The Expression And Transformation Mechanism Of BRD4 And PD-L1 In Tongue Squamous Cell Carcinoma

Background and ObjectiveTongue squamous cell carcinoma(TSCC)is the most common malignant tumor of the oral and maxillofacial region,accounting for 43.4%of the incidence of oral squamous cell carcinoma(OSCC).It is aggressive and has a high lymph node metastasis rate.The current treatment option is mainly surgery-based comprehensive treatment,but the 5-year survival rate is still not satisfactory.How to improve the effect of adjuvant therapy after surgery,and to improve the survival rate have been urgently to be solved.In recent years,breakthroughs have been made in tumor immunotherapy.Among them,PD-L1 is one of the most widely used targets.In tumor cells,highly expressed PD-L1 can cause tumor antigens to break down T cells and allow tumors to escape immune surveillance.In OSCC,the increased expression of PD-L1 is closely related to the occurrence and development of OSCC,and related drugs have been used in clinical replacement,but the therapeutic effect remains to be improved.Bromine domain protein 4(BRD4)is a member of the bromine domain and the super-tip structure(BET)family.It contains two tandem bromine domains and a super-terminal domain.In the cell cycle,BRD4 recruits different transcription regulators and plays an important role in regulating DNA replication,cell cycle,gene transcription and other cellular activities.Recent studies have shown that BRD4 plays an important role in the occurrence and development of a variety of tumors,and the research on targeted drugs of BRD4 has achieved good results in the treatment of malignant tumors.Among them,JQ1 is widely used in BRD4 related research because of its high affinity and specificity for BRD4.In some other sites,BRD4 can achieve immune escape by increasing PD-L1.Therefore,we consider that BRD4 and PD-L1 are also elevated in the occurrence and development of TSCC,and there is a close relationship between the two.Based on the above ideas,we intend to analyze the association between BRD4 and PD-L1 in oral and maxillofacial squamous cell carcinoma by bioinformatics technology to clarify the feasibility of the study;In clinical specimens and TSCC cells,the regulatory relationship between them was further verified,and the regulatory mechanism between the two was explored.In summary,this study combines of clinical indicators and basic research,clarifies the interaction between BRD4 and PD-L1 in TSCC,and explores the regulatory mechanism of the two in TSCC cells,providing new insights into TSCC-related collaborative immunotherapy.And provide new ideas for the combined use of immunotherapy.Materials and Methods1.Bioinformatics analysis of BRD4 and PD-L1 expression in oral and maxillofacial squamous cell carcinoma.(1)The UALCAN and GEPIA databases were used to analyze the expression of BRD4 and PD-L1 in oral and maxillofacial squamous cell carcinoma,and the correlation between the two was analyzed using the GEPIA and HNCDB databases.(2)The relationship between the expression of BRD4 and PD-L1 and clinicopathological factors using the UALCAN database,and the prognosis of patients using the GEPIA and HNCDB databases.2.Expression of BRD4 and PD-L1 in TSCC tissue samples and analysis of clinicopathological factors.(1)The immunohistochemistry(IHC)method was used to detect PD-L1 expression in 46 TSCC tissues and 41 normal adjacent cancer tissue specimen sections;the real-time quantitative reverse transcription polymerase chain reaction(QRT-PCR)and Western blot experiments were used to detect the mRNA and protein expression of BRD4 and PD-L1 in 15 fresh TSCC tissues and normal tongue mucosa tissues.(2)Statistics the expression of BRD4 and PD-LI in TSCC specimens,calculate the statistical correlation between the two,and analyze the expression and clinical pathological factors.Study on the regulation of PD-L1 expression by BRD4 in TSCC cell line.(1)Transfect siRNA into Ca127 and Osc19 cells to silence the BRD4 gene,select the most significant group by using QRT-PCR and Western blot,and use the same method to measure the expression of PD-L1 in this group;use CCK8 to screen for JQ1 Optimal concentration,mRNA and protein expression of PD-L1 after 12,24,and 48 hours of action at this concentration.(2)The blank control virus was used to screen out the optimal MOI value for transfection in Ca127 and Oscl9 cells.Transfection of the virus under this condition increased the expression of BRD4.QRT-PCR and Western blot were used to detect the expression of PD-L1 mRNA and protein(3)Immunofluorescence method and Flow cytometry was used to observe the expression of PD-L1 on Cal27 and Osc19 cell membranes after BRD4 was increased or decreased.4.Study on the mechanism of BRD4 regulating PD-L1 in TSCC cells.(1)Use String database to analyze the correlation between BRD4,C-MYC,CDK9 and PD-L1.(2)After using the same method to reduce or increase BRD4,QRT-PCR and Western blot was used to detect the expression of C-MYC and CDK9.(3)C-MYC and CDK9 siRNAs were screened by the same method,and then transferred to Cal27 and Osc19 cells to detect PD-L1 mRNA and protein expression using QRT-PCR and Western blot.(4)C-MYC or CDK9 was increased by transfection.RT-PCR and Western blot were used to detect the mRNA and protein expression of PD-L1 in Cal27 and Osc19 cells.5.Effects of inhibiting BRD4 on the growth of TSCC in nude mice and detection of related genes.(1)After calcination of nude mice with Cal27 and Oscl9 cells,JQ1 was injected into the tail vein regularly to record the tumor-bearing volume and final weight and perform statistical analysis.(2)Immunohistochemical examination was performed on the isolated tumor-bearing tissue sections,and the expressions of mRNA and protein of BRD4,PD-L1,C-MYC and CDK9 in tumor-bearing tissues were detected by QRT-PCR and Western blot.Result1.Bioinformatics analysis of BRD4 and PD-L1 expression in oral and maxillofacial squamous cell carcinoma.(1)In the UALCAN database,BRD4 and PD-L1 were analyzed separately,and the results showed that both expressions were elevated in oral and maxillofacial squamous cell carcinoma,which was statistically significant.Subsequently,the correlation between the two in the TCGA database was analyzed through the correlation in the GEPIA database,and the results showed no statistical significance.However,in the HNCDB database,by integrating the database of the disease site in the oral cavity,the analysis showed that BRD4 was positively correlated with PD-L1 expression in OSCC.(2)In the UALCAN database,the expressions of BRD4 and PD-L1 and clinicopathological factors were analyzed,and the results showed that the expression of BRD4 was significantly increased in men,and the expression of patients with low differentiation was higher,Age,cancer stage,and lymph node metastasis were not related;PD-L1 expression was higher in stage Ⅰ patients than in stage Ⅳ,and higher in older patients(61-80 years)than in younger patients(21-40 years).There were statistically significant differences in lymphatic metastasis(N1 vs N2)and were associated with multiple lymphocyte infiltration,but were not statistically related to other factors.Analysis using GEPIA and HNCDB databases showed that PD-L1 expression is related to disease-free survival(DFS)of patients with oral and maxillofacial squamous cell carcinoma,and that BRD4 and PD-L1 can affect patients’progression-free survival(PFS).2.The expression of BRD4 and PD-L1 in TSCC tissue samples and the analysis of clinicopathological factors.(1)The IHC results showed that BRD4 was mainly expressed in the nucleus,and PD-L1 was mainly expressed on the cytoplasm and cell membrane surface,and the expression was significantly increased in TSCC tissue samples.With normal tissues as controls,the mRNA and protein levels of BRD4 and PD-L1 in TSCC tissue was elevated significantly.(2)Statistics on the expression of PD-L1 and BRD4 in TSCC tissues,and the use of chi-square test results showed that the expression of PD-L1 and BRD4 in TSCC was related;PD-L1 expression and lymph node metastasis in TSCC There is a statistical correlation with tumor TNM staging,and has nothing to do with the patient’s age,gender,smoking history,and tumor differentiation;BRD4 expression in TSCC is slightly different.In addition to being related to patient lymph node metastasis,it is also related to tumor differentiation.However,factors such as patient age,gender,smoking history,and tumor TNM stage were not related to BRD4 expression.3.Regulation of PD-L1 expression by BRD4 in TSCC cell lines.(1)First,siRNA was transfected into Cal27 cells and Osc19 cells to silence the BRD4 gene.By QRT-PCR and Western blot detection,the first group(siBRD4#1)had the most significant silencing effect.The same experiments showed that the expression levels of PD-L1 mRNA and protein in Cal27 and Osc 19 cells were significantly reduced under the action of siBRD4#1.CCK8 was used to detect the effect of BRD4’s targeted drug JQ1 concentration gradient on cell proliferation.A minimum dose of 5 pM 1 ml of inhibitory cells was selected to act on Cal27 and Osc19 cells.The results showed that with the extension of time,JQ1 inhibited BRD4 and the PD-L1 expression was significantly suppressed from the 24th hour.(2)After screening,the lowest doses with satisfactory transfection efficiency were:MOI=10,Polybrene=4 μg/ml in Cal27 cells;MOI=30,Polybrene=4 μg/ml in Osc19 cells.Transfection into Cal27 cells and Osc19 cells under the above conditions.The test results showed that at the same time that BRD4 was significantly increased,the expression of PD-L1 was also significantly increased.(3)It was found by IHC that when BRD4 was decreased or increased,the expression of PD-L1 also changed,which confirmed the regulation effect of BRD4 on PD-L1 in TSCC cells.At the same time,the expression of PD-L1 on the surface of cell membrane also changes with the change of PD-L1 expression.The same result was confirmed by detecting the fluorescence intensity of PD-L1 antibody on the cell membrane surface by flow cytometry.4.The results of research on the mechanism of BRD4 regulating PD-L1 in TSCC cells.(1)The analysis results of the String database database show that the four proteins,BRD4,C-MYC,CDK9 and PD-L1,are related in the human body.Among them,BRD4,C-MYC and CDK9 are closely related.Not only textual data supports the relationship between the three proteins,but also experimental results confirm.Moreover,the genes of BRD4 are closely related to the genes of C-MYC and CDK9 in the expression process.PD-L1 is only related to C-MYC in text data,and has no direct relationship with CDK9 and BRD4.(2)Inhibition of BRD4 expression using siRNA and targeted drug JQ1.The results showed that the mRNA and protein expression levels of C-MYC and CDK9 in Ca127 and Osc19 cells were significantly reduced;meanwhile,RT-PCR and Western blot were used to detect BRD4 lentivirus transfection.The expression of C-MYC and CDK9 in Cal27 and Oscl9 cells showed that the mR:NA and protein levels of both were significantly increased.(3)Screen the group that has the best effect of inhibiting C-MYC and CDK9 in siRNA,and suppress the expression of C-MYC and CDK9 by siRNA respectively.The results show that the mRNA and protein expression of PD-L1 after inhibiting C-MYC are both significantly decreased,but the expression of PD-L1 did not change after inhibition.Lentiviruses were transfected into Cal27 and Osc19 cells and stably expressed C-MYC or CDK9.The results showed that the expression of mRNA and protein of PD-L1 increased significantly after C-MYC increased,and the decrease of BRD4 with JQ1 could reverse PD-L1 expression increased;after CDK9 increased,PD-L1 expression did not significantly change in mRNA and protein levels.5.The effect of inhibiting BRD4 on the growth of TSCC and the detection results of related genes in nude mice.(1)Two weeks after the subcutaneous injection,a hard tumor-bearing nodule was formed subcutaneously in the injection site of the nude mouse,which was round or oval in shape and gradually increased.The measurement of tumor volume after injection of JQ1 showed that the tumor-bearing volume of nude mice in the JQ1 injection group increased slower than that of the control group.The final weights were Cal27 group:705.5±38.1 mg VS 545 ± 27.1 mg,P<0.05;:567.8±117.8 mg VS 342.2±30.2 mg,P<0.05,all were statistically significant.(2)Immunohistochemical examination of tumor tissues in nude mice showed that in TSCC tumor tissues,the expressions of BRD4,CDK9,C-MYC,and PD-L1 in the JQ1 injection group were lower than those in the control group.Subsequent results were obtained at the mRNA and protein levels.Conclusion1.Bioinformatics analysis showed that BRD4 and PD-L1 were up-regulated in oral and maxillofacial squamous cell carcinoma,and the expression of both was positively correlated in it.At the same time,they were related to various clinical pathologies or prognosis of oral and maxillofacial squamous cell carcinoma.2.In TSCC tissues,the expression of BRD4 and PD-L1 was significantly increased,and the expression of the two was statistically related.The expression of BRD4 is related to the degree of lymphatic metastasis and differentiation,and the expression of PD-L1 is related to the lymphatic metastasis and TNM stage.3.In TSCC cells,BRD4 can regulate the expression of PD-L1 at the mRNA and protein levels,and significantly affect the expression of PD-L1 in the cell membrane.4.In TSCC cells,BRD4 can regulate the expression of C-MYC and CDK9 at the mRNA and protein levels;in addition,BRD4 can regulate the expression of PD-L1 through C-MYC.5.In nude mice,inhibition of BRD4 can significantly slow the growth of TSCC tumors and inhibit the expression of PD-L1,C-MYC and CDK9.