Effects And Potential Mechanism Of Tristetraprolin Regulate Senescence-associated Secretory Phenotype Of Gastric Cancer Cells

Background: Gastric cancer(GC)is one of the most common gastrointestinal carcinoma in China,and more than 90% of patients were diagnosed with advanced GC.Clinically,due to advanced GC is not sensitive to neoadjuvant chemotherapy(NAC).It is not widely carried out.The study shown that: chemotherapeutic drugs,such as paclitaxel,can induce tumor cell senescence and senescence cells secrete large amounts of inflammatory cytokines,which is called cell senescence-associated secretory phenotype(SASP)leading to resistance to chemotherapy.Our previous study on GC tissue specimens with NAC reported: the effect of NAC for GC is association with the tristetraprolin(TTP)expression,those who have high expression of TTP,always be more sensitivity on NAC.TTP,as a RNA binding protein,is able to bind to the AU-rich element(ARE)of the 3 ’UTR region of mRNA,such as a large number of cytokines,thus degrading the target RNA.TTP is able to degrade the target factor with a large number of inflammatory factors secreted by SASP.Thus,we hypothesized that TTP may increase the sensitivity of NAC in GC by inhibiting SASP induced by NAC.TTP may be a candidate sensitivity biomarker for NAC in GC.On this basis,we investigated the effect and mechanism of the expression of TTP,which can increase the sensitivity of NAC by inhibiting SASP.It is significant for a further discussion on how to improve the sensitivity of NAC by TTP and find a personalized therapy for gastric cancer,also provide a theoretical support for TTP as NAC effective biomarkers.Methods:In the first part,we constructed a stabilized model of senescence GC cell induced by Paclitaxel in vitro.According to the related experimental results,BGC-823 cell line was selected to further exprement.Paclitaxel was used to induce GC cell line for 24 h,48h,72 h with the concentration of 0,25 nM,35nM,45 nM,respectively.Stabled the GC cell senescence model was confirmed in vitro by SA-beta-gal galactosidase staining.In the second part,we constructed the TTP overexpression and TTP interference cell lines of GCcell line BGC-823 to screen the stable expression and stable interference cell line.The experiment was divid into three groups: TTP overexpression group,TTP interference group,control group.BGC-823 cells induced by Paclitaxel for a certain period of time,the cells were harveste and the mRNA levels of SASP related factors was measured by qPCR.In addition,the cell culture was collected to detect the secretion level of SASP related factors by ELISA.In the third part,the expression level of p65 in TTP overexpression,TTP shRNA and control cell line were determined by Western blot.Next,cells were stimulated by a small amount of TNF-α to activate NF-κB signaling pathway,and the expression of p65 was detected by nuclear magnetic resonance.Finally,we did the immunofluorescence assay to determine the level of p65 expression both in and out of the nucleus,and image pro plus software was used to analyze the fluorescence intensity.Results:The first part: when BGC-823 cells induced by paclitaxel at 35 nM for 72 h,senescence related SA-beta-gal staining is positive,indicating BGC-823 senescence model successfully constructed in vitro,and in 72 h,the BGC-823 cell induced by paclitaxel show the highest proportion of cell senescence.The second part: to construct the TTP overexpression and TTP interference cell lines of BGC-823 cells,respectively,the TTP expression was measured by qPCR and Western blot.Then,the BGC-823 cell line is stable overexpression of TTP and stable interference of TTP screened by Puromycin in 2mg/ml.On this basis,we found that overexpression of TTP could decrease the expression of SASP related factors in the model of GC cell line BGC-823 induced by paclitaxel.The third part: in order to study the mechanism of the expression of TTP on the SASP in senescence of BGC-823 cells model induced by paclitaxel,Western blotting results showed that the expression of p65 in the nucleus in overexpression of TTP group were significantly decreased compared with control.The p65 in the nucleus in the TTP interference group was significantly higher than that in the control group.In order to verify that if the abnormal expression of TTP have influence on p65 into the nucleus.Further,we performed immunofluorescence to observe the fluorescence of p65 in vitro and in vivo on over expression of TTP group,TTP interference group,and control group under the condition of paclitaxel induced senescence model.Statistical analysis showed that the fluorescence of p65 in the nucleus in the overexpression of TTP group was significantly different compared with the control group and the TTP interference group.Conclusions: We successfully constructed the model of GC cell line BGC-823 induced by paclitaxel in vitro and found that overexpression of TTP could inhibit the expression of SASP related cytokines in GC cell line.Next,TTP can directly degrade the expression of SASP related factors through ARE binding pathway,and also can inhibit the activation of NF-κB signaling pathway by inhibiting the p65 entry into the nucleus.This may suggest that TTP may increase the sensitivity of NAC to GC by reducing the expression of SASP related factors.