Ezrin Promotes The Progression Of Non-small Cell Lung Cancer By Regulating The Expression Of PD-L1 In Human Non-small Cell Lung Cancer

Objective:To investigate the expression of Ezrin protein in non-small cell lung cancer(NSCLC).To investigate whether Ezrin protein regulates the mechanism of YAP and PD-L1 involved in infiltration and metastasis of non-small cell lung cancer.Methods:1.A total of 164 NSCLC patients that underwent radical surgical treatment in Shanxi Tumour Hospital were enrolled.And the expression of Ezrin,YAP and PD-L1 was detected by immunohistochemical staining.2.The Ezrin and PD-L1 protein expression was detected by western-blot in three lung cancer cell lines A549,H1299 and H520,and the cell line with high expression of Ezrin were selected for follow-up experiment.The experiment was repeated for 3 times.The Ezrin si RNA transfection model was established.Two Ezrin si RNA transfection groups with different sequences,negative control group,transfection reagent control group and blank control group.The 6-well culture plates were used for transfection.Firstly,si RNA stability and transfection efficiency were analyzed through pre-experiment to obtain the optimal transfection conditions: reagent dose and cell density,the experiment was repeated for 6 times;cells were collected within the appropriate time for further observation of transfection efficiency through western-blot and q RT-PCR,and sequences with high interference efficiency were screened.The experiment was repeated for 5 to 6 times.3.After transfection,the expression of Ezrin and PD-L1 were quantitatively and qualitatively detected by western-blot,q RT-PCR and immunofluorescence staining.The experiment was repeated for 5 times.4.Using CCK-8 method to detect the cell proliferative capacity.There are six groups in the experiment: Three different sequences of ezrin si RNA transfection group,one negative si RNA transfection group,one transfection reagent control group,one blank control group.The experiment was repeated for 5 times.5.In vitro cell migration and invasion assay.Using transwell assay to detect the migration and invasion ability of cells in each group.The experiment was repeated for 5times.6.Graphpadprism8.0 and SPSS26.0 software was used for statistical analysis,and independent sample t-test was used to test the differences between the groups.P<0.05 was defined as statistically significant.Results:1.The positive expression rates of the three kinds of proteins in NSCLC were detected by immunohistochemistry: Ezrin was 43.9%(72/164),YAP was 54.3%(89/164)and PD-L1 was 47.6%(78/164),which were significantly higher than those in normal lung tissue: Ezrin 18.8%(3/16)、YAP 6.3%(1/16)、PD-L1 12.5%(2/16),and the differences were statistically significant.Moreover,the expression of YAP and Ezrin was positively correlated with the expression of PD-L1.2.The cell lines with high Ezrin expression were screened out: Among the three cell lines,the expression of Ezrin and PD-L1 in H1299 cell line was the highest,and the expression of Ezrin in A549 and H520 cell lines was low,the difference was statistically significant.3.Ezrin knockout resulted in decreased expression of PD-L1 at both protein and RNA levels: Compared with the control group,the expression of Ezrin PD-L1 protein RNA in the Ezrin interference sequence transfected group was significantly decreased,P<0.05.Immunofluorescence showed that the number of fluorescence in gene knockout group was less than that in control group.4.The knockdown of Ezrin decreased the cell proliferative capacity of lung cancer cells: CCK-8 results showed that compared with control cells,The cell proliferative capacity of Ezrin knockdown group was reduced,P<0.05.5.The knockdown of Ezrin attenuated the migration and invasion ability of H1299cells: Compareing with the control group,the number of transmembrane cells decreased in the Ezrin knockdown group,P<0.05.Conclusion:Knockdown of Ezrin gene in non-small cell lung cancer can down-regulate the levels of YAP and PD-L1,and inhibit the invasion and metastasis of lung cancer cells.