The Role And Mechanism Of EZH2 In The Regulation Of PD-L1 Expression In Non-Small Cell Lung Cancer Cells

Background and objectivesLung cancer is the most commonly diagnosed cancer in global and accounts for the most cancer-related mortalities.Lung cancer can be simply divided into two categories: non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).NSCLC accounts for about 80% to 85% of the total.Although some big achievements have been made in the early diagnosis and treatment of NSCLC due to the continuous breakthroughs in surgery,radiotherapy and chemotherapy,targeted therapy and immunotherapy,the overall survival rate,especially the survival rate of patients with advanced NSCLC,is still facing great challenges.Therefore,it is still urgent to further elucidate the pathogenesis of lung cancer and explore new therapeutic targets.Studies have shown that immune escape is an important mechanism of tumorigenesis and development,which can be roughly divided into two aspects: the escape mechanism of tumor cells themselves and the escape mechanism of immune cells in tumor microenvironment.The activation of PD-1/PD-L1 signaling pathway is an important mechanism of tumor immune escape.At present,immune checkpoint therapy based on blocking PD-1/PD-L1 signal pathway has achieved certain curative effect in cancer treatment.However,a series of clinical trials have shown that the objective response rate of PD-1/PD-L1 inhibitor therapy is still low.Therefore,further exploring the regulation mechanism of PD-L1 expression in NSCLC cells can benefit of further inhibition of PD-1/PD-L1 signaling pathway and improve the immunotherapeutic effects.There are two main pathways in the regulation of PD-L1 expression: "endogenous" and "exogenous".Among them,the continuous up-regulation of PD-L1 expression caused by proto-oncogene mutation or deletion is the main mechanism of "endogenous" regulation.On the other hand,the specific microenvironment in which tumor cells are located is the "exogenous promotion" mechanism of high expression of PD-L1.For example,in hypoxia environment,the up-regulated hypoxia inducer-1 ?(HIF-1?)can promote the expression of PD-L1 by binding the hypoxia response element of PD-L1 promoter.However,the precise molecular mechanism of the increased expression of PD-L1 in NSCLC is not completely clear,so it is expected to inhibit the expression of PD-L1 from the "source" by further elucidating the molecular mechanism of the high level of PD-L1 basic expression in NSCLC cells.Zeste enhancer homologue 2(EZH2)is an important epigenetic regulatory molecule,which has histone methyltransferase activity and can catalyze lysine trimethylation at position 27 of histone H3,thus mediating gene silence.EZH2 is overexpressed in a variety of tumor tissues,and its inhibitor has achieved curative effect in some early clinical trials,indicating that EZH2 is a potential therapeutic target.At present,the mechanism of EZH2 involved in the occurrence and development of NSCLC is not completely clear.EZH2 has a wide range of downstream signaling pathways and can inhibit the production of Th1 chemokine CXCL9 and CXCL10,thus establishing immunosuppressive microenvironment in human ovarian cancer.Therefore,it is necessary to study whether EZH2 is involved in the immunosuppression of NSCLC,and whether this process is associated with the expression of PD-L1 in NSCLC cells.In summary,this study aims to analyze the expression of EZH2 in NSCLC and its correlation with PD-L1 and the poor prognosis of NSCLC patients by downloading the gene chip data of lung cancer in TCGA database and collecting tissue samples of NSCLC patients.By further inhibiting EZH2 in vivo and in vitro,we had investigated the effect of EZH2 on the expression of PD-L1 in NSCLC and its intrinsic molecular mechanism.Methods1.The expression of EZH2 in lung cancer and its correlation with PD-L1 and the poor prognosis of NSCLC patients.The gene chip data of lung cancer in TCGA database were downloaded online to analyze the expression of EZH2 in lung cancer and normal lung tissue.The tissue samples of NSCLC patients were collected and the expression of EZH2 in cancer tissues and normal tissues was detected by qPCR.The relationship between EZH2 and prognosis of patients with lung cancer was analyzed using ProgGene V 2 and Kaplan-Meier plotter.The correlation between EZH2 expression and PD-L1 was analyzed by combining the results of gene chip in GEO DataSets and qPCR detection in lung cancer tissues,so as to reveal the expression of EZH2 in NSCLC and its relationship with PD-L1 and poor prognosis of NSCLC patients.2.The effect of EZH2 on the expression of PD-L1 in lung cancer cells in Vitro.Sh-EZH2 lentivirus knockdown and EZH2 inhibitor GSK343 were used to inhibit the expression of EZH2 in mouse lung cancer cell line LLC.Flow cytometry and qPCR were used to detect the expression of PD-L1 in lung cancer cells.By these in vitro experiments,we intend to verify the effect of EZH2 on the expression of PD-L1 in lung cancer cells.3.In vivo experiments confirmed the effect of EZH2 inhibition on PD-L1 expression and tumor immune response in lung cancer cells.The tumor volume and weight of C57 mice were observed by subcutaneous injection of LLC with low EZH2.The expression of PD-L1 and the changes of tumor immune response in lung cancer cells were detected by flow cytometry et al.4.Determine the mechanism of EZH2 regulating PD-L1 expression in Lung Cancer cells.The regulation of EZH2 on non-small cell lung cancer HIF-1? expression was defined by qPCR and Flow cytometry Analysis after the knockdown of EZH2.Furthermore,the expression of HIF-1? in LCC was up-regulated by hypoxia stimulation to observe whether HIF-1 ? was the upstream regulator of PD-L1.Then,combined with hypoxia stimulation and EZH2 knockout,the expression of PD-L1 in LLC cells was detected to confirm whether the up-regulation of HIF-1? was the exact molecular mechanism of EZH2 regulating PD-L1 expression in NSCLC.Results1.The expression of EZH2 in NSCLC was positively correlated with the expression of PD-L1 and the poor prognosis of NSCLC patients.The analysis of gene chip data from TCGA database showed that EZH2 was overexpressed in lung cancer tissues.Similarly,in the 20 NSCLC tissue samples collected in our hospital,the mRNA level of EZH2 in lung cancer tissue was significantly higher than that in normal lung tissue,indicating that the expression of EZH2 in NSCLC was significantly increased,suggesting that EZH2 is a potential marker of NSCLC.The results of online database survival analysis of ProGgene V 2 and Kaplan-Meier Plotter showed that the overall survival time and non-recurrent expression period of patients with high expression of EZH2 were significantly lower than those with low expression of EZH2.It can be seen that the level of EZH2 is negatively correlated with survival prognosis.Further analysis of the results of lung cancer gene chip in GEO DataSets database showed that the expression of EZH2 in lung cancer tissue was positively correlated with PD-L1,which was consistent with the results of 20 NSCLC tissue samples.These results suggest that the abnormal overexpression of EZH2 in NSCLC is positively correlated with the expression of PD-L1 and the poor prognosis of patients with lung cancer.2.The inhibition of EZH2 could reduce the expression of PD-L1 in NSCLC in vitro.shEZH2 lentivirus interference experiment showed that the shEZH2-2 group was the most significant.The expression of PD-L1 in EZH2 was observed by qPCR,WB and flow cytometry.PD-L1 was significantly decreased,and the level of PD-L1 descent closely with the knockdown efficiency of EZH2.Similarly,after 24 hours of GSK343 intervention,the flow cytometry showed that the mean fluorescence intensity of PD-L1 in the GSK343 treatment group was significantly reduced compared to the control group and exhibited a good dose-dependent effect.The above in vitro experimental results show that EZH2 can regulate the expression of PD-L1 in LLC.3.The knockdown of EZH2 can inhibit the expression of PD-L1 in NSCLC cells,thus enhancing the anti-tumor immune response and delaying the progress of lung cancer in vivo.tumor bearing experiment showed that the growth rate,volume and weight of shEZH2 group were significantly lower than those of control group.Flow cytometry showed that the average fluorescence intensity of PD-L1 in shEZH2 knockout group was significantly lower than that in control group.After further isolation of tumor tissues,the proportion of infiltrating CD8 T cells in shEZH2 group was significantly higher than that in control group,and the proportion of functional IFN-? secreted by CD8 T cells was also significantly increased,which indicated that the expression of PD-L1 in NSCLC decreased and the anti-tumor immune response mediated by CD8 T cells increased after EZH2 knockdown,thus delaying the progress of lung cancer.4.EZH2 regulates the expression of PD-L1 by up-regulating the HIF-1? level in NSCLC.After transfecting with shEZH2,the expression level of HIF-1? in LLC was decreased significantly,indicating that EZH2 played an important role in the maintenance of HIF-1? expression.Furthermore,LLC was induced with hypoxia.Both qPCR and flow cytometry analysis suggested that HIF-1?,which was highly expressed in hypoxia,could up-regulate the expression of PD-L1 in LLC.In addition,the PD-L1 expression of LLC decreased with the intervention of HIF-1? inhibitor IOX2.As a result,by positive stimulation and negative intervention,it is proved that HIF-1? is an important maintenance factor for the high expression of PD-L1 in NSCLC.In order to further explore the correlation between EZH2,HIF-1? and PD-L1,shEZH2 stimulation was combined with hypoxia induction.And we found that the transcription level of PD-L1 in hypoxia EZH2 silent group was also significantly lower than that in hypoxia group,indicating that EZH2 is the upstream regulator of PD-L1 expression induced by HIF-1?.In conclusion,EZH2 can regulate the expression of PD-L1 in NSCLC cells through HIF-1?.Conclusion1.The expression of EZH2 in NSCLC was increased,which was positively correlated with the level of PD-L1 and the poor prognosis of patients with NSCLC.2.EZH2 knockdown can inhibit the expression of PD-L1 in NSCLC,thus delaying the progress of NSCLC by enhancing the anti-tumor immune response.3.EZH2 regulates the expression of PD-L1 by up-regulating the HIF-1? level of NSCLC cells.