| Breast cancer is a malignant tumor that occurs in the mammary epithelial tissue and ranked as the first female malignant tunor.Triple negative breast cancer(TNBC)is negative for the expression of genes of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER-2).There is no effective targeted therapy for TNBC in clinical practice due to the high tumor heterogeneity,low degree of differentiation,strong invasion and metastatic ability of the tumor cells,and thus,it is neccessary to find noval targets for TNBC therapeutics.Elucidation the mechanism of TNBC progression will provide a theoretical basis for success TNBC treatments.LNK(lymphocyte adaptor protein,SH2B3)is a member of the SH2B family.It plays an important regulatory role in growth and development,metabolic balance and immune regulation in vivo.LNK can suppress multiple downstream signal transduction pathways by inhibition of JAK2 activity and plays an important role in maintaining the homeostasis of hematopoietic system.We first used the database to analyze the expression level of LNK in clinical samples of breast cancer with different receptor expression,and found that LNK is highly expressed in TNBC.Then we confirmed the high expression of LNK in paraffin-embeded human TNBC samples,as well as in human TNBC cell lines.The effects of LNK on the growth of human breast cancer were studied using in vitro proliferation and apoptosis assay,and the mechanism of LNK function in TNBC was also studied.Our studies provide a potential new molecular target for the treatment of TNBC.Methods:1.Paraffin-embedded tumor sections of TNBC and non-TNBC patients were collected and analyzed for LNK expression by immunohistochemistry(IHC).2.Western blot and qRT-PCR were used to analyze the expression of LNK in human breast cancer cell lines(TNBC and non-TNBC).3.The plasmids pLKO-shLnk-83#,pLKO-shLnk-84#were co-transfected into 293T cells with the corresponding lentivirus packaging plasmids(VSVG,pMDL,pREV)to generate lentiviruses,respectively.pLKO-shLnk-puro was used to generate control virus.Lentivirus was used to infect human breast cancer cell lines:MB-231(ER-,PR-,HER2-)、Hcc1937(ER-,PR-,HER2-)、MCF-7(ER+,PR-,HER2-),to construct stable transfer cells lines with the scilencing of LNK.Western blot and qRT-PCR experiments were used to confirm the LNK silencing in these cell lines in both protein and mRNA levels.The effect of knockdown of LNK on the proliferation of human breast cancer cells was detected by plate cloning formation assay and CCK-8 assay.Flow cytometry was used to analyze the apoptosis and cell cycle of MCF-7 and MB-231 with the scilencing of LNK.Transwell chamber assay was used to detect the invasive ability of MB-231 after scilencing of LNK.4.The plasmid pOZ-hLnk and the packaging plasmid pCLAmpho were co-transfected into MB-231 cells to construct 231-hLNK stable cell line with the overexpression of LNK.Cells were cultured after being sorted twice by flow cytometry based on the screening marker IL-2R on the retriviral vector.The overexpression of LNK was detected by western blot.The effect of overexpressed LNK on the proliferation of MB-231 was examined by plate cloning formation assay and CCK-8 assay.Invasive ability of MB-231 with enforced LNK expression was measured by the transwell chamber invasion assay.5.To explore the mechanism of high expression of LNK in tumorigenesis of TNBC,western blot was used to analyze the signaling molecules such as JAK2,STAT3,p-STAT3,STAT5,p-STAT5,p38,AKT and 14-3-3 proteins to find the molecular pathways involved in the role of LNK in TNBC.Results:1.IHC staining showed that LNK is highly expressed in paraffin-embedded TNBC tissues.2.Western blot and qRT-PCR showed LNK was highly expressed in both mRNA and protein levels in human TNBC cell lines.3.With the analysis of western blot and qRT-PCR,LNK silenced stable transfected cell lines(MCF-7,MB-231,Hcc1937)were successfully constructed with the decreased LNK mRNA and protein levels.Plate cloning formation assay and CCK-8 proliferation assay showed that the proliferation of MCF-7 cells were accelerated after the sciencing of LNK while the cell proliferation of TNBC cell lines MB-231 and Hee1937 cells were suppressed.The cell cycle assay showed that the G0/G1 phase and S phase were decreased and the G2 phase was increased after the knockdown of LNK in MB-231.Apoptosis assay showed that apoptosis level is increased after knockdown of LNK.The results from transwell chamber invasion assay also showed that cell invasive ability was severely decreased with the knockdown of LNK.All these data indicates that scilencing LNK can greatly suppress the growth in TNBC cells.4.MB-231 cells with the overexpression of LNK were successfully constructed with the infection of retrovirus containing LNK gene.Plate cloning formation assay and CCK-8 proliferation assay confirmed that the proliferation of MB-231 was promoted with the overexpression of LNK.Transwell chamber invasion assay also showed that the invasive ability was enhanced after the overexpression of LNK in MB-231 cells.Noticably,in TNBC cell lines,the overexpression of LNK highly promoted cell proliferation and invasion,suggesting that the high level of LNK may drive tumorigeneis and progression of TNBC patients.5.By western blot analysis,STAT3 and p-STAT3 levels were dramatically up-regulated,while p-STAT5,p38,AKT and 14-3-3 protein levels were not significantly changed in MB-231 with the overexpression of LNK.This suggests that LNK may work as an oncogene via up-regulating the activity of JAK2-STAT3 signaling pathway.Conclusion:LNK promotes the tumorigenic activities of triple-negative breast cancers,possibly by up-regulating the JAK2-STAT3 signaling pathway. |
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