Study On The Role And Mechanism Of Adapter Protein LNK On ATC Progression

Objective: Our previous clinical study showed that the elevated peripheral platelet(PLT) and white blood cell(WBC) counts may be adverse prognostic factors of anaplatic thyroid carcinoma (ATC) patients,suggesting that hematopoietic relative genes may involved in ATC progression. The adapter protein LNK(also known as SH2B3) has been reported as an upstream regulator of many hematopoietic factors and closely related to hematopoietic carcinomas, thus we suppose LNK may take part in ATC progression. This study aims to explore the role of LNK on ATC cells and clarify its mechanism.Methods: To exam the LNK mRNA expression in thyroid carcinoma tissue samples and cell lines, real-time PCR reaction was performed in a thermocycler. Molecular cloning technique was used to construct the LNK eukaryotic expression vector:pLKO.1-shLNK and pMSCV-LNK-flag, pcDNA3.1-LNK-flag. LNK stable knockdown/overexpression cell lines were established with above plasmids.Real-time PCR and weatern blot were performed for examming the expression of LNK mRNA and protein in stable cell lines. MTT, clone formation, and transwell assays were employed to assess the abilities of proliferation, clonality, and migration of ATC cells, respectively. To determine the interacting protein of LNK, flag-pull down assay was applied, and the result was confirmed by co-IP assay.LNK-interacting protein was knocked-down in LNK-overexpression cell lines to determine the malignant biological behavior of tumor cells. Western blot was used to detect the relative pathway proteins expression.Results: LNK mRNA levels were higher in ATC compared with differentiated thyroid caicinoma both in cell lines and tissue samples. LNK stable knockdown/overexpression cell lines were successfully constructed. Knock-down of LNK suppressed the abilities of proliferation, clonality, and migration of ATC cells,and LNK overexpression enhanced these abilities. Flag-pull down and co-IP assay both confirmed that 14-3-3? and 14-3-3? were interacting proteins of LNK.14-3-3?/14-3-3? knock-down inhibited above cell functions enhanced by LNK-overexpression, followed with the change of apoptosis pathway ERK1/2-Akt-NF-?B-Bcl-2/Bcl-xL proteins expression.Conclusion: We demonstrated that LNK enhanced the anti-apoptosis ability of ATC cells by interacting with 14-3-3? and 14-3-3?. Then, the LNK-14-3-3?/14-3-3?complex promoted ATC cells proliferation,clonality,and migration via the 14-3-3?/14-3-3?-ERKl/2-Akt-NF-?B-Bcl-2/Bcl-xL signaling pathway. This discovery could lead to a new development of prognostic tool as well as potential drug targets.