| Purpose:Chromobox2（CBX2）is widely regarded as a oncogene in many studies.However,the exact role of CBX2 in gastric cancer is still unclear.Yes-associated protein（YAP）,as a transcription coactivator of Hippo family,is a mechanical transduction protein.YAP plays a key role in proliferation,differentiation and organ size.More importantly,YAP has the ability to induce the localization and accumulation of β-catenin.Therefore,we aim to explore that cbx 2 gene knockout may inhibit the expression of gastric cancer cells by promoting β-catenin signaling pathway,YAP phosphorylation and its transport to cytoplasm.Research methods:1.the correlation between the expression of cbx2 in gastric cancer and clinicopathological factors.Clinical samples were collected from 167 patients with gastric tissues,and CBX2 expression was used WB and PCR to analyze.At the same time,we used Wb and PCR to analyze the expression of cbx2 protein and m RNA in gastric cancer cells.2.preliminary study on the effect and mechanism of inhibiting the expression of cbx2 on gastric cancer.CBX2 was upregulated in GC cell lines,and CBX2 knockdown inhibited the activation of β-catenin signaling pathway.The CBX2 interference plasmid was constructed and transfected into MFC cells to down-regulate the expression of CBX2.RT-q PCR was used to detect the expression of CBX2 to determine the transfection efficiency of sh RNA-CBX2.After transfection,β-catenim and downstream factors c-myc and cyclin D1 were tested by western blotting.expression.We further explored that CBX2 knockout could inhibit the proliferation and spread of MFC cells by inactivating the β-catenin signaling pathway.Since DKK1 is a negative regulator of Wnt/β-catenin signaling,β-catenin signaling is activated by silencing DKK1.The transfection efficiency of sh RNA-DKK1-1 was detected by RT-q PCR.MFC cell viability and proliferation transfected with sh RNA-CBX2-1 or sh RNA-DKK1-1 were tested by colony formation assay and CCK-8 method.3.To verify that CBX2 knockdown inhibited the migrate and invade cells by inactivating the β-catenin signaling pathway.Transwell and Scratch assay analysis were used to evaluate the migration and invasion ability of MFC cells transfected with sh RNA-CBX2-1 or sh RNA-DKK1-1.It has been confirmed that MMP7 and MMP13 are significantly up-regulated,Continue to detect MMP7 and MMP13 proteins in MFC cells transfected with sh RNA-CBX2-1 or sh RNA-DKK1-1.YAP plays a key role in the growth,proliferation,and various tumor differentiation types,and YAP has the ability to induce nuclear localization and accumulation of β-catenin.Transfection of MFC cells with CBX2 interfering plasmids,and the expression of P-YAP and YAP in the cytoplasm and nucleus was tested by western blotting.Transduced by overexpressing YAP plasmid.MFC cells were stained,and the show of YAP was checked by WB.Wb was used to check the show of β-catenin、c-myc and cyclin D1 after overexpression of yap plasmid.4.The effect of knockdown of CBX2 on the growth of xenograft tumors in nude mice.Continuing to take in vivo tumorigenic experiments in nude mice also verified that CBX2 has the ability to promote tumor formation.Results:1.Immunohistochemistry was carried out on the tissues,and the feedback of the detection results showed that CBX2 was significantly related to progress of gastric cancer.The expression of cbx2 was not significantly adjacent with gender,age,tumor growth site,vascular and nerve invasion.The Kaplan-Meier method was used to analyze the connection between CBX2 expression and survival in 167 patients was no difference between each other.The expression of cbx2 protein and m RNA in gc MFC,hgc-27,AGS,mkn-45 cell s and normal GES-1 cells were significantly added by Wb and RT q PCR.The presention level of cbx2 was the highest in MFC cells.2.sh RNA-CBX2 was constructed to downregulate the expression of CBX2.The sh RNA-CBX2-1 with low CBX2 expression was selected for subsequent experiments.The expression of β-catenin were significantly decreased by western blotting,and the results indicated that CBX2 knockdown inhibited the activation ofβ-catenin signaling pathway.Since DKK1 is a negative regulator of Wnt/β-catenin signaling,a DKK1 interfering plasmid was constructed to transfect MFC cells,and RT-q PCR was used for detection,and sh RNA-DKK1-1 with higher transfection efficiency was choosed for forward experiments.Using sh RNA-DKK1-1 to transfect sh RNA-CBX2-1,Scratch experiments and Transwell analysis were performed to verify that CBX2 knockout inhibited gc cell migration and invasion。3.YAP protein in the cytoplasmic matrix was noteworthy descended,and the emergence of YAP in the nucleus was raised,impling that CBX2 has a regulatory influence on the emergence of YAP.The YAP higher expression plasmid was constructed.It was verified that YAP overexpression weakened the inhibitory effect of CBX2 gene knockout on protein expression.4.The two groups of nude mice were injected subcutaneously with gastric cancer cell MFC that stably silenced CBX2 and gastric cancer cell MFC with high CBX2 expression,and then observe the occurrence of tumor in the armpit of nude mice.MFC cells expressing gastric cancer cells were weak,indicating that CBX2 has the ability to enhance tumor growth in nude mice.Conclusion:Studies have shown that cbx2 is significantly up-regulated in GC cells,and cbx2 knockdown can be inhibited β-Catenin pathway promotes Yap phosphorylation and translocation.Cbx2 may represent a new and promising therapeutic mark for gastric cancer... |
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