UCHL1 Regulates The Mechanism Of PD-L1 Expression In Non-small Cell Lung Cancer Cells

BackgroundsLung cancer is the most common cause of cancer-related deaths worldwide,and non-small cell lung cancer(NSCLC)accounts for about 80-85%of the total number of lung cancers.Its high incidence rate,high metastasis rate and high recurrence rate seriously affect human health.Important advancements in the treatment of non-small cell lung cancer(NSCLC)have been achieved over the past two decades,increasing our understanding of the disease biology and mechanisms of tumour progression,and advancing early detection and multimodal care.However,the overall cure rate and survival rate of NSCLC are still very low,especially in advanced or metastatic disease.In recent years,the development of immune checkpoint inhibitors(ICIs)has brought hope to the treatment of advanced or metastatic non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).Clinical studies have shown that non-small cell lung cancer is in PD-1/PD-L1 has good results in the treatment of immune checkpoint inhibitors.Immune checkpoint blockade is an effective immunotherapy that has changed the treatment mode of many cancers.Immune checkpoint agents include antibodies against cytotoxic T lymphocyte-associated protein 4(CTLA-4)or programmed cell death 1(PD-1).Programmed cell death ligand 1(PD-L1)is an immune checkpoint protein expressed on tumor cells and tumor infiltrating immune cells.The binding of PD-L1 to PD-1 changes the activity of T cells,resulting in inhibition of its proliferation,survival,cytokine production and cytoxicity.PD-L1 is expressed on the surface of many immune cells,including macrophages,dendritic cells and endothelial cells.The expression of PD-L1 on the surface of cancer cells due to some oncogenic signals may promote tumor escape.Previous studies show that post-translational modification,such as ubiquitination,phosphorylation,glycosylation and palmitoylation,can regulate the expression and function of PD-L1.The strategy of targeting PD-L1 post-translational modification has made important progress in activating anti-tumor immunity,especially through ubiquitination modification.The ubiquitination modification of proteins is mainly carried out through three ubiquitin-related enzymes:E1 ubiquitin-activating enzyme,E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase(ubiquitin protein ligase).In particular,the E3 ubiquitin ligase plays an critical role in the modification process.Through E3 ubiquitin ligase,the ubiquitin molecule can be accurately coupled to the target protein molecule.The process of protein ubiquitination modification is reversible.The reverse process is called deubiquitination,which can maintain protein stability through deubiquitination enzymes(DUBs).Deubiquitinating enzyme can reverse the modification catalyzed by ubiquitin ligase,cleave the lysine residue on the target protein and the peptide bond on the C-terminus of the ubiquitin molecule.thereby blocking the function of E3 ubiquitin ligase,The connection between the substrate protein and the ubiquitin chain is blocked,and the role of deubiquitination is exerted,so it is considered to be an important regulator of various cellular processes.The ubiquitin carboxyl terminal hydrolase UCHL1 in the deubiquitinase family has the function of removing ubiquitin in ubiquitinated proteins and prevents ubiquitinated proteins from being targeted by proteasome degradation.It is unknown whether UCHL1 affect the expression of PD-L1 on NSCLC cell surface.It is not clear whether the regulation of UCHL1 on PD-L1 expression affect activity of the T cell cocultured with the cancer cells.Moreover,it is also unknown how UCHL1 regulates PD-L1 expression in NSCLC cells.Answering all the questions is the main purpose of our research.ObjectivesTo investigate whether and how the deubiquitinase UCHL1 regulates the expression of PD-L1 molecule on the surface of NSCLC cellsMethods and resultsMethods and results?:The deubiquitination enzyme UCHL1 can regulate the expression of PD-L1(?)UCHL1 promotes PD-L1 protein expression1.In NSCLC cells,silencing the expression of UCHL1 can inhibit the expression of PD-L1 proteinFirst,synthesize siRNA of UCHL1,and conduct gene silencing experiment in NCI-H460 cell line.The cells transferred into siNC or siUCHL1 were collected,and the changes in protein level of PD-L1 molecule were detected by flow cytometry and Western Blot.The experimental results show that silencing UCHL1 can reduce the expression of PD-L1 at the protein level.2.In NSCLC cells,overexpression of UCHL1 can promote the expression of PD-L1 proteinFirst,the pRK5-UCHL1-Flag plasmid was constructed on pRK5 empty and overexpressed in A549 cell line.The cells transferred into pRK5 or pRK5-UCHL1-Flag were collected,and the changes in protein level of PD-L1 molecule were detected by flow cytometry and Western Blot.The experimental results show that overexpression of UCHL1 can make PD-L1 molecules highly expressed at the protein level.3.In NSCLC cells,UCHL1 inhibitors can inhibit PD-L1 protein expression UCHL1 inhibitor LDN-57444 was used to inhibit the expression of UCHL1 in H460 cell line.The inhibitor of UCHL1 was added to NCI-H460,and the changes in protein level of PD-L1 molecule were detected by flow cytometry and Western Blot.The experimental results show that the protein level of PD-L1 molecule has a significant downward trend after the inhibitor inhibits the activity of UCHL1.(?)UCHL1 regulates PD-L1 expression at the gene level1.UCHL1 promotes PD-L1 mRNA expressionFor siRNA experiment in H460 cell line,first transfer siNC or siUCHL1 into the cells,and then collect RNA from siNC or siUCHL1 cells after 24h,and then perform reverse transcription to synthesize cDNA.The RT-PCR method was used to identify the effect of UCHL1 on PD-L1 at the mRNA level.The experimental results showed that the down-regulation of UCHL1 expression at the mRNA level can result in low expression of PD-L1 molecules.Perform protein overexpression UCHL1 experiment in A549 cell line,first transfect the overexpression plasmid,collect RNA of pRK5 or pRK5-UCHL1-Flag cells after 24h,and perform reverse transcription to synthesize cDNA,use RT-PCR method to identify the UCHL1 pair at the mRNA level The effect of PD-L1,the experimental results show that overexpression of UCHL1 at the mRNA level can make PD-L1 molecules highly expressed.Add inhibitor of UCHL1 to NCI-H460 cells,collect RNA after 24h,and perform reverse transcription to synthesize cDNA.Use RT-PCR to identify the effect of UCHL1 on PD-L1 at the mRNA level.The experimental results show that the inhibitor of UCHL1 can lower the expression of PD-L1 molecule at the mRNA level.2.UCHL1 promotes the transcription of PD-L1 geneThe laboratory constructed a plasmid pGL3-PD-L1 containing the promoter region of the PD-L1 gene.siUCHL1 was transfected in NCI-H460 cells,and plasmids pGL3-Basic+pRL-TK(20:1)and pGL3-PD-L1+pRL-TK(20:1)were transfected 24h later,and the supernatant was lysed after 24h The double luciferase reporter gene detected the activity of the PD-L1 gene promoter region,and the results showed that silencing UCHL1 expression led to a decrease in the transcriptional activity of PD-L1 gene.A549 cells were transfected with pRK5-UCHL1 plasmid,and plasmids pGL3-Basic+pRL-TK(20:1)and pGL3-PD-L1+pRL-TK(20:1)were transfected after 24 hours.Cells were collected after 24 hours of lysis In the supernatant,the activity of the PD-L1 promoter region was detected by the double luciferase reporter gene,and the results showed that overexpression of UCHL1 resulted in increased transcriptional activity of PD-L1 gene.3.Correlation analysis of expression of UCHL1 and PD-L1 in human non-small cell lung cancerWe purchased two serially sliced human non-small cell lung cancer tissue chips from Shanghai Super Biotech Co.,Ltd.One of them was stained with UCHL1 antibody and the other was stained with PD-L1 antibody.Mirror observation and taking pictures.The results showed that the local PD-L1 expression in cancer tissues with higher expression of UCHL1 was stronger,with a correlation trend.However,there is no obvious statistical significance through statistics,so we plan to expand the amount of specimens for further demonstration.?:Functional detection of UCHL1 regulating PD-L1(I):Stimulate Jurkat cells to express PD-1 moleculesWe used PHA to stimulate Jurkat cells to express PD-1 molecules.The concentration of PHA with the highest PD-1 expression by flow cytometry was 10 ug/ml.(?):NSCLC cells with high expression of UCHL1 can inhibit the activity of PD-1 expressing Jurkat cellsThe PHA-induced Jurkat cells and NSCLC cells were co-cultured in U-shaped 96-well plates,and CD3/CD28 antibody was added to activate Jurkat cells.After 48 hours,the level of IL-2 in the culture supernatant was detected by ELISA.First determine the optimal ratio of Jurkat cells and NSCLC cells co-cultured.The results show that when the ratio of the two is 1:1,NCI-H460 cells can inhibit the activity of Jurkat cells.Jurkat cells were co-cultured with NCI-H460(silent UCHL1),the results showed that NSCLC cells with low expression of UCHL1 would not reduce the activity of Jurkat cells.Jurkat cells were co-cultured with A549(over-expressing UCHL1)and A549(over-expressing UCHL1 while siPD-L1).The results show that NSCLC tumor cells with high expression of UCHL1 can inhibit the activity of Jurkat cells,and this inhibition depends on the tumor cells expressing PD-L1 molecules.?:Mechanism of UCHL1 regulating PD-L1 expression(I):Identify signal pathways that affect PD-L1 expression in NSCLC cellsThrough literature review,we found that the signal pathways that affect PD-L1 expression are:NF-?B,STAT1,STAT3,ERK,and PI3K/AKT pathway.First,we synthesized small interfering RNAs of P65,STAT1,STAT3,and AKT,and then transferred siP65,siSTAT1,siSTAT3,and siAKT into NCI-H460 cells,respectively.After 24h,the RNA was collected and reverse transcribed to synthesize cDNA.The expression of PD-L1 mRNA was detected by RT-PCR.At the same time,the expression of PD-L1 protein was detected by Western Blot after 48h.The experimental results show that after the transcription factors STAT1,STAT3 and ERK are silenced,the effects of mRNA and protein levels on the expression of PD-L1 molecules are not significant.However,the use of siP65 and siAKT to inhibit the related signaling pathways NF-?B and PI3K/AKT can significantly reduce the expression of PD-L1 at the mRNA and protein levels.(?):UCHL1 regulates the expression of PD-L1 in NSCLC cells through the NF-kB pathway1.UCHL1 promotes P65 signaling pathway activation in NSCLC cellssiUCHL1 was transferred into NCI-H460 cells,and the protein was received 48 hours later.Detect the phosphorylation level of Ser536 at the transcription factor P65 by Western Blot.The results showed that down-regulation of UCHL1 expression can lead to a decrease in P65 activation.The pRK5-UCHL1 plasmid was transfected in A549 cells,and the protein was received 48 hours later.Detect the phosphorylation level of Ser536 at the transcription factor P65 by Western Blot.The results showed that high expression of UCHL1 could increase the activation level of transcription factor P65.UCHL1 inhibitor LDN-57444(10uM)was added to the NCI-H460 cell culture system,which continued to inhibit protein collection after 48 hours.Detect the phosphorylation level of Ser536 at the transcription factor P65 by Western Blot.The results showed that inhibition of UCHL1 function can lead to decreased P65 activation.2.UCHL1 promotes PD-L1 gene expression through P65 signaling pathwayA549 cells were transfected with pRK5 or pRK5-UCHL1 plasmids,and plasmids pGL3-PD-L1+pRL-TK(20:1)and pGL3-PD-L1-MutP65+pRL-TK(20:1)were transfected 24h later,24h After lysing the cells,the supernatant was collected,and the dual luciferase reporter gene was used to detect the activity of the PD-L1 promoter region.The results showed that UCHL1 overexpression increased the activity of PD-L1 promoter region,but did not increase the activity of PD-L1 promoter region with P65 binding site mutation.In A549 cells,UCHL1 was overexpressed while P65 was silenced,and a rescue experiment was performed.After 48 hours,cells were collected and detected for changes in the protein level of PD-L1 by Western Blot.The results showed that the overexpression of UCHL1 promoted the expression of PD-L1,but at the same time silencing P65 attenuated the up-regulation of PD-L1.3.UCHL1 promotes PD-L1 expression through AKT-P65 signal axis in NSCLC cells.1)UCHL1 promotes AKT signaling pathway in NSCLC cellssiUCHL1 was transferred into NCI-H460 cells and the protein was collected after 48 hours.Detect the phosphorylation level of Ser473 at the transcription factor AKT by Western Blot.The results showed that down-regulation of UCHL1 expression can lead to a decrease in AKT activation.The pRK5-UCHL1 plasmid was transfected in A549 cells,and the protein was collected after 48 hours.Detect the phosphorylation level of Ser473 at the transcription factor AKT by Western Blot.The results showed that the high expression of UCHL1 can increase the activation of the transcription factor AKT.UCHL1 inhibitor LDN-57444(10uM)was added to the NCI-H460 cell culture system,which continued to inhibit protein collection after 48 hours.Detect the phosphorylation level of Ser473 at the transcription factor AKT by Western Blot.The results showed that inhibition of UCHL1 function can lead to decreased AKT activation.2)UCHL1 promotes PD-L1 gene expression through AKT signaling pathwayUCHL1 was overexpressed in A549 cells while AKT was silenced,and rescue experiments were performed.The RNA was collected after 24 hours,and cDNA was synthesized by reverse transcription.The effect of mRNA on PD-L1 at the mRNA level was identified using RT-PCR and collected after 48 hours.Cells were detected by PD-L1 at the protein level by Western Blot.The results showed that the overexpression of UCHL1 promoted the expression of PD-L1,but at the same time silenced AKT attenuated the up-regulation of PD-L1.3)UCHL1 promotes PD-L1 expression through the AKT-P65 signal axissiAKT was transfected into NCI-H460 cells,and the protein was collected 48 hours later.Detect the phosphorylation level of Ser536 at the transcription factor P65 by Western Blot.The results showed that down-regulation of AKT expression could lead to P65 activation.A549 cells were transfected with AKT plasmid at the same time and P65 was silenced.The RNA was collected 24 hours later and cDNA was synthesized by reverse transcription.The effect of mRNA level on PD-L1 was identified using RT-PCR.At the same time,the cells were collected after 48 hours Western Blot was used to detect changes in PD-L1 molecules at the protein level,and activation and phosphorylation of P65 and AKT.The results showed that after the plasmid AKT was transferred,the phosphorylation level of AKT increased and the expression of PD-L1 increased;while the plasmid AKT and siP65 were transferred simultaneously,the expression of PD-L1 was down-regulated,indicating that AKT promotes the expression of PD-L1 dependent on P654.UCHL1 regulates PD-L1 expression through Enhancers1)Selection and construction of EnhancersThrough bioinformatics analysis and literature reports,the location of the PD-L1 gene on the chromosome was determined(chr9:5,450,503-5,470,567).On the UCSC website,we have analyzed the following six regions of transcription of PD-L1 gene containing P65 regulation(Chr9:5,586,200-5,587,000),(Chr9:5,459,189-5,459818),(Chr9:5479406-5480560),(Chr9:5,493,000-5,494.000),(Chr9:5,478.000-5,479.000),(Chr9:5582885-5583869)),we named them A,B.C.D.E,F.We constructed them on PGL3-Promoter vectors and named them pGL3-Promoter-A,pGL3-Promoter-B,pGL3-Promoter-C,pGL3-Promoter-D,pGL3-Promoter-E,pGL3-Promoter-F.2)Detection of Enhancers regulation of PD-L1First,the activity of the enhancer was detected in 293T cells,and the recombinant plasmid was transferred to the dual luciferase reporter gene experiment.The results showed that enhancer C,enhancer E,and enhancer F were active and regulated by transcription factor P65;Then transfect siUCHL1 and siP65 in NCI-H460 cells,and simultaneously transfect plasmids pGL3-Promoter+pRL-TK(20:1)and pGL3-Promoter-C(F)+pRL-TK(20:1),after 24h The supernatant was collected from the lysed cells,and the dual luciferase reporter gene experiment was used to detect the enhancer activity.We found that UCHL1 in NSCLC cells can regulate the enhancer activity of Enhancer-C and Enhancer-F.PD-L1 expressionConclusions?:Deubiquitinase UCHL1 up-regulates the expression of PD-L1 in NSCLC cells.?:UCHL1 may contribute to immune evasion of NSCLC cell by promoting PD-L1 expression.?:UCHL1 promotes the expression of PD-L1 through the AKT-P65 signal pathway axis.?:UCHL1 can promote the expression of PD-L1 by affecting the activity of enhancersInnovation and significance?:This study demonstrates for the first time that UCHL1 regulates PD-L1 expression in non-small cell lung cancer.The results of this study show that UCHL1 mainly regulates PD-L1 expression through the AKT-P65 signaling pathway axis,and UCHL1 can also regulate PD-L1 genes through Enhancers.?:The results of this research reveal the regulatory mechanism of UCHL1 on PD-L1 molecules in non-small cell lung cancer,indicating that UCHL1 may become a therapeutic target for non-small cell lung cancer,and provide a new way to prevent tumor immune escape of non-small cell lung cancer.