| Objective:PD1/PD-L1 blockade therapy is a novel cancer treatment strategy.In recent years,immune checkpoint blockade(ICB)targeting PD-L1/PD-1 has achieved certain success in many malignant tumors.However,only a small proportion of advanced hepatocellular carcinoma(HCC)patients benefit from PD1/PD-L1 blockade therapy.The expression level of PD-L1 on HCC cells is a determining factor for the therapeutic effect of PD-L1/PD-1 blockade.Therefore,understanding the regulation mechanism of PD-L1 in HCC is of great significance for identifying new immune prognostic factors.NUAK1 is a serine/threonine kinase that is highly expressed in various malignant tumors such as HCC.NUAK1 can promote tumor cell proliferation,migration,invasion,and drug resistance.However,the role of NUAK1 in tumor immune escape is unknown.This project is important for understanding the function of NUAK1 in immune escape and improving the response rate of PD1/PD-L1 blockade therapy by interpreting the transcriptional mechanism of PD-L1 regulation in HCC.Methods:1.To explore whether NUAK1 mediated immune escape of hepatocellular carcinoma:The expression of NUAK1 and infiltration of CD8~+T cell in 20 clinical specimens of hepatocellular carcinoma were detected by immunohistochemical staining,and the correlation analysis was conducted.NUAK1overexpression and knockdown H22 cell lines were constructed to establish mouse transplant tumor model.The effects of NUAK1 on survival time and NUAK1 on growth of subcutaneous transplanted tumor were detected,and the expression of NUAK1 and infiltration of CD8~+T cell in tumor tissues were detected by immunohistochemical staining.Flow cytometry was used to detect the proportion of CD8~+T cell in the xenografted tumors.2.To explore the mechanism of NUAK1 mediating immune escape in hepatocellular carcinoma: transcriptome sequencing was used to search for differential genes after overexpression of NUAK1,and through enrichment of differential genes,genes with obvious differences in immune-related signaling pathways were identified.Immunohistochemical staining was used to detect the expression of NUAK1 and PD-L1 in clinical specimens of hepatocellular carcinoma,and the correlation was analyzed.A rat model of hepatocellular carcinoma was established,and the m RNA and protein expressions of NUAK1 and PD-L1were detected by Real-time PCR and Western Blot.The expressions of NUAK1 and PD-L1 in mouse transplant tumor model were detected by immunohistochemical staining.3.To explore whether NUAK1 mediates the transcription regulation of PD-L1 in hepatocellular carcinoma cells:NUAK1-overexpressed hepatocellular carcinoma cell lines(Huh-7,Hep G2,SNU-368,SNU-739)and NUAK1-knocked hepatocellular carcinoma cell lines(Huh-7,Hep G2)were constructed.The expressions of CD274 m RNA and PD-L1 protein were detected by Real-time PCR and Western Blot.4.To explore whether GSK3βmediated the transcriptional regulation of PD-L1 by NUAK1 in hepatocellular carcinoma:Western Blot assay was used to detect the phosphorylation of GSK3βat Ser9 in NUAK1-overexpressed hepatocellular carcinoma cell lines(Huh-7 and Hep G2).The expression of NUAK1and p-GSK3βin clinical specimens of hepatocellular carcinoma was detected by immunohistochemistry,and the correlation was analyzed.Western Blot assay was used to detect the expression of p-GSK3βin rat hepatocellular carcinoma model.The expressions of NUAK1 and p-GSK3βin the mouse transplant tumor model were detected by immunohistochemical staining.In addition,NUAK1-overexpressed hepatocellular carcinoma cell lines(Huh-7 and Hep G2)were cultured for 24 h with the GSK3βinhibitor AR-A014418(which inhibits GSK3βphosphorylation at Ser9).The Real-time PCR and Western Blot experiments testing CD274 m RNA and PD-L1 protein expression changes.5.To explore whether GSK3β-mediatedβ-catenin nuclear translocation is involved in NUAK1 transcriptional regulation of PD-L1 in hepatocellular carcinoma:Western Blot assay was used to detect the expression of totalβ-catenin protein in NUAK1-overexpressed hepatocellular carcinoma cell lines(Huh-7and Hep G2).The expression of NUAK1 andβ-catenin in clinical specimens of hepatocellular carcinoma was detected by immunohistochemical staining,and the correlation was analyzed.Western Blot assay was used to detect the expression ofβ-catenin in rat hepatocellular carcinoma model.The expressions of NUAK1 andβ-catenin in mouse transplant tumor model were detected by immunohistochemical staining.The expression ofβ-catenin in the nucleus was detected by Western Blot.Immunofluorescence assay was used to locateβ-catenin in cells.NUAK1-overexpressed hepatocellular carcinoma cell lines(Huh-7 and Hep G2)were cultured with GSK3βinhibitor AR-A014418 for 24 h,and the expression ofβ-catenin was detected by Western Blot assay.β-catenin gene knockout in NUAK1-overexpressed hepatocellular carcinoma cell lines(Huh-7 and Hep G2)and overexpressed in NUAK1-down-knocked hepatocellular carcinoma cell lines(Huh-7 and Hep G2),The changes of CD274 m RNA and PD-L1 protein expression were detected by Real-time PCR and Western Blot.Results:1.In clinical specimens of hepatocellular carcinoma,the proportion of NUAK1 and CD8~+T cell was negatively correlated;In mouse transplant tumor model,overexpression of NUAK1 significantly shortened the survival time of mice and promoted tumor growth,and the infiltration of CD8~+T cell in the xenografted tumors was significantly reduced,while knockdown of NUAK1 could prolong the survival time of mice and inhibit tumor growth,and the infiltration of CD8~+T cell in the xenografted tumors was significantly increased.These results suggest that NUAK1 mediates immune escape of hepatocellular carcinoma by inhibiting infiltration of CD8~+T cells.2.Transcriptome sequencing analysis showed that NUAK1 was involved in the regulation of PD1/PD-L1 signaling pathway and was related to adaptive immune response.Among the differential genes in the two pathways,CD274 m RNA expression level was the highest.In clinical specimens of hepatocellular carcinoma,NUAK1 was positively correlated with PD-L1 expression.The m RNA and protein expression levels of NUAK1 and PD-L1 were significantly increased in the liver of DEN group.In the mouse transplant tumor model,overexpression of NUAK1 significantly promoted the expression of PD-L1 in tumor tissue,while knockdown of NUAK1 inhibited the expression of PD-L1 in tumor tissue.These results suggest that NUAK1 mediates immune escape from hepatocellular carcinoma by inducing transcription expression of PD-L1.3.The expression levels of CD274 m RNA and PD-L1 protein were significantly increased in NUAK1-overexpressed tumor cell lines(Huh-7,Hep G2,SNU-368,SNU-739);After knockdown of NUAK1,the expression levels of CD274 m RNA and PD-L1 protein were significantly reduced.These results suggest that NUAK1 mediates the transcriptional regulation of PD-L1 in hepatocellular carcinoma cells.4.In NUAK1-overexpressed hepatocellular carcinoma cell lines(Huh-7 and Hep G2),the phosphorylation level of GSK3βat Ser9 was significantly increased,while knockdown of NUAK1 inhibited the phosphorylation level of GSK3βat Ser9.The expression level of NUAK1 was positively correlated with p-GSK3βin clinical specimens of hepatocellular carcinoma.The expression level of p-GSK3βin the liver of DEN group was significantly increased.NUAK1 overexpression significantly increased p-GSK3βexpression,while knockdown of NUAK1 significantly decreased p-GSK3βexpression.In addition,AR-A014418,a GSK3βinhibitor,could significantly inhibit NUAK1-induced up-regulation of CD274 m RNA and PD-L1protein expression.These results suggest that GSK3βmediates the transcriptional regulation of PD-L1 by NUAK1 in hepatocellular carcinoma cells.5.Totalβ-catenin protein was up-regulated in NUAK1-overexpressed hepatocellular carcinoma cell lines(Huh-7 and Hep G2);There was a positive correlation between the expression of NUAK1 andβ-catenin in clinical specimens of hepatocellular carcinoma.The expression level ofβ-catenin in the liver of DEN group was significantly increased.In the mouse transplant tumor model,NUAK1 overexpression significantly promoted the expression ofβ-catenin in tumor tissue,while knockdown of NUAK1 inhibited the expression ofβ-catenin in tumor tissue.NUAK1 overexpression significantly increased the expression ofβ-catenin in nucleus,while knockdown of NUAK1 significantly decreased the expression ofβ-catenin in nucleus.Immunofluorescence showed that NUAK1 overexpression promotedβ-catenin expression in the nucleus,while knockdown of NUAK1 inhibitedβ-catenin expression in the nucleus.In addition,AR-A014418,a GSK3βinhibitor,inhibited NUAK1-induced up-regulation ofβ-catenin.NUAK1-induced up-regulation of CD274 m RNA and PD-L1 protein was significantly inhibited after knockdown ofβ-catenin in NUAK1-overexpressed hepatocellular carcinoma cell lines(Huh-7 and Hep G2).However,ifβ-catenin is overexpressed in NUAK1-knockout hepatocellular carcinoma cell lines(Huh-7,Hep G2),β-catenin overexpression reverses NUAK1-knockdown induced CD274 m RNA and PD-L1 protein.These results suggest that NUAK1 may mediate transcription expression of PD-L1 in hepatocellular carcinoma via the GSK3β/β-catenin pathway.Conclusion:NUAK1 promoted the phosphorylation of GSK3βSer9 and inhibited its kinase activity,thereby reducing the GSK3β-mediated degradation ofβ-catenin,makingβ-catenin migrate to the nucleus to promote the transcription of PD-L1,and inhibiting the infiltration of CD8~+T cells to enable immune escape of hepatocellular carcinoma. |
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