Inhibition Of Girdin Gene Silencing On Malignant Behavior Of Colorectal Cancer Cells And Its Related Mechanism

Background:Colorectal Cancer(CRC)is a malignant tumor originating from the colorectal mucosal epithelium,which is prone to metastasis.Studies have shown that Gridin is expressed in a variety of malignant proliferating tumor tissues,and its expression accelerates the process of tumor proliferation,erosion and metastasis,and also regulates autophagy that inhibits tumor cell apoptosis.However,the effect and mechanism of Girdin protein on malignant behavior in CRC cells has not been studied.Purpose:In order to investigate the biological function and mechanism of Girdin protein in the development of CRC,this research used RNAi technology to down-regulate the expression of Girdin protein in cells,and investigated the effect of downregulation of Girdin protein on cell proliferation,apoptosis and migration and its related signaling pathways.Finally,the effect of Girdin protein in vivo on tumor cell proliferation was verified.Method:1.Construction of transfected cell lines with down-regulated expression of Girdin.The Girdin protein high expression cell line was screened by Western Blot in human CRC cell lines,Caco-2,HCT-15,Lo Vo and DLD-1.The cell line with the highest expression of Girdin protein was used for subsequent studies.The sh RNA expression vectors Girdin-p GCH1/Neo and NC-p GCH1/Neo were constructed based on the Girdin gene sequence,and the transfection reagent Lipofectamine was used for cell transfection.The NC and Girdin si RNA cells were screened by G418.Identification was performed by fluorescence microscopy,flow cytometry,QPCR andWestern Blot methods.2.Effect of Down-regulation of Girdin Protein on Proliferation and Apoptosis of CRC Tumor Cells.The cell proliferation activities of Lo Vo cells,NC cells and Girdin si RNA cells were detected by MTT assay at 12 h,24 h,48 h,72 h and 96 h.Flow cytometry was used to detect the cell cycle distribution and the apoptosis of Lo Vo cells,NC cells and Girdin si RNA cells.The apoptosis-related proteins cleaved caspase-3,Bax and Bcl-2in Lo Vo,NC and Girdin si RNA cells were detected by Western Blot.3.Effect of down-regulation of Girdin protein expression on tumor cell migration and invasion.The migration ability of Lo Vo cells,NC cells and Girdin si RNA cells was examined by cell scratch assay.Transwell assay was used to detect the invasive ability of each kind of cells.Western Blot was used to detect the expression of migration-invasive related proteins,and the function of Girdin protein in tumor cell migration and invasion was further explored.4.Relationship between Girdin and JAK/STAT signaling pathway.Western blot was used to detect the expression of JAK/STAT signaling pathway protein and phosphorylation levels and pathway-associated inflammatory factors in Lo Vo cells,NC cells and Girdin si RNA cells.NC cells and Girdin si RNA cells were treated with JAK inhibitor LY2784544 to investigate the effect of Girdin down-regulation on cell invasion ability after blocking JAK/STAT pathway.Transwell assay was used to detect the invasive ability of LY2784544-treated NC cells and Girdin si RNA cells.5.In vivo proliferative activity of Girdin protein expression down-regulated tumor cells.BALB/c nude mice were inoculated with Lo Vo,NC and Girdin si RNA cells,and the proliferation ability of each cell was examined.The apoptosis of tumor cells was detected by TUNEL method.Western Blot assay was used to detect the expression of invasion related proteins in tumor tissues.Result:1.Western Blot assay showed high expression of Girdin protein in Lo Vo cells for subsequent studies.The sh RNA expression vectors Girdin-p GCH1/Neo and NC-p GCH1/Neo were successfully constructed and confirmed to be transfectedsuccessfully.The m RNA and protein contents of Girdin protein in Girdin si RNA cells were significantly decreased.2.The MTT assay results showed that the proliferation activity of Girdin si RNA cells was significantly lower than that of Lo Vo cells and NC cell.The Flow cytometry results showed that Girdin si RNA cells accumulated in G0/G1 phase,and the percentage of S phase and G2/M cells decreased.Compared with Lo Vo group,the apoptotic cells in Girdin si RNA group increased significantly,the apoptotic rate was23.68%,which was 1.87 times higher than that in Lo Vo group.The apoptosis-related proteins cleaved caspase-3,Bax and Bcl-2 in Lo Vo,NC and Girdin si RNA cells were detected by Western Blot.The results showed that the expression of cleaved caspase-3and Bax in Girdin si RNA group was significantly higher than that in Lo Vo cells(P <0.01),which was 2.18 and 2.45 times of Lo Vo cells,respectively;the expression of Bcl-2 in Girdin si RNA group was significantly decreased,only 63% of Lo Vo cells.It is indicated that the down-regulation of Girdin protein expression can arrest cell cycle arrest,decrease proliferation and proliferation activity,reverse tumor cell anti-apoptosis and reduce tumor survival rate.3.The results of cell scratch test showed that the migration distance of Girdin si RNA cells was significantly lower than that of Lo Vo cells(P<0.05),indicating that the migration ability was significantly decreased.The average number of invading cells in the Girdin si RNA group in the Transwell experiment was only 37.00,which was significantly lower than that in the Lo Vo group(85.40)and the NC group(84.40)(P < 0.001).The expression of MMP-2,MMP-9 and Vimentin in Girdin si RNA group was significantly lower than that in Lo Vo cells by Western Blot(P < 0.01),which were only 41%,41% and 51% of Lo Vo cells,respectively.These results indicate that down-regulation of Girdin protein expression can reduce the expression of migration-invasive-related proteins in tumor cells and effectively inhibit the migration and invasion of tumor cells.4.Western Blot detection of JAK/STAT signaling pathway protein showed that the relative expression levels of p-JAK and p-STAT3 in Girdin si RNA group were reduced by 37% and 31%,respectively,compared with Lo Vo and NC groups.The expression levels of IFN and interleukin-6 were reduced by 31% and 45%,respectively.After the JAK/STAT pathway was blocked by the JAK inhibitor LY2784544,the phosphorylation of JAK and STAT3 was decreased in NC cells,while there was no significant change in Girdin si RNA cells.There was no difference in p-JAK and p-STAT3 protein expression levels between NC cells treated with LY2784544 and untreated Girdin si RNA cells.The results of Transwell assay showed that the invasiveness of NC cells was significantly decreased after treatment with LY2784544,which was similar to that of Girdin si RNA group.However,the cell invasion ability of Girdin si RNA cells was not significantly changed after treatment with LY2784544.This indicates that down-regulation of Girdin protein expression can reduce the activation level of JAK/STAT signaling pathway,thereby inhibiting a series of cell activities such as proliferation and invasion mediated through this pathway.5.In vivo experiments showed that the tumor growth rate,mean tumor volume at each time point and mean tumor weight were significantly lower in the Girdin si RNA group than in the Lo Vo group.The results of TUNEL showed that the apoptotic cells in the tumor tissues of the Girdin si RNA group were significantly increased.Western Blot assay was used to detect the down-regulation of invasion related proteins in tumor tissues.It indicates that the down-regulation of Girdin protein expression can effectively inhibit the growth of tumor cells in the in vivo environment.Conclusion:The Lo Vo cell line with down-regulated Girdin protein expression was successfully constructed using RNAi technology.The results of cell proliferation,cell cycle,apoptosis and expression of apoptotic proteins indicated that Girdin protein is associated with tumor cell proliferation,and its down-regulation can significantly reduce cell proliferation activity and promote cell apoptosis.Cell scratch test,Transwell test and cell invasion-related protein detection indicated that Girdin protein is involved in the migration and invasion of tumor cells,and its down-regulation can effectively inhibit cell migration and invasion.Girdin protein regulates tumor cell proliferation and invasion mainly through JAK/STAT3 pathway.In vivo experiments showed that down-regulation of Girdin protein expression significantly inhibited the proliferation of tumor cells in vivo and induced apoptosis.In summary,Girdin protein plays an important role in the malignant activities of CRC tumor cells and can be used as a potential new therapeutic target in the clinical treatment of CRC.