Down-regulation Of RCC1 Sensitizes Immunotherapy By Upregulating PD-L1 Via P27^kip1/CDK4 Axis In Lung Adenocarcinoma

PART1 THE EXPRESSION OF RCC1 IN LUNG CANCER AND ITS EFFECT ON ITS PROGNOSIS AND FUNCTIONObjective: Lung cancer is still the cancer with the highest morbidity and mortality in China,and non-small cell lung cancer is the most common type of lung cancer.Regulator of chromosome condensation-1（RCC1）is a guanosine nucleotide exchange factor on the Ras-related nuclear protein（Ran）,which might participate in biological functions.In this part of the study,we mainly explore the expression level of RCC1 in lung cancer and its effect on prognosis,and elucidated the effect of RCC1 on the proliferation and apoptosis of lung adenocarcinoma cells in vitro.Methods: The mRNA expression data of various human cancers were downloaded and analyzed in TCGA database.The mRNA expression of RCC1 in Lung Adenocarcinoma（LUAD）and Lung squamous cell carcinoma（LUSC）was verified in Oncomine database,and the protein expression level of RCC1 in LUAD tissue was further verified in HPA database.The relationship between the expression level of RCC1 and the prognosis of patients with lung cancer was analyzed in Kaplan-Meier plotter,Prognoscan and TISIDB databases,respectively.We used siRNA to inhibit the expression of mRNA and protein of RCC1 in A549 and H1299 human lung adenocarcinoma cell lines.The proliferation ability and the apoptosis ratio of A549 and H1299 cells were detected by CCK-8 assay and flow cytometry,respectively.Results: The mRNA expression of RCC1 in lung adenocarcinoma and squamous cell carcinoma was higher than that in normal tissues in TCGA and Oncomine database,and the protein expression of RCC1 in lung adenocarcinoma was also higher than that in normal lung tissue.Survival analysis showed that the high expression of RCC1 was related to the poor prognosis of patients with lung cancer.Low expression of RCC1 might inhibit the proliferation of A549 and H1299 lung adenocarcinoma cells and increase the proportion of apoptosis of lung adenocarcinoma cells.Conclusion: Upregulation of RCC1 correlated with lung cancer,which might be related to the poor prognosis of patients with lung adenocarcinoma.RCC1 might promote the proliferation of lung adenocarcinoma cells and promote the occurrence and development of lung adenocarcinoma.PART2 THE EFFECT OF RCC1 ON THE EXPRESSION OF DOWNSTREAM MOLECULES p27^kip1/ CDK4/ PD-L1 IN LUNG ADENOCARCINOMA CELLSObjective: RCC1 could catalyze the transformation of RanGDP to RanGTP,so as to regulate the material exchange between nucleus and cytoplasm and affect the downstream molecules.In this part of the study,we explored the effects of RCC1 on the expression of downstream molecules p27^kip1/CDK4/PD-L1 and immune cell infiltration.Methods: We used siRNA to inhibit the expression of RCC1 in A549 and H1299 lung adenocarcinoma cell lines.The expression levels of mRNA and protein of p27^kip1/CDK4/PD-L1 were detected by q RT-PCR and WB.The relationship between the expression of RCC1 and immune cell infiltration was analyzed in TISIDB database.Results: By RCC1 knockdown,the mRNA and protein expression levels of p27^kip1 were increased in both lung adenocarcinoma cell lines,and the mRNA expression level of CDK4 was slightly increased to some extent in A549 cell line,but the protein expression level decreased to both lung adenocarcinoma cell lines.On the other hand,at the transcription level,the expression level of PD-L1 was increased only in H1299 cell line.At the translational level,low expression of RCC1 significantly increased level of PD-L1.In the TISIDB database,we found that the expression level of RCC1 was inversely proportional to the degree of immune cell infiltration.Conclusion: RCC1 might change the expression level of PD-L1 through p27^kip1/CDK4 axis at protein level.The high expression of RCC1 was related to the decrease of immune cell infiltration and the expression level of PD-L1.RCC1 might be used as a marker to predict the efficacy of immunotherapy.PART3 STUDY ON THE ANTI-TUMOR EFFECT OF LOW EXPRESSION OF RCC1 COMBINED WITH PD-L1 MONOCLONAL ANTIBODY IN MURINE CANCER MODELSObjective: The relationship of PD-L1 expression level and ICIs resistance has aroused our interest.Among the patients treated with Atezolizumab,the efficacy of PD-L1 positive patients is better than those with negative expression of PD-L1.In vitro,it was found that the low expression of RCC1 might increase the protein level of PD-L1.In this part of the study,we explored whether the combination of low expression of RCC1 and PD-L1 monoclonal antibody could enhance the anti-tumor effect.Methods: We constructed the lentiviral expression LV3-shRNA-RCC1 and transformed into lewis lung adenocarcinoma cell line.The tumor models were established in C57BL/6J mice.The tumor proliferation ability was assessed,and the protein level of p27^kip1,CDK4 and PD-L1 in the models were detected by immunohistochemistry.Results: In vivo experiments,the growth rate of tumor after RCC1 knockdown combined with PD-L1 monoclonal antibody was further inhibited than other groups.Moreover,we proved that the low expression of RCC1 might increase the expression of PD-L1 through the p27^kip1/CDK4 axis in vivo.Conclusion: Low expression of RCC1 combined with PD-L1 monoclonal antibody had better anti-tumor effect.It might enhance the efficacy of immune checkpoint inhibitor（ICIs）by regulating the abundance of PD-L1 through p27^kip1/CDK4 axis,which provides a new therapeutic direction for patients with negative PD-L1 expression or poor efficacy of PD-L1 monoclonal antibody.