A Mechanism Study Of Non-coding RNA Combined With Interferon Gamma In Regulating The Expression Of PD-L1 In Head And Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma(HNSCC)is the most common malignant tumor in the head and neck.The 5-year overall survival rate of patients is about 4050%.Although the level of clinical treatment(surgery,radiotherapy and chemotherapy)has been improved in recent years,the prognosis of advanced head and neck squamous cell carcinoma is still very poor.Especially in patients with recurrent or metastatic head and neck squamous cell carcinoma,the 5-year survival rate is even less than 5%.How to improve the survival rate of patients with recurrent or metastatic head and neck squamous cell carcinoma has become a hotspot and difficult issue.PD-1/PD-L1 blocking therapy is one of the most significant advances in tumor immunotherapy in recent years.By blocking the combination of PD-1 on T cells and PD-L1 on tumor cells,it reactivates the anti-tumor function of T cells and achieves the therapeutic effect.At present,the blocking therapy has been applied to the clinical treatment of melanoma,non-small cell lung cancer and other malignant tumors.In patients with recurrent and metastatic head and neck squamous cell carcinoma,pd-1/pd11 inhibitors combined with cisplatin have also initially shown encouraging effects,but the overall remission rate is unsatisfactory,only 13%-18%in the initial clinical trials.The reason is not clear.Therefore,to explore the role of PD-L1 in head and neck squamous cell carcinoma and study its regulatory mechanism is of great significance to improve the survival rate of patients with head and neck squamous cell carcinoma.In the tumor microenvironment(TME),tumor cells can express PD-L1,and its expression is negatively correlated with the clinical prognosis of patients with a variety of malignant tumors,but this conclusion remains controversial.Tumor infiltrating lymphocytes(TILs)are immune cells that exist in the tumor microenvironment,mainly T lymphocytes.They play an important role in antigen-specific host immune response in tumor tissues.Previous studies have confirmed the positive role of CD8+TILs in the tumor microenvironment in the immunotherapy of head and neck tumors,while there are relatively few research data on CD3+and CD4+TILs.Therefore,more studies are needed to clarify the expression of PD-L1 and TILs subsets(CD3+and CD4+)in head and neck squamous cell carcinoma and their role in disease prognosis.At present,the mechanism of PD-L1 expression in tumor cells is not clear.Studies have found that this may be related to the disordered expression of non coding RNA and the regulatory effect of interferon γ in the tumor microenvironment.Non-coding RNAs are the products of a group of genome transcription processes.Although they do not encode proteins,they can regulate the functions of related genes,thereby widely regulating the physiological or pathological processes of the body.The most studied non-coding RNAs include microRNAs(miRNA),lncRNAs and circular RNAs.MiRNA,as a non-coding RNA,can bind to the 3’UTR region of mRNA of its target genes,inhibit its translation by degrading mRNA,and regulate gene expression at the transcriptional level.CircRNA is a closed circular endogenous non-coding RNA whose 3’ and 5’ ends are covalently linked.Current studies have found that it can specifically bind to miRNA as a competitive endogenous RNA(ceRNA),inhibiting the function of miRNA,thereby indirectly regulating the expression of target genes.In recent years,with the deepening of research,it has been found that non-coding RNA plays an important and complex role in human cancer occurrence and tumor metastasis.miRNAs and circRNAs can be used as regulators of immune checkpoint molecules and are expected to become potential biomarkers and therapeutic targets.Interferon γ,as a key regulator of cellular immune response,it can not only promote immune regulation,but also have antiviral and anticancer activities.However,recent studies have found that IFN also plays a tumorigenic role in the process of tumor immune escape.Studies in melanoma and glioma have shown that IFN can up regulate the expression of PD-L1,thus promoting the immune resistance and immune escape of tumor cells.There are few studies on the relationship between interferon and PD-L1 in head and neck squamous cell carcinoma,so it still needs to be further clarified.In this study,using PD-L1 as the entry point,we first detected the expression of PD-L1 and TIL(CD3+,CD4+)in head and neck squamous cell carcinoma tissue samples,and analyzed their correlation with patient prognosis.Then,siRNA was used to transfect and detect the effect of silencing PD-L1 on the cell proliferation,migration and invasion ability,apoptosis and colony formation ability of head and neck squamous cell carcinoma cell lines in vitro.Interferon γ(IFN-γ)stimulation was used to clarify the regulation of PD-L1 expression through JAK/STAT signal transduction pathway mediated by interferon γ receptor(IFNGR).Finally,in the mechanism study,bioinformatics analysis was used to predict the miRNAs and circrnas targeting PD-L1.The direct targeting effect of miR-382-3p on PD-L1 and circ-0000052 on miR-382-3p were verified by a series of molecular biological methods,such as qRT-PCR,Western blot,especially the double luciferase reporter gene experiment.Focus on the regulatory relationship between the three.To provide targets and basis for PD1/PD-L1 immunotherapy.This study will be discussed and demonstrated from four chapters:Chapter Ⅰ Expression of PD-L1 and tumor infiltrating lymphocytes in head and neck squamous cell carcinoma and their correlation with disease prognosis Objective:To investigate the expression levels of PD-L1,CD3 and CD4 in head and neck squamous cell carcinoma and their correlation with prognosis.Methods:1.Collect laryngeal and hypopharyngeal cancer samples and detect the expression of PD-L1,CD3 and CD4 by immunohistochemical method.The RNA in the sample was extracted and the expression of PD-L1 was quantitatively detected by qRT-PCR.2.Statistically analyze the correlation between the expression levels of PD-L1,CD3 and CD4 and the prognosis of patients with head and neck squamous cell carcinoma.Result:1.Immunohistochemistry showed that the score of PD-L1 was≥1 in 86.0%of head and neck squamous cell carcinoma samples,and nearly half of the samples(45.2%)showed high expression(score>5).2.Immunohistochemistry showed that CD3 and CD4 were expressed in 100%of the samples,and CD3 was high expression in tumor infiltrating lymphocytes,while CD4 was relatively low.3.qRT-PCR showed that the expression of PD-L1 mRNA was detected in 81.7%of the samples and was positively correlated with the expression of PD-L1 protein.4.PD-L1 overexpression was associated with the deterioration of OS and DFS.Compared with patients with low infiltration,patients with high CD4+T lymphocyte infiltration had significantly improved OS and DFS.5.Univariate and multivariate analysis showed that age,tumor differentiation,PDL1 expression and CD4 expression significantly affected the overall survival and disease-free survival of patients.6.The expression level of PD-L1 was related to the patient’s age,but had no significant correlation with other clinicopathological features.Conclusion:1.PD-L1 is expressed in most head and neck squamous cell carcinoma tissues.High expression of PD-L1 is negatively correlated with the prolongation of OS and DFS.2.CD3+ and CD4+ T lymphocytes exist in the tumor microenvironment of head and neck squamous cell carcinoma,and the expression of CD3 is higher than that of CD4.3.The high expression of CD4 in tumor infiltrating lymphocytes was positively correlated with the prolongation of OS and DFS.Chapter Ⅱ Antitumor effect of silencing PD-L1 on head and neck tumor cells in vitroObjective:To investigate the effects of silencing PD-L1 on the proliferation,migration,invasion and apoptosis of head and neck squamous cell carcinoma cell line.Methods:1.Screening of head and neck squamous cell carcinoma cell lines with high expression of PD-L1 by qRT-PCR.2.Construction of silencing PD-L1 cell line by liposome transfection of siRNA.3.qRT-PCR and Western Blot were used to detect the mRNA and protein expression of silenced PD-L1 cell line.4.MTS method,Transwell chamber and Matrigel,Caspase 3 activity detection and other methods were used to detect the effect of silencing PD-L1 on the proliferation,migration,invasion and apoptosis of head and neck squamous cell carcinoma cells.Result:1.qRT-PCR showed that the expression levels of PD-L1 mRNA in FaDu and SCC-9 cells were significantly up-regulated compared with normal oral epithelial cell lines.2.After silencing PD-L1,qRT-PCR and Western Blot showed that the expression level of PD-L1 in FaDu cell line decreased significantly.3.After silencing PD-L1,MTS showed that the proliferation activity of FaDu and SCC-9 cells decreased,especially at 48 hours.4.After silencing PD-L1,Transwell migration and invasion experiment showed that the migration and invasion ability of FaDu and SCC-9 cells decreased significantly.5.After silencing PD-L1,caspase-3 enzyme activity in FaDu and SCC-9 cells increased,indicating that silencing PD-L1 can induce tumor cell apoptosis.Conclusion:1.PD-L1 is highly expressed in head and neck squamous cell carcinoma cell lines.2.Transfection of siRNA with liposome can effectively silence the expression of PD-L1 in tumor cells.3.Silencing PD-L1 can inhibit the proliferation of head and neck squamous cell carcinoma cell line,reduce the migration and invasion of tumor cells,and induce tumor cell apoptosis.Chapter Ⅲ Study on the regulatory mechanism of miRNA on PD-L1 and the transduction pathway of IFN-γ regulating PD-L1Objective:To explore the targeting relationship between miRNA-382-3p and PD-L1,and the regulation of IFNGR/JAK/STAT signal transduction pathway on PD-L1.Methods:1.Bioinformatic target gene prediction software was used to find potential miRNAs that target and regulate PD-L1,and their expression levels in head and neck squamous cell carcinoma cell lines were detected by qRT-PCR.2.FaDu and SCC-9 cells were transfected with pre miR-375-5p and pre miR-3823p,respectively,and miRNA was detected by qRT-PCR to verify the transfection efficiency.qRT-PCR and Western Blot were used to detect the effect of up-regulated miRNAs on the expression level of PD-L1.3.PD-L1 3’UTR wild-type and mutant-type recombinant plasmids were constructed,and the direct targeting effect of miR-375-5p and miR-382-3p on PD-L1 was verified by dual-luciferase gene reporter assay.4.The effect of transfection of PD-L1-siRNA,pre miR-382-3p and anti miR-3823p on the colony formation ability of FaDu cells was observed by clonogenic assays.5.After FaDu and SCC-9 cells were treated with IFN-γ,qRT-PCR was used to detect the expression of related factors in the JAK/STAT signaling pathway,and Western Blot was used to detect the expression level of PD-L1 protein.Result:1.Five potential tumor suppressor miRNAs that may target PD-L1 were found by bioinformatics target gene prediction software,namely:miR-34c-5p,miR-23a-3p,miR-197-5p,miR-375-5p,miR-382-3p.The expression of the above miRNAs was detected by qRT-PCR in FaDu and SCC-9 cells.The results showed that the expression levels of miR-375-5p and miR-382-3p in FaDu and SCC-9 cells were significantly lower than those in NOE cells.Finally,miR-375-5p and miR-382-3p were selected.Carry out the next experiment.2.The results of qRT-PCR showed that the expression of miR-375-5p and miR382-3p in FaDu cells was significantly increased after transfection of pre miRNAs,indicating that the transfection was successful.The expression of PD-L1 was downregulated after transfection,and the down-regulation of PD-L1 after transfection with miR-382-3p had significant differences.3.The results of the dual luciferase reporter assay of miR-375-5p and miR-382-3p with PD-L1 3’UTR showed that cells transfected with pre miR-382-3p could interact with wild-type PD-L1 3’UTR.The luciferase activity value decreased significantly.However,the luciferase activity of cells transfected with pre miR-375-5p and wild-type PD-L1 3’UTR recombinant plasmids was similar to that of control group,and there was no significant difference.It shows that miR-382-3p has a direct targeting relationship with PD-L1.4.Western Blot results showed that PD-L1 protein expression was significantly decreased after siRNA silencing of PD-L1 and transfection of pre miR-382-3p.In contrast,cells transfected with anti miR-382-3p showed no significant change in PDL1 expression.5.The results of clonogenic assays showed that the colony formation rate of cells in the siRNA silencing PD-L1 group and pre miR-382-3p group was significantly lower than that in the control group.6.The results of qRT-PCR showed that the expressions of JAK2,STAT1 and PDL1 were significantly up-regulated in FaDu cells after the treatment of IFN-γ.Western Blot results showed that the expression level of PD-L1 protein in FaDu and SCC-9 cells was significantly increased after IFN-γ treatment.After silencing STAT1,the expression of PD-L1 in the experimental group(IFN-γ+siRNA STAT1)was significantly lower than that of FaDu stimulated by IFN-γ alone.Conclusion:1.miR-382-3p is under-expressed in head and neck squamous cell carcinoma cell lines(FaDu,SCC-9)2.miR-382-3p has a direct targeted regulatory effect on PD-L1.The low expression of miR-382-3p in head and neck squamous cell carcinoma cells relieved the targeted inhibition of PD-L1,resulting in the overexpression of PD-L1.3.Silencing PD-L1 or up regulating miR-382-3p can inhibit the colony forming ability of head and neck squamous cell carcinoma cell lines.4.Interferon γ can induce head and neck squamous cell carcinoma cells to upregulate the expression of PD-L1 through the IFNGR/JAK/STAT signaling pathway.Chapter Ⅳ Study on the regulatory mechanism of circRNA/miRNA/PD-Ll axis in head and neck squamous cell carcinomaObjective:To study the regulatory mechanism of circRNA-0000052 and miRNA-382-3p on PD-L1.Methods:1.The circRNA database was used to search for potential circRNAs targeting miRNA-382-3p.2.The expression level of circ-0000052 in head and neck squamous cell carcinoma cell lines was detected by qRT-PCR and the stability of circ-0000052 was verified by RNase R and Actinomycin D treatment.3.FaDu cells were transfected with pre miR-382-3p and miR-NC,respectively,and the expression levels of circ-0000052,miR-382-3p and PD-L1 in FaDu cells were detected by qRT-PCR.4.The circ-0000052 3’UTR wild-type and mutant-type recombinant plasmids were constructed,and the direct targeting effect of miR-382-3p and circ-0000052 was verified by dual-luciferase gene reporter assay.5.FaDu cells were transfected with pre miR-382-3p,siRNA targeting circ0000052(si-circ-0000052),and co-transfected with si-circ-0000052 and anti miR-3823p.The expression of PD-L1 was detected by Western Blot and explore the regulation between molecular axes.Result:1.Through the functional analysis of the KEGG pathway in head and neck squamous cell carcinoma,circ-0000052 was selected as a candidate circRNA for functional study.2.qRT-PCR results showed that the expression of circ-0000052 in FaDu and SCC9 was significantly higher than that in NOE cell line.Among them,the FaDu cell line has the highest expression level.After treatment with RNase R,the PCR results showed that the expression of circular circ-0000052 had no significant change compared with the control group,indicating that its closed circular structure could resist the degradation of RNase R.After treatment with actinomycin D,the mRNA of circular circ-0000052 was more stable than that of its linear sequence AGO1.3.FaDu cells were transfected with pre miR-382-3p,and the results of qRT-PCR showed that miR-382-3p was significantly increased,and PD-L1 expression was decreased,while the expression of circ-0000052 had no significant change.4.The results of the dual-luciferase gene reporter assay showed that the luciferase activity of the experimental group transfected with pre miR-382-3p and circ-0000052 wild-type was significantly reduced,indicating that there is a target regulating effect between circ-0000052 and miR-382-3p.5.Western Blot results showed that transfection of pre miR-382-3p or si-circ0000052 alone could reduce the expression of PD-L1.Simultaneous transfection of sicirc-0000052 and anti miR-382-3p resulted in increased PD-L1 protein expression compared with transfection of si-circ alone.This indicated that the effect of si-circ0000052 could be reversed by anti miR-382-3p,which further confirmed that miR-3823p or circ-0000052 could directly or indirectly regulate the expression of PD-L1.Conclusion:1.circ-0000052 is highly expressed in head and neck squamous cell carcinoma cell lines(FaDu,SCC-9).2.circ-0000052 has a direct targeted down-regulation effect on mir-382-3p.3.The up-regulated expression of circ-0000052 in head and neck squamous cell carcinoma cells can reduce the expression level of miR-382 through the direct targeted sponge adsorption,thereby releasing the inhibition of PD-L1 expression,resulting in the up-regulation of PD-L1..