The Effect Of Endocan Knockdown On The Migratory Ability Of Gastric Cancer Cells And Its Potential Mechanism

Objective To investigate the effect of endocan knockdown on the migration capacity of gastric cancer cells SGC7901 and its potential molecular mechanism.Methods The endocan interference sequence was designed,lentiviral vector was prepared,and then lentiviral packaging was carried out to infect human gastric cancer SGC7901 cell line,and a stable human gastric cancer SGC7901 cell line with endocan knockdown was constructed,which carried a fluorescent signal,and the cells were observed with fluorescence microscopy to determine the transfection efficiency,and the expression level of endocan protein and RT-q PCR were detected by Western blot experiment m RNA expression levels to determine their transfection results.The effect of low endocan expression on the migration ability of gastric cancer SGC7901 was detected by scratch experiment.We found that the expression level of endocan was negatively correlated with the expression level of TGF-α,and the addition of EGFR-specific inhibitor and TGF-α neutralizing antibody was added to detect the cell migration ability of each cell group again.Western blot detects the expression of migration-associated proteins and phosphorylation levels of MAPK signaling pathway-associated proteins.We also found that the expression level of endocan was negatively correlated with the expression level of Cav-1,and the expression of Cav-1was closely related to the level of cholesterol,LSS gene regulates cholesterol synthesis,we transfected sh RNA specific to LSS gene into endocan knocked gastric cancer cell SGC7901 to make LSS gene low expression,detect migration-related protein and expression and phosphorylation level of MAPK signaling pathway-related protein.Results The transfection efficiency was found to be good under a fluorescence inverted microscope,and the RT-q PCR results showed that the m RNA expression level of endocan was significantly reduced compared with the wild-type SGC7901 cell endocan in the group of SGC7901 cells.The Western blot results showed that the protein expression level of endocan in the group of SGC7901 cells knocked down by endocan was significantly reduced compared with that of wild-type SGC7901 cells,and the protein level of TGF-α was significantly increased.It was found that the expression levels of Claudin1 and E-cadherin proteins in each endocan knockdown cell group were significantly lower than those in each wild-type cell group.The levels of ERK phosphorylation and Src phosphorylation were higher than those of wild-type cell groups.The expression levels of Cav-1 and N-cadherin proteins in the endocan knockdown cell group with low LSS expression were lower than those in the endocan knockdown cell group,and the phosphorylation levels of Src and ERK were also relatively reduced.The results of scratch experiments showed that the migration ability of SGC7901 cells knocked down by endocan was enhanced compared with that of SGC7901 cells of each wild type,and the migration ability was weakened after the addition of EGFR-specific inhibitors.Conclusion Low expression of endocan enhances the migration ability of gastric cancer cell SGC7901.The potential mechanism is: endocan low expression upregulates TGF-α expression and then activates TGF-α/Src/ERK signaling pathway down-regulation E-cadherin and Claudin1 expression,and may also upregulate Cav-1expression,activate Cav-1/Src/ERK signaling pathway,upregulate N-cadherin expression,and ultimately enhance its migration ability.