| Objective: Worldwide,gastric cancer is the fifth most common cancer and the third leading cause of cancer-related death.In China,most of patients were diagnosed with an advanced gastric cancer at the time of treatment.Even though the comprehensive treatment based on surgery has been developing,the prognosis of patients with gastric cancer has not yet reached the expected level.Although the response rate to multi-agent chemotherapy of recurrence or metastatic gastric cancer can be 50% or greater,there are nearly all patients became chemotherapy resistance,and the median survival is extended only 10-12 months.Therefore,the researches on the occurrence,development,and chemotherapy resistance of gastric cancer are quit important for improving the patients’ prognosis.In recent years,more and more researches were focus on the cancer stem cells(CSCs),and the gastric cancer stem cells(GCSCs)were gradually been confirmed.Gastric cancer stem cells are a kind of malignant tumor cell group with self-renewal ability and high tumorigenicity,and have differentiation potential and high drug resistance.They are closely related to biological behaviors such as drug resistance,recurrence and metastasis for gastric cancer.CDK5RAP3 is a binding protein of the cyclin dependent kinase 5 activator protein.The role and mechanism of CDK5RAP3 in the biological characteristic of gastric cancer stem cells has not been reported.In the current study,we firstly examined the expression of CDK5RAP3 and the GCSCs marker CD44 in gastric cancer patients,and the relationship between them and clinicopathological factors and prognosis.Secondly,the in vitro and in vivo cell function expriments were used to explore the relationship between CDK5RAP3 and GCSCs spheriods growth ability,invasion and migration ability,organoids formation,tumorigenic ability and chemoresistance.Finally,molecular biology studies were used to investigate the potential molecular mechanism of CDK5RAP3 for GCSCs.So the aim of this study was to investigate the effect and molecular mechanisms of CDK5RAP3 in GCSCs,and provide new theories and ideas for the study of GCSCs,and also can provide an important evidence for targeted therapy of gastric cancer.Methods: 1.Using the immunohistochemical to detect the expression of CDK5RAP3 and CD44 in gastric cancer tissues,then to explore the correlation between CDK5RAP3 and CD44.Additionally,the correlation between these two protein and the clinical pathological characteristics and prognosis were investigated.2.We used western blot method to detect the CDK5RAP3 and the GCSCs markers in monolayer and spheriod cells.3.The packaged lentivirus were used to build the stable overexpressed or down-regulating CDK5RAP3 in gastric cancer cell lines to obseve the effect of CDK5RAP3 on the growth of GCSCs.4.Transwell test was used to investigate the effect of CDK5RAP3 on the invasion and migration of GCSCs.5.Construct murine gastric cancer cells with stably overexpressing CDK5RAP3,and observing their effects on the organoids formation of GCSCs.6.By building the nude mice subcutaneously into tumor model,validating the influence of CDK5RAP3 in the formation of GCSCs.7.The vitro cell function assay to observe the effect of CDK5RAP3 on chemoresistance of spheroidal gastric cancer cells,and futher verify the resistance of CDK5RAP3 to chemotherapy in nude mice.8.Using molecular biology techniques to explore the mechanism of CDK5RAP3 in GCSCs.By using the PI3 K inhibitor and overexpressing CDK5RAP3 to analyse the effect of CDK5RAP3 on PI3K/AKT signalling pathway and Epithelial-mesenchymal transition(EMT)in GCSCs.9.Immunochemistry was used to detect the expression of EMT transcription factor Snail in gastric cancer tissue,and we combined with the expression of CD44 and CDK5RAP3,to observe its effect on the prognosis of patients.Results: 1.The results of IHC showed that CDK5RAP3 protein expression in adjacent normal tissues was significantly higher than that in gastric cancer tisssues,and the CD44 protein were lower in adjacent normal tissues than that in tumor tissues.There were negatively correlated expression between CDK5RAP3 and CD44.2.CDK5RAP3 and CD44 were both the independent factors on the prognosis for gastric cancer patients.However,only in CD44 high expression patients,the CDK5RAP3 high expression patients had a better survival than low expression paitents.3.In vitro expriments,CDK5RAP3 suprresed the spheroid growth,invasion and migration,and organoids formation of GCSCs.4.The nude mice subcutaneous tumor formation model showed that CDK5RAP3 can reduce the tumorigenic ability and chemoresistance of GCSCs.5.The PI3 K inhibitor can inhibit the invasion and migration of GCSCs,and increase the expression of CDK5RAP3,while the phosphorylation level of AKT,the expression of EMT transcription factor Snail,and the mesenchymal marker N-cadherin were decreased.6.When overexpression of CDK5RAP3 can inhibit the phosphorylation of S473 at AKT and affect the expression of EMT transcription factor Snail,affecting the activity of EMT in GCSCs.7.High expression of CDK5RAP3 can improve the long-term survival of patients with high expression of Snail in gastric cancer patients with high expression of CD44.Conclusion: 1.CDK5RAP3 is negatively correlated with the expression of GCSCs marker CD44 in gastric cancer patients.They were both the independent prognostic factors.They were expected to be a joint predictor of gastric cancer prognosis.2.CDK5RAP3 not only suppressed the spheroid growth,invasion and migration,and organoid format ability of GCSCs,but also inhibited the tumorigenic ability and chemoresistance of GCSCs.3.CDK5RAP3 can reduce the expression of EMT transcription factor Snail by inhibiting the phosphorylation level of AKT,and reduce the EMT activity of GCSCs.4.In patients with high expression of CD44,the expression of CDK5RAP3 can improve the long-term survival of patients with high expression of Snail,and can be a potential trarget for the treatment of gastric cancer. |
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