The Mechanism Study Of Carboplatin Induced PD-L1 Expression Via PVR In Non-small Cell Lung Cancer Cells

Background and objectLung cancer is a disease with high morbidity and mortality in China,about 85%of which are non-small cell Lung cancer(NSCLC).Platinum-based combination chemotherapy is the most widely used chemotherapy regimen in clinical practice.However,long-term use of platinum-based drugs often leads to ototoxicity,nephrotoxicity,and drug resistance,which seriously affect the effect of treatment and the life quality of patients.Recent studies have shown that chemotherapy agent could inhibit tumor immune response by reshaping the composition of immune cells in the tumor microenvironment and regulating the expression of immune molecules such as PD-L1 in tumor cells,thereby leading to tumor immune escape.But whether platinum-based chemotherapy agent can induce tumor immunosuppression in lung cancer remains largely unknown,which is a clinically worthy issue.At present,there were few studies focus on platinum drugs on the expression of PD-L1 in NSCLC cells,and its intrinsic regulatory mechanism is still unclear.This study intends to observe the effect of carboplatin on the expression of PD-L1 in NSCLC cells,so as to provide new theory basis for the regulation of PD-L1 in tumor cells,propose new evidence for the participation of platinum drugs in tumor immune escape,and provide support for the combination of platinum chemotherapy and immunotherapy.MethodsHuman non-small cell lung cancer cell lines H292,A549 and H1299 were cultured in vitro.The expression of PD-L1 in those three cell lines were detected by real-time fluorescence quantitative PCR(RT-PCR),Western blotting,cytoimmunofluorescence,and flow cytometry,and the functional significance was detected by T cell proliferation test.The pathway protein-specific small-molecule inhibitors were used to verify the role of EGFR,PI3K/AKT and ERK signaling pathways in the regulation of PD-L1 by carboplatin in NSCLC cells,which was analyzed by RT-PCR and Western blotting.Subsequently,we used the whole transcriptome sequencing analysis and gene silencing to identify the key regulatory molecules'involved in the upregulation of PD-L1 induced by carboplatin in NSCLC cells.We further used Western blotting and RT-PCR to analyse EGFR,PI3K/AKT and ERK signaling pathways in the regulation of PVR induced by carboplatin in NSCLC cells.Results1.Carboplatin could significantly upregulate the expression of PD-L1 in NSCLC cells.CCK8 test showed that carboplatin had a dose-dependent cytotoxic effect on H292,A549 and H1299 cells.Different dose of carboplatin was respectively used to treat H292,A549 and H1299 cells for 48h prior to RT-PCR and Western blotting analyses.The RT-PCR results showed that mRNA levels of PD-L1 were increased in a dose-dependent manner,PD-L1 protein levels were also significantly upregulated at different dose of carboplatin administration.2.NSCLC cells could significantly inhibit the proliferation capacity of T cells after treatment with carboplatin.In detail,H292 cells were treated with 50?M carboplatin for 48h prior to co-cultured with T cells for 72h.And then we used flow cytometry to detect the proliferation capacity of T cells.The results showed that the proliferation ratio of T cells co-cultured with NSCLC cells in carboplatin treatment group was significantly lower than that in control group,indicating that the NSCLC cells treated by carboplatin could inhibit the proliferation capacity of T cells.3.EGFR?PI3K/AKT?ERK signaling pathways were involved in carboplatin induced PD-L1 in NSCLC cells.H292 cells were treated with 50?M carboplatin for for 0 h as negative control,and the experimental group for 10min,30min,60min,and 48h.The RT-PCR results showed that the phosphorylated and total protein levels of EGFR,PI3K and ERK were significantly increased in H292 cells.In addition,the phosphorylated protein of AKT were also significantly increased,suggesting that carboplatin could activate the signaling pathways of EGFR,PI3K/AKT and ERK in H292 cells.We used protein inhibitors to block these signaling pathways,Western blotting results showed that carboplatin induced PD-L1 was suppressed in H292cells.4.PVR mediated carboplatin to upregulate PD-L1 expression in NSCLC cells.The results of whole transcriptome sequencing and RT-PCR showed that PVR played a key role in upregulation of PD-L1 induced by carboplatin.Western blotting results showed that carboplatin could significantly increase the expression of PVR and PD-L1 simultaneously.Blocking the EGFR,PI3K/AKT,ERK pathway by inhibitors,we found that carboplatin dramatically promoted the expression of PVR and PD-L1 via activating the PI3K/AKT signaling pathway.The results of functional experiments showed that the treatment with siPVR in NSCLC cells significantly reverse the inhibitory effect of carboplatin induced NSCLC cells on T cell proliferation.ConclusionCarboplatin increased the expression of PD-L1 in NSCLC cells,thus inhibiting the proliferation capacity of T cells.Further more,we found that PVR was a key molecule in the upregulation of PD-L1 induced by carboplatin.In terms of mechanism,our results revealed that carboplatin upregulated PVR through PI3K/AKT signal axis to promote the expression of PD-L1.