The Role And Mechanism Investigation Of Integrin-Linked Kinase In The Proliferation Of Breast Cancer Cells

ObjectiveBreast cancer is one of the most common malignant tumor in women,its incidence ranks the second in women worldwide.Although the major therapy strategy for breast cancer is surgical treatment combined with radiotherapy or chemotherapy.In recent years,molecular targeted therapy,a new kind of treatment,has gradually been known and accepted,especially,widely used in the patients who are not appropriate for the treatments mentioned above.However,the molecular mechanism of regulating the progression of breast cancer remains largely unknown,resulting in the lack of effective therapy targets of molecular targeted therapy.Thus it is necessary to explore the mechanism of regulating the breast cancer cells growth for improving the efficiency of molecular targeted therapy and providing the potential treatment targets.Loss of adhesion between tumor cells and extracellular matrix,which is required during the invasion process and dissemination of cancer cells,is closely controlled by Integrins,and Integrin-linked kinase(ILK)is a major serine-threonine kinase of Integrins and widely expressed in a broad range of human tissues.Previous studies have reported that up-regulation of ILK expression is frequently observed in multiple types of carcinomas.Moreover,ILK participates in regulating some physical functions of cancer cells,including cells growth,cell-cycle progression,invasion and migration,cell motility and tumor angiogenesis.In breast cancer,activation of integrin-linked kinase can promote cell motility and metastasis by binding to LIMD2.However,the roles of integrin-linked kinase in breast cancer cells growth and the corresponding mechanisms are yet to be explored.The aim of the current study is to explore the role and mechanism of overexpression and silence ILK on breast cancer cell proliferation by using the gene transfection and RNAi assays.The current study can help people to further understand the mechanism of breast cancer,and provide important reference basis for the establishment of breast cancer biomarkers and targets.Methods1?Stable transfection of ILK in MCF-7 breast cancer cells was constructed by gene transfection method;WST-1,BrdU incorporation and LDH release assays were utilized to investigate the effects of ILK over-expression on the proliferation,viability and apoptosis of MCF-7 cells;Western blot assays were explored to test whether ILK over-expression could influence the protein expression of Akt,p-Akt1(Thr308)and PCNA genes in MCF-7 cells.2?Low expression of ILK in MDA-MB-231 breast cancer cells was constructed by RNAi method;WST-1,BrdU incorporation and LDH release assays were utilized to investigate the effects of low expression of ILK on the proliferation,viability and apoptosis of MDA-MB-231 cells;Western blot assays were explored to test whether low expression of ILK could influence the protein expression of Akt,p-Akt1(Thr308)and PCNA genes in MDA-MB-231 cells.3?The Akt inhibitors LY294002 was used to suppress expression of p-Akt1(Thr308)in ILK over-expression MCF-7 cells,WST-1,BrdU incorporation and LDH release assays were utilized to investigate the effects of blocking PI3K/AKT pathway on the proliferation,viability and apoptosis of ILK over-expression MCF-7cells;Western blot assays were explored to test whether blocking PI3K/AKT pathway could influence the protein expression of PCNA genes in ILK over-expression MCF-7 cells.4?The PI3K/AKT pathway in ILK low expression MDA-MB-231 cells was activated by transient transfection of HA-AKT;WST-1,BrdU incorporation and LDH release assays were utilized to investigate the effects of activating PI3K/AKT pathway on the proliferation,viability and apoptosis of ILK low-expression MDA-MB-231 cells;Western blot assays were explored to test whether activating PI3K/AKT pathway could influence the protein expression of PCNA genes in ILK low-expression MDA-MB-231 cells.Results1?WST-1,BrdU incorporation and LDH release experiments showed thatover-expression of ILK can promote proliferation and viability,inhibit apoptosis of MCF-7 cells;Western blot assays indicated that over-expression of ILK in MCF-7cells can up-regulate the protein expression of p-Akt1(Thr308)and PCNA genes,however,the protein expression of Akt was not affected by the transfection of ILK gene.2?WST-1,BrdU incorporation and LDH release experiments showed that low-expression of ILK can inhibit proliferation and viability,promote apoptosis of MDA-MB-231 cells;Western blot assays indicated that low-expression of ILK in MDA-MB-231 cells can down-regulate the protein expression of p-Akt1(Thr308)and PCNA genes,however,the protein expression of Akt was not affected by the silencing of ILK gene.3?10 ?M LY294002 significantly inhibited the expression of p-Akt1(Thr308),but did not influence the expression of Akt in ILK over-expression MCF-7 cells,suggesting LY294002 can effectively block PI3K/Akt pathway in MCF-7 cells;WST-1,BrdU incorporation and LDH release assays indicated that blocking PI3K/Akt pathway inhibited proliferation and viability,promote apoptosis of ILK over-expression MCF-7 cells;In addition,Western blot assays indicated that blocking PI3K/Akt pathway could inhibit the expression of PCNA at protein level in ILK over-expression MCF-7 cells.4?Transient transfection of HA-AKT significantly up-regulated the expression of p-Akt1(Thr308),but did not influence the expression of Akt in ILK low-expression MDA-MB-231 cells,suggesting transient transfection of HA-AKT can effectively activate PI3K/Akt pathway in MDA-MB-231 cells;WST-1,BrdU incorporation and LDH release assays indicated that activating PI3K/Akt pathway promoted proliferation and viability,inhibited apoptosis of ILK low-expression MDA-MB-231 cells;Moreover,Western blot assays indicated that activating PI3K/Akt pathway could up-regulate the expression of PCNA at protein level in ILK low-expression MDA-MB-231 cells.ConclusionIn conclusion,the present study have demonstrated that ILK facilitates cell proliferation and inhibits apoptosis through activating the PI3K/Akt pathway in breast cancer cells,suggesting a vital role of ILK as an oncogene in the progression of breast cancer.The current study can help people to further understand the mechanism of breast cancer,and provides an effective potential targets for molecular targeted therapy of breast cancer.