| Inhibitors of apoptosis proteins(IAPs)are important regulators of apoptosis,and their overexpression inhibits apoptosis and promotes tumor cell growth.Inhibitors of apoptosis protein-like protein-2(ILP-2),a member of the IAPs family,is highly expressed in breast cancer cells and is a potential target for breast cancer therapy.Knockdown of ILP-2 expression can induce apoptosis and inhibit the proliferation and migration of breast cancer cells.XIAP and Livin in the IAPs family can promote tumor cell growth by regulating the occurrence of cellular autophagy,which in turn inhibits apoptosis.And ILP-2,as a member of the IAPs family,has a protein structure similar to XIAP and Livin.Therefore,we speculate that ILP-2 may promote the growth of breast cancer cells by regulating cellular autophagy.In order to clarify whether ILP-2 protein can promote the growth of breast cancer cells by regulating autophagy,this experiment were set five groups were : control(breast cancer cells),positive control(breast cancer cells + Rapamycin),negative control(breast cancer cells +NC + Rapamycin),breast cancer cells + si RNA-3 + Rapamycin,breast cancer cells + si RNA-5 +Rapamycin.In this study,we propose to knock down the expression of ILP-2 in MCF-7 and MX-1 of breast cancer cells by RNA interference technology,to clarify the association between ILP-2 and autophagy,and to further explore the mechanism of ILP-2 regulating autophagy to promote the growth of breast cancer cells,to provide a theoretical basis for the clarification of ILP-2 as a molecular target for clinical treatment of breast cancer and the combination of targeted therapy and chemotherapy.Results(1).The results of MDC fluorescence assay showed that the number of autophagic vesicles was significantly increased in the positive group compared with the blank group;while the number of autophagic vesicles was significantly lower in the positive group when rapamycin treatment was added after interference with ILP-2.(2).Western Blot results showed no significant difference in ILP-2 expression in the positive group compared with the control;compared with the positive group,the expression of ILP-2,ATG14,Beclin1 was down-regulated and the ILC3-II/LC3-I ratio decreased in breast cancer cells MCF-7 and MX-1 after knockdown of ILP-2,while the expression of P62 increased.(3).CCK-8,cell scratch,and AO-EB results showed that cell proliferation and migration activity decreased and apoptosis rate increased significantly with the downregulation of autophagy level after knockdown of ILP-2 compared with the positive group;WB assay showed that cleaved-caspase-3 expression was significantly increased and MMP-9 expression was significantly downregulated after knockdown of ILP-2 compared with the positive control.(4).CO-IP results showed that ILP-2 interacted with PI3K(P85).It is speculated that ILP-2 may regulate the occurrence of cellular autophagy by interacting with PI3 K to modulate the PI3K-Akt signaling pathway.(5).WB results showed that compared with the positive group cells,interfering with the expression of ILP-2,cellular autophagy was significantly inhibited,PI3 K,Akt,LC3-II/LC3-I ratio decreased and P62 expression was significantly up-regulated;Bcl-2,Beclin1 expression was down-regulated and Bax,Caspase-9,3 expression was significantly increased.JC-1 results showed that knockdown of ILP-2 expression resulted in a significant decrease in mitochondrial membrane potential and a significant increase in apoptosis rate.Conclusion(1).ILP-2 is closely associated with cellular autophagy.(2).High levels of ILP-2 can regulate the autophagy level of MCF-7 and MX-1 in breast cancer cells,inhibit apoptosis,and promote the growth and proliferation of tumor cells.(3).ILP-2 interacts with PI3K(P85),and the possible way for ILP-2 to regulate autophagy level to promote breast cancer cell growth is to activate PI3K/Akt signaling pathway.(4).ILP-2 can promote tumor cell growth by regulating Bcl-2/Bax,Caspase-mediated signaling pathway to activate autophagy to inhibit apoptosis. |
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