radiation.The results of Q-PCR showed that silencing p53 could decrease the expression of TP53INP1 m RNA after ionizing radiation,but WB results showed that the expression of TP53INP1 protein was not significant in silencing p53.These results indicate that p53 has no direct effect on the expression of TP53INP1 after ionizing radiation and may be regulated by interaction.5 the interaction between TP53INP1 and STAT3The expression of TP53INP1 Ri and p SUPER cells was collected 24 hours after 8Gy irradiation and the RNA expression gene was extracted by Gene expression profile chip.We found that the expression of STAT3 was also decreased after TP53INP1 silencing.Western blot experiments confirmed this conclusion.STAT3(signal transduction and transcriptional activator 3)acts as a transcription factor to regulate extracellular signaling pathways such as cytokines and growth factors by interacting with the polypeptide receptors on the cell surface.STAT3 proteins are mainly activated by tyrosine phosphorylation,STAT3 Nuclear transcription factors can regulate the expression of more than 1000 gene products.In order to confirm the relationship between the two gene,first confirmed by immunofluorescence TP53INP1 and STAT3 co-localization in the cell,and then through the IP(immunoprecipitation)experiments found that the relationship between the two gene,the interaction between TP53INP1 and STAT3 after 8Gy irradiation was significantly enhancedcompared with non-irradiated group.To confirm the presence of transcriptional regulation between the two gene,further application of CHIP chromatin immunoprecipitation technique revealed that STAT3 as a transcription factor can bind to the promoter sequence of TP53INP1.The expression of TP53INP1 in MCF-7 cells was transiently transfected with STAT3 si RNA,and WB was used to verify the transfected cells after 8Gy irradiation can reduce the expression of TP53INP1.The results show that there is interaction between TP53INP1 and STAT3 after ionizing radiation.Conclusion:1.Ionization radiation induced breast cancer MCF-7 cells autophagy and lead to autophagic death of cells.2.TP53INP1 involved in breast cancer MCF-7 cells autophagy caused by ionizing radiation.3.TP53INP1 may be involved in ionizing radiation-induced breast cancer MCF-7 cell autophagy through the STAT3 pathway.