Analysis Of EZH2 Regulating PD-L1 Expression Through TIMP2 In Non-small Cell Lung Cancer

Objective: To investigate the use of shRNA to reduce the levels of EZH2 and TIMP2 in human lung adenocarcinoma cells(A549 cells)and mouse lung adenocarcinoma cells(CCL cells)by cell transfection technology.The effect of the reduced levels of EZH2 and TIMP2 on the expression of PD-L1,thus providing a theoretical basis for immunotherapy and subsequent research.Methods:(1)The shRNA plasmids designed for EZH2 and TIMP2 genes were packaged and transfected into A549 cells and CCL cells through lipofectamine TM3000 liposomes to reduce the expression of the corresponding EZH2 and TIMP2 genes.The experiments were divided into single-gene experimental group(sh EZH2 group,sh TIMP2 group)and multi-genome experimental group(sh EZH2&TIMP2 group),as well as control group(primitive cells)and Nege group(empty RNA).(2)Puromycin was used to screen each group of cells,and the stably transfected A549 cells and CCL cells were screened and purified with the lowest concentration,and the transfection efficiency was verified by fluorescence microscope observation and PCR.(3)RT-PCR experimental analysis was performed to verify the expression changes of EZH2,TIMP2 and PD-L1 genes in each group of cells,and all data were statistically analyzed by the Graphpad Prism 9.0.Results:(1)The specific shRNA was designed to be introduced into A549 and CCL cell lines,and the PCR test was performed by observing with a fluorescence microscope and performing cell screening.The results indicated that the expression of EZH2 decreased in the cell group transfected with sh EZH2 RNA,the expression of TIMP2 in the cell group transfected with sh TIMP2 RNA decreased,and the corresponding genes in the cell group transfected with both RNAs decreased.(2)The detection results of RT-PCR in human lung adenocarcinoma cell lines and mouse lung adenocarcinoma cell lines indicated that reducing the expression of EZH2 with shRNA would increase the expression of TIMP2.Down-regulation of TIMP2 expression with shRNA increased EZH2 expression,as well as PD-L1 expression.(3)Through RT-PCR technology,we found that the expression of PD-L1 in cells decreased after the expression of EZH2 and TIMP2 were simultaneously reduced by shRNA.Inhibition of EZH2 expression using shRNA alone also reduced PD-L1;however,comparing the expression levels of PD-L1 in the two groups of cells,the former showed higher PD-L1 expression.Conclusions:(1)The expression of EZH2 and the expression level of TIMP2 in non-small cell lung cancer cells will interact with each other,indicating that there is a certain regulatory relationship between the two results.(2)The experimental results provide a theoretical basis for EZH2 to regulate PD-L1 expression through TIMP2,which will help to further explore the specific regulatory mechanism of intracellular PD-L1 expression.