| Breast cancer(BC)is a malignant tumor that occurs in the mammary epithelial tissue Triple negative breast cancer(TNBC)is the most lethal subtype of BC with the negative expression of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth receptor 2(HER-2).TNBC has poor prognosis because of its high recurrence rate and strong metastatic ability.At present,traditional chemotherapy and surgical treatment are still the preferred treatments for TNBC because of no effective clinical therapeutic targets available.Therefore,it is very urgent to explore new biological therapeutic targets and clarify the mechanism of the tumorigenesis in TNBC.Lymphocyte adaptor protein(LNK),also known as SH2B3,is a member of the SH2B family which has no enzymatic activity.It is a negative regulator of the hematopoietic system and mainly expresses in hematopoietic cells and endothelial cells,but has low expression in hematoiogical malignancy.However,there is few study on LNK in solid tumors.Our research group found that LNK highly expressed in TNBC in the previous study.On this basis,this study explores the new biological functions and mechanism of LNK in the occurrence and development of TNBC and would provide a new theoretical target for the clinical treatment of TNBC.Methods:1.Paraffin-embedded tumor sections of TNBC and non-TNBC patients were collected and analyzed for the expression of human LNK(hLNK)by immunohistochemistry(IHC).2.Western blot and qRT-PCR assays were used to analyze the expression of hLNK in human breast cancer cell lines.3.The lentivirus plasmids pLKO-shLNK83,pLKO-shLNK84 were transfected into 293T cells with the corresponding packing plasmids to generate recombinant lentivirus,respectively.Human TNBC cell line MDA-MB-231(MB-231)and human non-TNBC cell line(MCF-7)were infected by the recombinant lentivirus to construt stable transfected cell lines with the silencing of hLNK after puromycin screening.4.The hLNK cDNA was amplified by PCR and subcloned into the lentiviral vector pCDH to construct the recombinant plasmid pCDH-hLNK,which was transfected into 293T cells to generate recombinant lentivirus with the overexpression of hLNK.Human TNBC cell lines:MB-231,MDA-MB-453(MB-453)and non-TNBC cell lines:MCF-7,were infected and screened by puromycin to establish the cell lines:MB-231-hLNK(231-hLNK),MB-453-hLNK(453-hLNK)and MCF-7-hLNK with the stable overpression of hLNK.5.The expression level of hLNK in the breast cancer cell lines established were detected by fluorescence microscope and western blot assay.Then CCK-8 and clonal formation assay were used to detect the proliferative ability of the breast cancer cell lines established;Wound healing assay and transwell chamber assay were performed to analyze the migrating and invasive abilities of those LNK silenced/over-expressed breast cancer cell lines mentioned above.6.To explore the mechanism between hLNK and the tumorigenesis of TNBC,western blot was used to analyze the phospharated level of the signaling molecules:Jak2,Stat3,Stat5,AKT,PI3K,p38 and 14-3-3 family proteins.Results:1.The result of IHC staining indicated that hLNK is highly expressed in paraffin-embedded TNBC tissues;Western blot and qRT-PCR analysis showed that hLNK is highly expressed in TNBC cell lines,especially in MB-231.2.The hLNK silenced cell lines:231-shLNK83,231-shLNK84 and MCF-7-shLNK83,MCF-7-shLNK84,were successfully constructed.The results of clonal formation assay and CCK-8 proliferation assay showed that the proliferative ability of MB-231 was severely inhibited by the silencing of hLNK.At last,231-shLNK83,231-shLNK-84 lost their ability to growth.While the proliferative ability of MCF-7-shLNK83 and MCF-7-shLNK84 were greatly prompted,especially MCF-7-shLNK84 cell line.Indicating that hLNK is essential for the proliferation of MB-231.3.The TNBC cell lines(231-hLNK,453-hLNK)with hLNK overexpression were successfully constructed.The results of clonal formation assay,CCK-8 assay,wound healing assay and transwell chamber assay confirmed that overexpression of hLNK down-regulated the ability of proliferation,migration and invasion of 231-hLNK,453-hLNK,but all these cells still remained the ability of proliferation,migration and invasion,while MCF-7-hLNK completely lost those abilities.It indictes that a certain level of LNK is necessary for the proliferation of TNBC,LNK upregulation can still promote TNBC tumor progression,but its promoting effect may be decreased by the forced over-expression of LNK.4.The mechanism of overexpression of hLNK to promote the development of TNBC was analyzed by western blot analysis.5.The results of western blot showed that p-Jak2,p-Stat3 were down-regulated in 231-hLNK and 453-hLNK compared with its controls.It indicates that hLNK promotes the tumorigenesis and recurrence of TNBC mainly through Jak2-Stat3 pathway.While silencing of hLNK caused cell proliferation and invasion in MCF-7 also by up-regulating Jak2-Stat3 signaling pathway.Conclusion:LNK is essential for the proliferation of TNBC cells,the high expression of LNK in TNBC can inhibit the tumorigenesis and metastasis of TNBC,which mainly via activating Jak2-Stat3 signaling pathway. |
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