| Objective:Aberrantly expression of Annexin A2?Annexin II,ANXA2?is one of the common features of wide spectrum of cancers,including breast cancer,pancreatic cancer,liver cancer,prostate cancer and glioma.Accumulating data suggest that there is a significant correlation between ANXA2 and cancer progression.While,the molecular mechanism and potential intermolecular crosstalk for ANXA2 on tumor cells are not clear.Moreover there are significant different mechanisms between different cancer types.This study focus on the role of ANXA2 in glioma cells and try to find out the potential preliminary molecular mechanism.Methods:Human AnxA2 coding sequence was cloned,the lentiviral expression vector of ANXA2 overexpression and RNAi-knockdown were constructed and stable transfected in U251 cell by lentivirus infection.The effect of ANXA2 overexpression and RNAi-knockdown was detected by RT-PCR,Western-blot and immunofluorescence.The proliferation activity of glioma U251 cell was measured by MTT assay,clone formation assay,flow cytometry and the transplanted tumor experiment in nude mice.The potential mechanisms of proliferation were elucidated by Western-blot,EMSA and drug inhibition.For apoptosis,cascade relationship between NF?B-P65,Akt,Caspase-3,PTEN and ANXA2 were analyed by drug intervention experiment and Western-blot,apoptosis rate was measured by flow cytometry.Migration and invasion of U251 cells were investigated by scratch test and tranwell chamber containing matrix glue,the expression and function analysis of MMPs was performed by Western-bloting and Gelatin-Zymography.Results:U251 Cells with ANXA2-overexpression and RNAi-knockdown were successfully constructed by lentivirus infection.The rate of overexpression was42.7%±5.2%?p<0.05?,and the rate of knockdown was 72.2%±3.2%?p<0.01?.Knockdown of ANXA2 could significantly inhibit the proliferation of U251 cell.ANXA2-knockdown had no effect on the expression of STAT3,but decreased the level of pSTAT3?Y705?and transcriptional activity,downregulated the expression of CyclinD1,and led to the G1/S block;further results show that ANXA2 probably regulate pSTAT3?Y705?level through direct binding with STAT3,thereby affecting the pSTAT3-CyclinD1 pathway-mediated cell proliferation.The related experiments were repeated in the glioma U87,indicating that this mechanism in the glioma cell have a certain representation.However,overexpression of ANXA2 significantly promoted the proliferation of U251 in MTT and nude mice transplanted tumor experiment,but had no significantly effect on clone formation rate,cell cycle and pSTAT3-CyclinD1pathway.When introducing ANXA2 back to ANXA2-knockdown U251,we found the rescue of pSTAT3?Y705?expression,suggesting that the recombinant ANXA2 has a complete functional structure and that endogenous ANXA2 may be redundant;Overexpression of ANXA2 enhances the expression of pSTAT3?Y705?in the presence of EGF,suggesting that the promotion of ANXA2 in MTT and nude mice may be due to accumulation and synergistic effect of paracrine factors EGF.There was no significantly inhibition of apoptosis and acitve Caspase-3 in ANXA2-overexpressed U251 cell,suggesting that the abnormal growth of ANXA2-overexpressed transplanted tumor was not due to the promotion of proliferation or the inhibition of apoptosis.Significantly promotion of apoptosis and acitve Caspase-3 were observed in ANXA2-knockdown cells,suggesting that both inhibition of proliferation and promotion of apoptosis contribute to the growth inhibition of ANXA2-knockdown transplantd tumor.Further studies on the molecular mechanism showed that drug inhibition of pP65?Ser536?increased the expression of active Caspase-3 and apoptotic phenotype,drug inhibition of pAkt?Thr308?led to the downregulation of pP65?Ser536?and upregulation active Caspase-3,while ANXA2-knockdown suppressed both pP65?Ser536?and pAkt?Thr308?.Immunoprecipitation also confirmed that ANXA2 binds directly to PTEN,an inhibitor of PI3K-Akt pathway.So,a possible explanation for apoptotic promotion of ANXA2-knockdown is the absence of ANXA2 leads to release of PTEN,which blocks the PI3K-Akt pathway,inhibits pAkt?Thr308?,the lack of pAkt?Thr308?downregulates pP65?Ser536?by inhibition of IKK?and finally leading to the promotion of apoptosis.Possible explanation for the failure of apoptotic inhibition in ANXA2-overexpressed cells is that excess ANXA2 can not bind more PTEN,as a tumor suppressor gene,PTEN is scarce in tumor cells,so there is no more significant inhibition of apoptosis,although ANXA2 is redundant.ANXA2-overexpression can promote the secretion of active MMP2/9,whereas ANXA2-knockdown is opposite.It can be seen that ANXA2 can produce positive regulatory effect on the invasion and metastasis of U251 cells by affecting the secretion of active MMP2/9.Conclusion:ANXA2 affects proliferation of human glioma cells through pSTAT3-CyclinD1pathway by direct interaction with STAT3.ANXA2 on this pathway is in redundant state,while there is positive synergies bewteen ANXA2 and EGF.ANXA2 in human glioma cells may exert an effect on cell apoptosis through PTEN-AKT-P65-Caspase3signal chain.ANXA2 could also affect the invasion and metastasis of U251 cells by affecting the secretion of active MMP2/9. |
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