| Gastric cancer（GC）is a common gastrointestinal malignancy which contains approximately 7.7 percent of all cancer deaths each year.Although the level of diagnosis and treatment of GC has improved considerably in the past decades,the incidence and mortality rate of GC is still increasing due to the lack of early diagnosis and aggressive treatment.In China,the high incidence of GC is mainly due to people’s dietary habits,genetics,infections and other high-risk factors,coupled with the lack of attention,most patients are at the progressive and mid-to-late stages while they were diagnosed and commonly got a poor prognosis.It is very urgent to find a new tumour marker for the clinical research of GC.Lymphocyte adaptor protein（LNK）,also known as SH2B3,is a member of the SH2B protein family which do not possess enzymatic activity and are highly expressed in vascular endothelial cells,haematopoietic stem cells and their precursor cells.LNK is widely studied in haematological system and is generally considered to be a tumour suppressor in haematological system.The high expression of LNK inhibits the occurrence and development of haematopoietic malignancies by activating tyrosine kinase（TK）located on the cell surface.However,there are only several papers focused on LNK in solid tumours,suggesting that LNK acts as a tumour enhener in high-grade ovarian,melanoma and thyroid cancers,which is contradictory to the role of LNK in haematological system.Therefore,in order to further elucidate the mechanism of LNK in solid tumours and enrich its biological functions,we took GC as a representative of solid tumors in our study to analyse the expression level and functional mechanism of LNK in GC.It would provide theoretical basis and useful target for personalized treatment and clinic research on GC.Methods:1.Preliminary analysis of LNK expression in GC from TCGA database applying bioinformatics which was validated by datasets GSE54129 and GSE29272 for the consistency of the raw bioinformatics data.2.Construction of the GC cell line with hLNK stable overexpression.The lentiviral plasmids pCDH-hLNK,pCDH and its packaging plasmids were transfected into cell line 293 T to prepare recombinant lenti virus with hLNK overexpression.Then human GC cell line AGS was infected by the recombinant lentivirus,after puromycin resistance screening,the cell line stably overexpressing hLNK was obtained and named AGS-hLnk.The overexpression level of hLNK in AGS was verified by GFP green fluorescence intensity and western blot assay.3.Construction of the GC cell line with stable knockdown of hLNK.The plasmid（pLKO-shRNA,pLKO-shLNK83,pLKO-shLNK84）was co-transfected with their corresponding packaging plasmids in 293T cells respectively to obtain the recombinant lentiviruses with hLNK knockdown.GC cell line SGC-7901 were infected by lentivirus obtained above respectively.Followed by puromycin screening stable,GC cell line with the knockdown of hLNK were obtained and named as SGC-7901-shLNK83（7901-shLNK83）,SGC-7901-shLNK84（7901-shLNK84）,control strain SGC-7901-control（7901-control）,respectively.To verify the knockdown level of hLNK in SGC-7901,western blot assay was used.4.The effects of hLNK overexpression/knockdown on the proliferation ability of GC cell lines were analyzed by plate cloning formation assay and MTT method.The effects of hLNK overexpression/knockdown on the migrative ability of GC cell lines were analyzed by wound healing assay.The effects of hLNK overexpression/knockdown on the invasive ability of GC cell lines were evaluated by transwell chamber assay.5.The activation levels of signaling molecules such as Jak2,Stat3,Stat5 were analysed by western blot to investigate the main mechanism of hLNK affecting the progression of GC.Results:1.According to the bioinformatics results obtained from TCGA database,LNK was highly expressed in GC tissues.The results were subsequently validated by GSE54129 and GSE29272 datasets and the consistent results were obtained.2.The GC cell line AGS-hLNK stably overexpressing hLNK was successfully constructed.The results of plate cloning assay and MTT method showed that overexpression of hLNK boosted the proliferation of the cell line AGS-hLNK.Both wound healing assay and transwell assay revealed that the migrative and invasive ability of AGS-hLNK were enhanced after hLNK overexpression.It indicats that LNK exerts a role in promoting the progression of GC cell lines AGS.3.The GC cell line 7901-shLNK with stable knockdown of hLNK was successfully constructed.The results of plate cloning formation assay and MTT assay showed that the proliferaticve ability of 7901-shLNK was significantly inhibited after knockdown of LNK.However,the result of wound healing assay showed that the invasive ability of 7901-shLNK has no obvious change.4.The results of western blot analysis of Jak-Stats signaling pathway molecules showed that phosphorylated Jak2,Stat3 and Stat5 were up-regulated obviously,especially Stat3.It indicates that the main mechanism of LNK in promoting GC progression is mainly because of the activation of Jak2-Stat3 signaling pathway.Conclusion:LNK is relatively highly expressed in GC tissues and affects the disease free survival probability of GC patients.Overexpression of LNK facilitated the proliferation,invasion and metastasis of GC cells whereas knockdown of LNK inhibited the proliferation of GC cells.This mechanism of LNK on enhancing GC progression was mainly attributed to the activation of Jak2-Stat3 signaling pathway. |
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