The Effect Of HMGB1 On PD-L1 Induced By IFN-γ On Lung Cancer Cells

ObjectiveAccording to the 2018 Global Cancer Report issued by the International Agency for Research on Cancer(IARC)which under the World Health Organization(WHO),the incidence and mortality of cancer are still on the rise,and cancer will become the main cause of human death in the 21st century.The incidence and mortality of lung cancer are relatively high in both men and women,which has become an important risk factor threatening human health.The survival and prognosis of lung cancer patients still need to be improved.With the continuous development of medical science and technology and medical research field,there have been a variety of lung cancer treatment methods.In addition to traditional surgery,chemotherapy and radiotherapy,new cancer treatment methods such as molecular targeted drugs for mutant genes and cellular immunotherapy have emerged.The Nobel Prize in Physiology or Medicine in 2018 was awarded to James Allison of the United States and Tasuku Honjo of Japan in recognition of their"contribution to the discovery of negative immunomodulation therapy for cancer",and immunotherapy was promoted to the public.In cellular immunotherapy,immune checkpoint blockade(ICB)and genetically engineered T cells are gradually used in clinical trials,showing better therapeutic effect.The survival and prognosis of patients can be improved obviously,but due to the complexity of tumor microenvironment,the existence of immunosuppressive molecules or suppressive cells makes the therapeutic effect of immune therapy have obvious limitations.Current clinical experimental data show that only 20-30%of cancer patients can benefit significantly from immunosuppressive therapy.Therefore,exploring the key molecules and their regulatory mechanisms that affect the therapeutic effect of immunotherapy in tumors is the key to improve the anti-tumor effect of cellular immunotherapy,improve the prognosis of patients and prolong the survival time of patients.In the immune microenvironment of tumors,CD8~+T cells play an important role in anti-tumor effect.According to the infiltration of CD8~+T cells,tumors can be divided into"cold tumors"and"hot tumors".In"cold tumors",the key is to improve the infiltration of CD8~+T cells.In the"hot tumors"with sufficient effective CD8~+T cell infiltration,how to relieve the functional exhaustion of effective CD8~+T cells and improve their killing efficiency is the key problem to be solved urgently.This article mainly discussed that HMGB1 in lung cancer tissue combined with RAGE inhibited the phosphorylation of JAK/STAT3 pathway,thus inhibited the regulation of PD-L1 on the surface of lung cancer cells induced by IFN-γ,and further synergistically enhanced the ability of CD8~+T cells to secrete effector cytokines,enhanced their killing function,and provided a reference for immunotherapy and even combined therapy of lung cancer.MethodsThe correlation between HMGB1 and PD-L1 expression in lung cancer was analyzed by TCGA database;The correlation between HMGB1 and PD-L1 expression in paraffin sections of lung cancer tissue resected by thoracic surgery of the First Affiliated Hospital of Zhengzhou University from 2013 to 2015 was detected by immunohistochemical staining;Flow cytometry was used to detect the expression of PD-L1 in the existing lung cancer cell lines in our laboratory.A549 and NCI-H322 cell lines with relatively high expression of PD-L1 were screened for subsequent experimental study;In the process of culturing A549 and NCI-H322 cell lines,recombinant human HMGB1 protein was added to the cell line.Flow cytometry,real-time fluorescent quantitative PCR and Western-blot were used to detect the effect of HMGB1 on the expression of PD-L1;The A549 cell line overexpressing PD-L1 was constructed in vitro.The recombinant human HMGB1 protein was added into the cell line.The effect of HMGB1 on the expression of PD-L1 was detected by flow cytometry and real-time fluorescent quantitative PCR;After the increase of PD-L1 expression induced by human IFN-γand human recombinant HMGB1 protein were added,the expression of PD-L1 was detected by flow cytometry and real-time fluorescent quantitative PCR;The expression of HMGB1 in A549 was knocked down in vitro,and the expression of PD-L1 was detected by flow cytometry,real-time fluorescent quantitative PCR and immunofluorescence assay after adding exogenous IFN-γ.In the process of culturing A549 and NCI-H322 cell lines,the expression of PD-L1 was elevated by adding human IFN-γand then recombinant human HMGB1protein was added.The phosphorylation level of JAK/STAT3 pathway was detected by Western-blot assay;The expression of HMGB1 in A549 was knocked down in vitro,and the protein was extracted after adding human IFN-γ.The phosphorylation level of JAK/STAT3pathway was detected by Western-blot assay;After adding TLR4 or RAGE inhibitors,the expression of PD-L1 and the phosphorylation level of JAK/STAT3 pathway were detected by flow cytometry and Western-blot assay;The supernatant of A549 cells treated with HMGB1 was incubated with CD8~+T cells.The expression of Granzyme B and Perforin was detected by flow cytometry.Results1.The results of TCGA database analysis showed that HMGB1 expression was negatively correlated with PD-L1 expression in lung cancer tissues,and the same result was confirmed in clinical samples of lung cancer patients by immunohistochemical.2.HMGB1 did not affect the expression of PD-L1 in lung cancer cell lines.3.Cytological results showed that HMGB1 did not affect the expression of PD-L1 in the PD-L1 overexpressed A549 lung cancer cell line.4.HMGB1 can reduce the induction of IFN-γon PD-L1 on the surface of lung cancer cell lines.5.HMGB1 may decrease the phosphorylation of JAK/STAT3 pathway by binding with its classical receptor RAGE.6.The presence of HMGB1 can synergistically enhance the anti-tumor function of CD8~+T cells.ConclusionThe expression of HMGB1 in lung cancer cells can affect the phosphorylation of JAK/STAT3 pathway by binding with RAGE,and then affect the regulation of IFN-γon PD-L1.When more HMGB1 is released from lung cancer tissues,the up-regulation effect of PD-L1 by IFN-γcan be inhibited on the surface of lung cancer cells.Under this condition,the activity of CD8~+T cells infiltrated in tumors can be enhanced by secreting more effector cytokines,and the survival of patients can be improved.It provides a new reference for immunotherapy and even combination therapy of lung cancer.