Bioinformatics Analysis And Preliminary Verification Of TP53INP2 Regulation Of PD-L1 Expression In Laryngeal Carcinoma Through TICAM1

Background:The commonly used treatment methods for advanced(recurrent)laryngeal cancer include radiotherapy,chemotherapy,concurrent chemoradiotherapy,molecular targeted therapy,immunotherapy and combination therapy,but the effect is still poor and the cure rate is low.Since the mutual regulation of autophagy and tumor immune escape is one of the intrinsic factors in the progression of tumor malignancy.It is a worthy research direction to study the interaction between autophagy and immune checkpoint and improve the anti-tumor efficacy by regulating the autophagy and immune checkpoint pathways.Objective:The differential genes of autophagy pathway and immune checkpoint pathway in laryngeal cancer were screened and verified,and the mechanism of action between autophagy pathway and immune checkpoint pathway in laryngeal cancer was preliminarily discussed.Methods:(1)Differential gene screening:The original expression profile data of laryngeal cancer RNA-seq were downloaded from the TCGA database(https://tcga-data.nci.nih.gov/tcga/),and Spearman Correlation Analysis was conducted.After difference analysis of the screened genes with correlation >0.5,gene selection with P < 0.05,| log2(foldchange)| < 1 is the threshold value,namely DESeq2 method selected three groups of differential genes.(2)Plasmid preparation:The RNAi interference plasmid vector and the overexpression vector of the differential genes were constructed.The sequence of the plasmid was consistent with the preset sequence,and the sequencing result was a single peak,and the plasmid vector was successfully constructed.(3)Verification of differential genes:After the successful construction of the plasmid vector,the lentivirus was packaged to infect laryngeal cancer cell lines TU686 and AMC-HN-8 cells.Respectively by Western Blot,Real-time PCR detection of STAT1、TP53INP2 and EGF gene knock on low efficiency,CCK-8 test cell proliferation activity,streaming double standard detection,apoptosis,and will be after the data analysis of gray level,draw a bar chart,and finally target genes were screened.(4)To explore the role of TP53INP2-TICAM1-PDL1 signal axis in autophagy and PD-L1TP53INP2 knockdown plasmid and TICAM1 overexpression plasmid were constructed to infect AMC-HN-8 cells.The key proteins on the TP53INP2-TICAM1-PDL1 signal axis were detected by scratch assay,Transwell migration assay,autophagy activator rapamycin,and Western Blot.The interaction of four key proteins TP53INP2,LC3 AB,TICAM1 and PD-L1.Results:(1)TP53INP2-TICAM1 、 STAT1-CTSL 、 EGF-PRKAA2.Three pairs of differential genes in the autophagy pathway and PD-L1 pathway in laryngeal cancer are TP53INP2-TICAM1,STAT1-CTSL and EGF-PRKAA2,respectively.(2)After the knockdown of STAT1,the proliferation level of laryngeal cancer cells was significantly decreased.While the apoptosis level was significantly increased,LC3 and PD-L1 protein levels were significantly decreased.(3)After EGF knockdown,the proliferation level of laryngeal cancer cells was significantly decreased,while the apoptosis level was significantly increased.LC3 and PD-L1 protein levels in laryngeal cancer cells were significantly decreased.(4)After TP53INP2 knockdown,the protein expression levels of LC3-II/I and PD-L1 were significantly decreased,and the activity of cell proliferation was significantly increased.As for the apoptosis level,the apoptosis level of TU686 cells was significantly increased after TP53INP2 knockdown,while that of AMC-HN-8cells was significantly decreased.(5)Only TP53INP2 knockdown virus obtained satisfactory double targets in both cell lines with significant knockdown efficiency.TP53INP2-TICAM1 was finally selected as the pair of target genes for the study.(6)After TP53INP2 knockdown,the expression of TP53INP2,LC3 AB and TICAM1 was significantly down-regulated,and the cell proliferation and migration levels were also significantly decreased,but the cell proliferation and migration levels were significantly increased after the use of autophagy activator.After TICAM1 knockdown,the expressions of PD-L1 and LC3 AB proteins were down-regulated.However,when TICAM1 was overexpressed while TP53INP2 knockdown,the expression of LC3 AB protein was significantly up-regulated,and the expression of PD-L1 was up-regulated.The changes of TP53INP2 protein were different in each experiment.Conclusion:(1)After knocking down STAT1,the proliferation level of laryngeal cancer cells was significantly decreased,the apoptosis level was significantly increased,LC3 and PD-L1 protein levels were significantly decreased.After EGF knockdown,the proliferation level of laryngeal cancer cells was significantly decreased,the apoptosis level was significantly increased,and the expression levels of LC3 and PD-L1 were significantly decreased.(2)After TP53INP2 knockdown,the proliferative activity of laryngeal cancer cells was significantly increased,apoptosis levels were significantly decreased,LC3 and PD-L1 protein levels were significantly decreased,but further experiments were needed to confirm.(3)The TP53INP2--TICAM1--PD-L1 signaling axis may be one of the mechanisms between autophagy and PD-L1 networks in laryngeal cancer.TP53INP2 and TICAM1 both have positive regulatory effects on autophagy and PD-L1 pathways.TP53INP2 can further regulate autophagy and PD-L1 through positive regulation of TICAM1,while autophagy activator rapamycin can up-regulate autophagy by promoting the nucleation of TP53INP2.