The Effect Of Immunomodulatory Molecules PD-L1 And ADCY7 On The Prognosis Of Renal Clear Cell Carcinoma And The Regulation Mechanism Of PD-L1

Objective: Renal carcinoma is the second most common tumors in the urinary system and renal clear cell carcinoma accounts for 80-90% of all renal cancers.Due to its insidious onset,people can’t pay high attention to renal clear cell carcinoma.Most cases are discovered during physical examination,and about 30% of the patients are diagnosed to be advanced cancer,the prognosis outcome of which is often poor.The conventional treatment for early renal cancer is surgical operation.As it is not sensitive to radiotherapy and chemotherapy,how to improve the quality of life and prolong the survival time of patients with advanced renal cancer has become the mainstream of current research.With the development of molecular biology,a variety of targeted drugs against renal cancer have emerged,such as sutent Pfizerof anti-vascular endothelial growth factor receptor VEGFR,and ivermox,tersilomos of the MTOR receptor blocker targeting the MTOR pathway.They have achieved enormous curative effect.They have greatly improved the quality of life and survival time of patients with advanced renal cancer.With the development of tumor interdisciplinary in recent years,tumor immunology has become a hot research field,and brought a new Gospel for patients with renal carcinoma.The PD-L1 / PD-1 pathway has been discovered early,relatively mature and highly concerned immunotherapy target.Programmed cell death molecules-1 PD receptor 1(also called CD279)is a kind of immune inhibitory receptor,belonging to the members of the CD28 family,and it is a type of 55 KDa Ⅰtransmembrane proteinit consisting of chromosome 2 long arm 2 q27.3 gene coding.PD-1 is expressed on the surface of activated T cells,B cells,monocytes,dendritic cells and natural killer cells.PD-1 is combined with its main ligand PD-L1,which is mainly expressed on the surface of hematopoietic cells,tumor cells and so on.The combination of PD-1 and PD-L1 produces inhibitory signals which inhibits the activity of T cells,thus allows the tumor to escape the host’s immune surveillance causing disease progression.At present,the anti-PD-L1 / PD-1 treatment drug Nivolumab and so on have been approved for the treatment of a variety of clinical solid tumors,and derived good clinical feedback.However,whether the expression of PD-L1 on the surface of tumor cells can be used as a molecular marker for anti-PD-1 therapy and its relationship with survival and prognosis of tumor patients have been controversial.Since a tremendous of energy and angiogenesis is required for tumor growth,most solid tumors are exposed to deficient oxygen environment.Renal clear cell carcinoma tends to be large in size which has a natural hypoxic environment.Previous studies report that,PD-L1 in renal clear cell carcinoma is mainly regulated by HIF-2α(hiif-2 α)in a hypoxic environment,but other mechanisms that may up-regulate PD-L1 in a hypoxic environment has not been discussed.Previous studies report that hypoxia can acetylate histones,leading to the activation of various oncogenes.Is there a mechanism by which histone acetylation regulates the transcriptional expression of PD-L1 in renal clear cell carcinoma.We want to know the causes of histone acetylation levels to increase,the relationship between regulatory histone acetylation levels and prognosis of patients,whether there are any other genes that are abnormally expressed in renal clear cell carcinoma that are associated with immune regulation,and the relationship with the prognosis of patients.In this study,we aimed at exploring the expression and controversy of PD-L1 in renal clear cell carcinoma in histological level.We analyze the PD-L1 in hypoxia environment besides HIF-2 alpha regulatory mechanism,study relationship with the prognosis and the PD-L1 control in renal clear cell carcinoma,explore the expressed genes between renal clear cell carcinoma and normal kidney tissues differentially and analyze their relationship with the immune adjustment and prognostic value.Our study will provide new scientific evidence for the diagnosis and prognosis evaluation of renal clear cell carcinoma.Materials and methods: 1.Detect expression of PD-L1,HIF-2,Actin,acetyl-histone and other proteins by western blotting.2.Test the m RNA level of PD-L1 by q-rt-pcr.3.Detect expression of PD-L1,HIF-2 and Histone H3K9 ac in renal clear cell carcinoma tissue samples by immunohistochemistry.4.Use the geo database to download the renal clear cell carcinoma differential and express dataset.5.Apply Robust Rank Aggregation to obtain consistent and expressed genes from various geo data sets.6.Apply DAVID to conduct go term analysis of differentially expressed genes,and conclude a false discovery rate(FDR)<0.05 defined as significant difference.7.Construct the protein action network on the basis of string database,and analyze module analysis on cytoscape.The rules to false degree cutoff: 2,k-core: 2,Node Score cutoff: 0.2,haircut: true,Fluff: false,Max the Depth from seed: 100).8.From TCGA database(https://cancergenome.nih.gov/)download the renal clear cell carcinoma gene expression quantity(FPKM)files,analyze the relationship between genetic variations and the clinical information.Timer database was used to analyze the relationship between differentially expressed genes and immune infiltrating cells in tumor tissues.9 Statistical processing:apply chi-square test,Spearman rank correlation test and Fisher’s precise probability calculation by SPSS 22.0 statistical software,T test was used for group comparisons.Each experiment was repeated 3 times,and the data were expressed as mean standard deviation(s).P< 0.05 was statistically significant.Univariate and multivariate survival analyses were performed by Cox model.Results: 1.PD-L1 is an independent adverse factor for patients with renal clear cell carcinoma.(1)Through the bioinformatics website,we analyzed the prognosis of patients with PD-L1 and renal clear cell carcinoma,and the results showed that there were differences in the prognosis evaluation of PD-L1,and several databases draw opposite conclusions.(2)The positive expression rate of PD-L1PD-L1 in 102 patients with renal clear cell carcinoma was 56/102(54.9%).(3)The expression rate of PD-L1 in female patients was higher than that in male patients(P=0.005),and the furhman grade in patients with positive PD-L1 expression was higher than that in patients with negative PD-L1 expression(P<0.001).(4)Single-factor survival analysis results showed that the total OS of patients with positive PD-L1 was worse(HR=4.61,95%CI was 1.54-13.8,P value<0.05).In multivariate cox analysis,PD-L1 was the independent prognostic factor of the patients(HR=3.38,95%CI 1.14-13.09,p<0.05).In addition,T stage,fuhrman nuclear grading and age were independent prognostic factors of the patients in this group.2.Hypoxia can enhance the acetylation degree of histone H3K9 ac and up-regulate the expression of PD-L1 by inhibiting the activity of histone deacetylase.(1)In ACHN and 786-o cell lines of renal clear cell carcinoma cells treated with 2% hypoxia at 24,the expression levels of PD-L1 to HIF-2 in PD-L1 were detected by western blot,and the PD-L1 mrna level was detected improved by q-rt-pcr.(2)We knocked out HIF-2 in ACHN and 786-o cell lines,and found that there was no significant change in the expression of PD-L1 in the two cell lines under hypoxia.At the same time,PD-L1 transcription level improved.(3)Western blot was used to detect the changes of histone acetylation under hypoxia,and it was found that the acetylation levels of histone H3K9 and H3K27 under hypoxia were increased.Histone deacetylase activity kit was used to detect the overall activity of HDAC,and it was found that the overall activity of histone deacetylase was inhibited.(4)PD-L1 protein and transcription level increased after the addition of histone deacetylase inhibitors.H3K9 ac has the same activation site as hypoxia.(5)The relationship between H3K9 ac and PD-L1 gene CD274 in the kidney was detected by using DNA element encyclopedia ENCODE and UCSC genome browser,and H3K9 ac was found to be largely concentrated in the transcription initiation region of PD-L1,indicating that H3K9 ac was closely related to the transcription activation of PD-L1 in renal tissue cells.3.PD-L1 expression in renal carcinoma is positively correlated with HIF-2 and histone H3K9 ac,which Is an independent prognostic factor of renal clear cell carcinoma.(1)Using tissue samples from 102 patients with clear cell carcinoma of the kidney who had the same prognosis as PD-L1,HIF-2,a known regulatory factor,and H3K9 ac,a factor verified by our experience,were used to verify the tissue level.The positive rate of histone-H3K9 ac was 62/102(60.8%),and the positive rate of HIF-2 expression in 102 patients was 53/102(51.9%).(2)Histone H3K9 ac was associated with the fuhrman and grade of renal clear cell carcinoma,and the positive rate of histone H3K9 ac expression in higher fuhrman grade was higher(P<0.01).HIF-2 was expressed more positively at higher T stages in this patient population.(P = 0.02).In addition,HIF-2 positive rate was higher in pathological specimens with necrotic tissue in tumor center(P=0.03).(3)Spearman correlation analysis showed that the expression of H3K9 ac and HIF-2 in patients’ tissues was positively correlated with PD-L1,and the r values were 0.321 and 0.469,respectively,p<0.01.(4)Univariate analysis showed that patients with histone-H3K9ac(HR=7.34,95%CI 1.7-31.68,p=0.014)with high expression of histone-H3K9 ac OS had been worse,and HIF-2 had nothing to do with OS.COX multivariate analysis including PD-L1 and its related regulatory factors showed that age(p=0.029),PD-L1 expression(p=0.016),H3K9 ac expression(p=0.028),and furhman nuclear grading(p=0.013)were independent prognostic factors.In the lasso regression of dimension-reduction treatment,the results showed that age,PD-L1,histone-H3K9 ac,T stage and fuhrman nuclear grading were independent prognostic factors in our patients with renal clear cell carcinoma.4.Bioinformatics comprehensive analysis showed that there were 6 significant differentially expressed genes in renal clear cell carcinoma compared with adjacent tissues.ADCY7 and LPAR5 were up-regulated,while CXCL12,BDKRB2,CASR and KNG1 were down-regulated in renal clear cell carcinoma.(1)Gene expression datasets based on gene expression omnibus(GEO).(2)We used the RAA method to define a total of 980 significant DEG,including 429 up-regulated and 551 down-regulated genes.(3)These differential genes were significantly enriched in tumor-related biological functions and pathways through biological function analysis.(4)A STRING database was used to establish the PPI network,and Cytoscape’s MCODE plug-in was applied to conduct module analysis in the whole network.Six differentially expressed genes were identified in renal clear cell carcinoma.Among them,ADCY7 and LPAR5 are up-regulated genes in renal clear cell carcinoma,while CXCL12,BDKRB2,CASR and KNG1 are down-regulated genes.5.ADCY7 and LPAR5 are immune-related genes,which are associated with poor prognosis of patients.(1)Applying the TIMER of Harvard University(Tumor Immune Estimation Resource)database to detect the relationship between the differentially expressed genes and tumor infiltrating Immune cells.It is found that the up-regulated differentially expressed genes ADCY7 and LPAR5 are positively correlated with the number of Tumor infiltrating lymphocytes,which may be immunomodulator genes.(2)Analysis of the relationship between ADCY7,LPAR5 and immune infiltrating cells using the renal clear cell carcinoma dataset in the TCGA database suggests that high expression of macrophages suggests a good prognosis,while ADCY7 is a poor prognostic factor for renal clear cell carcinoma.Conclusion: 1.Hypoxic microenvironment can inhibit the overall activity of histone deacetylase,increase the acetylation level of histone H3K9,and promote the up-regulation of PD-L1 in renal clear cell carcinoma.Patients with high PD-L1 expression have worse prognosis than those with low expression,and together with their regulatory factor histone H3K9 ac.2.CXCL12,BDKRB2,CASR and KNG1 are independent adverse prognostic factors for patients with renal clear cell carcinoma.3.CXCL12,BDKRB2,CASR and KNG1 are significantly down-regulated genes in renal clear cell carcinoma,suggesting a good prognosis for patients with renal clear cell carcinoma.ADCY7 and LPAR5 are significantly up-regulated genes,which are associated with tumor infiltrating cells and can indicate poor prognosis.