| Objective:RNA editing is a research hotspot in the field of molecular biology in recent years.The most characteristic and ubiquitous type of RNA editing in higher eukaryotes is the conversion of adenosine(A)to inosine(I)in double-stranded RNA(dsRNA)at specific sites,catalyzed by Adenosine deaminases acting on RNA(ADARs)enzymes.A-to-I RNA editing is involved in a wide range of important biological processes,including mRNA translation,pre-mRNA splicing,microRNA(miRNA)silencing,and viral RNA mutation.ADAR1 is the most commonly expressed ADAR in mammals.ADAR1 has two subtypes:the interferon-inducible expression protein p150,and another is the constitutive expression protein p110.Several studies have shown that A-to-I RNA editing mediated by ADAR1 in humans most commonly target non-coding sequences of mRNA.Among them,more than 90%of RNA editing events occur in Alu repeats.Similar to the role of programmed cell death receptor-1(PD-1)as a checkpoint in immunity,the extensive A-to-I editing in dsRNA Alu repeats mediated by ADAR1 makes dsRNA recognized as "self" in vivo.This editing prevents dsRNA from binding to the dsRNA sensors and inhibits the activation of related pathways.Therefore,ADAR1 plays a vital role in regulating innate immunity and maintaining physiological homeostasis in the body.The abnormality of ADAR1 can lead to the occurrence of autoimmune diseases and disorders in immune cell development.In recent years,more and more attentions have been paid to the role of ADAR1 in tumors since many A-to-I RNA editing events have been found in tumor tissues.At present,the research on ADAR1 in tumors can be roughly divided into two directions:one focuses on the specific editing substrates of ADAR1 in tumors which act as a tumor suppressor or promoter;this has been reported in a large amount of literature.The other focuses on the immunosuppressive effect of ADAR1 in tumors,which has begun to be paid attention to in the past two years;this effect of ADAR1 may be related to the interferon-induced antiviral innate immune response pathways in the cell.Type Ⅰinterferon activates the intracellular Janus kinase(JAK)-signal transducer and activator of transcription(STAT)pathway by binding to the interferon receptor on the cell membrane.Then STAT1,which forms a dimer,translocates to the nucleus to assist in the transcription of some downstream effector genes,that is,interferon-stimulated genes(ISGs).Among ISGs,the most closely related to ADAR1 are dsRNA sensors,including retinoic acid-inducible gene Ⅰ(RIG-1),melanoma differentiation-associated gene 5(MDA5),dsRNA-dependent protein kinase(PKR),etc.These proteins have been previously reported to be involved in the ADAR1-related antiviral/pro-viral effects and the occurrence of autoimmune diseases caused by abnormal ADAR1.However,they are rarely reported to be involved in the role of ADAR1 in tumors,thus,the specific role of the interferon-related signaling pathways in ADAR1 and tumors and whether this effect is universal remain to be further elucidated.In addition,whether the immune environment plays a dominant role in the effects of ADAR1 on tumors has not yet been reported.From the perspective of ADAR1 immunosuppressive role,this study aims to explore whether knockout of Adarl performed by the CRISPR-Cas9 system has an inhibitory role on the growth of different mouse tumor cells in vivo and in vitro.After discovering that different tumor cells have different sensitivity to Adar1 deletion,while analyzing the regulation mediated by ADAR1 deficiency in tumors,we also horizontally compared the differences in the mechanism behind Adar1 knockout(KO)-sensitive and KO-insensitive cells to clarify whether the interferon-related signaling pathways are involved in mediating these effects.Finally,we discussed whether the immune environment in mice is involved in the tumor-suppressing effects mediated by ADAR1 deficiency,which shows whether ADAR1 mediates the immune tolerance of tumors in the body.Altogether these results provide a new basis and ideas for therapeutic interventions target ADAR1 in clinical.Materials and Methods:Stable transfected KO CT26,ID8,and B16 cell lines were constructed by the CRISPR-Cas9 system;In vivo,subcutaneous mouse models of Adar1 KO tumors and control tumors were constructed to compare the differences in tumor growth rates;In vitro,the differences in cell morphology between Adar1 KO tumor cells and control tumor cells were compared using an inverted microscope;In the phenotypic studies,Cell Counting Kit-8(CCK8)test and colony formation assay were used to detect the effects of Adar1 deletion on the proliferation and the colony-forming capacity of tumor cells,respectively.Flow cytometry was used to verify the effects of Adar1 knockout on tumor cell apoptosis,and enzyme-linked immunosorbent assay(ELISA)was used to detect the effects of Adar1 knockout on interferon-beta(IFNβ)and interleukin-6(IL-6)secretion from tumor cells;In the mechanism study,the effects of ADAR1 deficiency on the expression levels of proteins involved in apoptosis pathways and interferon-related signaling pathways were detected by Western blot;Stable transfected Adar1/Stat1 double knockout(DKO)CT26 and B16 cell lines.and Adar1/Eif2ak2 DKO CT26 and B16 cell lines were constructed using the CRISPR-Cas9 system;In vitro,comparisons of the cell proliferation in Adar1/Stat1 DKO tumor cells and control tumor cells,and in Adar1/Eif2ak2 DKO and control tumor cells,were performed by CCK8 tests.Comparisons of the apoptosis in Adar1/Stat1 DKO and control tumor cells,and in Adar1/Eif2ak2 DKO and control tumor cells,were performed through flow cytometry;In vivo,subcutaneous mouse models of Adar1/Stat1 DKO tumors,Adar1/Eif2ak2 DKO tumors,and control tumors were constructed,and the difference in tumor growth rates between them was compared;To further evaluate and compare the differences in the mechanism between Adar1 KO-sensitive and KO-insensitive cells,reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and Western blot were used to detect the differences in ISGs mRNA and protein expression levels between CT26,ID8,and B16 cells.RT-qPCR was also performed to detect the changes in ISGs mRNA expression levels in CT26 cells before and after Stat1 knockout,and in B16 cells before and after interferon stimulation and after Stat1 knockout.We downloaded the data,including ISGs signature,ADAR1 expression,and survival of patients with colon adenocarcinoma(COAD),skin cutaneous melanoma(SKCM),and head and neck squamous cell carcinoma(HNSCC),from FirebrowseR The Cancer Genome Atlas(TCGA)database and Gene Set Enrichment Analysis(GSEA)/MSigDB database to perform a prognostic analysis.RT-qPCR and Western blot were used to detect the changes in ISGs mRNA and protein expression levels in CT26 and ID8 cells before and after being treated with stimulator of interferon genes(STING)inhibitor C-176;Finally,a subcutaneous injection was performed to construct Adar1 knockout tumor and control tumor models in NCG mice to explore whether the immune environment is involved in mediating tumor growth inhibition by ADAR1 deficiency.The proprotions of activated spleen T cells and memory T cells in Adar1 KO tumor-bearing mice were detected by flow cytometry,the expression level of IFNy in peripheral blood of Adar1 KO tumor-bearing mice was measured by ELISA,and a rechallenge model was constructed in immunized normal mice to confirm whether T cell is involved in mediating antitumor effect and the specific immune memory is enhanced.Results:Using the CRISPR-Cas9 system,we constructed and isolated multiple Adar1 KO clonal stable transfected CT26,ID8,and B16 cell lines.Adar1 KO clonal homozygotes and heterozygotes were successfully isolated in CT26 and B16 cells,and Adar1 KO clonal heterozygote was successfully isolated in ID8 cells.Using these Adar1 KO clonal cells to construct subcutaneous tumor models,we found that both in CT26 and B16 tumor models,Adar1 KO clonal homozygotes lost tumorigenicity in vivo;those mice were tumor-free during the entire observation period.However,Adar1 KO clonal heterozygotes still had growth capacities in vivo but their growth rates were significantly lower than that of control tumors.In addtion,Adar1 KO clonal ID8 heterozygote also had a growth capacity in vivo.However,the tumors gradually became smaller and the mice also reached a tumor-free state.The observation of cell morphology showed that ADAR1 deficiency had no effects on the morphology of B16 cells but had a significant effect on the morphology of CT26 cells.The main changes of CT26 morphology can be concluded as follows:primary fibroblast-like spindle cell shape disappeared,the cell became round,shrunken,and transparent,and the intracellular particles increased.ID8 cells were more likely to grow in colony formation after Adar1 knockout,and the intracellular particles also increased.In the phenotypic studies,we set up three condition groups,including none stimulation,IFNβ stimulation,and interferon-gamma(IFNy)stimulation,to detect the effects of ADAR1 deficiency on tumor cells.CCK8 test and colony formation assay results showed that knocking out Adar1 inhibited the proliferation and the colony formation of CT26 and ID8 cells but had no effects on B16 cells.After further stimulation with IFNβ and IFNy,the proliferation rate and the colony formation capacity of Adar1 KO B16 cells were significantly reduced compared with control cells.Stimulation with IFNβ and IFNy also significantly expanded these phenotypes’ differences between Adar1 KO CT26 and control cells and between Adar1 KO ID8 cells and control cells.Flow cytometry with PI-Annexin V staining showed that deletion of Adar1 could induce the apoptosis of CT26 and ID8 cells,and this apoptosis-promoting effect is more significant under the stimulation of IFNβ and IFNy.However,loss of ADAR1 did not significantly promote the apoptosis of B16 cells,but Adar1 KO B16 cells could undergo significant apoptosis compared to control cells under the stimulation of interferons.Through detecting the expression levels of IFNβ and IL-6 in the cell culture supernatant by ELISA,we found that deletion of Adar1 promoted the secretion of IFNβ and IL-6 by CT26 cells,and this secretion-promoting effect is more significant under the stimulation of IFNβ and IFNy.Loss of ADAR1 did not significantly increase the expression levels of IFNβ and IL-6 in ID8 and B16 cell culture supernatant,but after further stimulation with interferons,the secretion of IFNβ and IL-6 was significantly higher in Adar1 KO ID8 and B16 cells than that of control cells.According to whether tumor cells need to be stimulated by interferons to change these phenotypes after Adar1 deletion,we classify CT26 and ID8 cells as Adar1 KO-sensitive cells and B16 as Adar1 KO-insensitive cells.Next,we used Western blot to detect the expression levels of proteins involved in the apoptosis pathways and the interferon-related signaling pathways,including the JAK-STAT pathway and ISGs,in Adar1 KO-sensitive and KO-insensitive tumor cells under different conditions(none stimulation,IFNβ stimulation).We found that Adar1 deletion slightly up-regulated the expression levels of Cleaved caspase-3 and Cleaved caspase-8 in CT26 cells,and this up-regulation was more pronounced under the stimulation of IFNβ.On the contrary,Adar1 deletion did not change the expression levels of Cleaved caspase-3 and Cleaved caspase-8 in B16 cells but increased the expression of these proteins under the combination of IFNβ stimulation.Western blot results of the JAK-STAT signaling pathway showed that loss of ADAR1 decreased the expression of total JAK1 and increased the expression of phospho-JAK1,phospho-STAT1,and total STAT1 in CT26 cells.While in B16 cells,knockout of Adar1 did not significantly change the expression levels of these proteins.Western blot results of ISG proteins showed that CT26 and ID8 cells significantly up-regulated the expression levels of RIG-I,MDA5,and PKR after Adar1 deletion,while in B16 cells,Adar1 deletion did not increase the expression of these proteins.However,B16 cells can activate the JAK-STAT pathway and up-regulate the expression levels of ISG proteins under IFNβ stimulation.To verify whether the activation of the JAK-STAT signaling pathway and downstream PKR(Eif2ak2)sensing pathway is involved in mediating the effects of ADAR1 deficiency on the tumor growth in vivo and in vitro,we used the CRISPR-Cas9 system to construct Adar1/Stat1 DKO tumor cells and Adar1/Eif2ak2 DKO tumor cells in Adar1 KO-sensitive cells(CT26)and Adar1 KO-insensitive cells(B16).CCK8 test and flow cytometry in vitro showed that knocking out stat1 completely rescued the growth arrest and apoptosis-inducing phenotypes of CT26 cells mediated by Adar1 deletion in the absence of stimulation and of B16 cells mediated by Adar1 deletion under IFNβ stimulation.Knockout of Eif2ak2 partially rescued the growth arrest phenotype of CT26 cells mediated by Adar1 deletion in the absence of stimulation and of B16 cells mediated by Adar1 deletion under IFNβstimulation.However,it completely rescued the apoptosis-inducing phenotype of CT26 and B16 cells mediated by Adar1 deletion under different conditions(none stimulation,IFNβ stimulation).In vivo,knocking out stat1 completely rescued the tumor growth inhibition mediated by Adar1 deletion in B16 cells,while knockout of Eif2ak2 partially rescued this phenotype in B16 cells.Next,we further explored the differences in the mechanism behind Adar1 KO-sensitive cells and KO-insensitive cells.RT-qPCR and Western blot results showed that the expression levels of PKR,RIG-I,MDA5,and 2’,5’-oligoadenylate synthetase 1(OAS1)were higher in Adar1 KO-sensitive cells(CT26 and ID8)than that in Adar1 KO-insensitive cells(B16).Knocking out Stat1 can down-regulate the expression levels of ISGs in CT26 cells.B16 cells can up-regulate the mRNA expression levels of the above ISGs under the stimulation of interferons,while knocking out Stat1 can block this phenotype.Afterward,we conducted a survival analysis of tumor patients in the TCGA database.The results showed that among COAD,SKCM,and HNSCC patients with high ISGs signature expression,patients with low ADAR1 expression had a better survival prognosis.Besides,after being treated with STING inhibitor C-176,Adar1 KO-sensitive cells(CT26 and ID8)significantly down-regulated the mRNA and protein expression levels of STAT1,PKR,RIG-1,MDA5,and OAS1.Finally,we want to clarify whether the immune environment in mice is involved in the tumor growth inhibition in vivo mediated by ADAR1 deficiency.After establishing a subcutaneous Adar1 KO tumor model in NCG mice,we found that both Adar1 KO CT26 and B16 cells restored tumorigenicity.However,the tumor growth rate was still lower than that of control cells.The activities of spleen CD4+and CD8+T cells,the expressions of spleen CD8+TCM and TEM,and the expressions of IFNγ in peripheral blood were all increased in Adar1 KO tumor-bearing mice compared with control tumor-bearing mice.Mouse rechallenge experiment showed that the tumor growth rate of mice inoculated with Adar1 KO CT26 cells was significantly lower than that of the control group under the challenge of wide-type(WT)CT26 cells 60 days later.Conclusions:In this study,we used the CRISPR-Cas9 system to successfully construct Adar1 KO clonal stable transfected cell lines based on three common mouse tumor cell lines CT26,ID8,and B16.Then we explored the effects of Adar1 deletion on the growth and function of these mouse tumor cells in vivo and in vitro and discussed whether the interferon-related signaling pathways and the immune environment are involved in mediating these effects.The conclusions of this study can be summarized as follows:(1)Loss of ADAR1 inhibits tumor growth by inhibiting the cell proliferation and colony formation,promoting apoptosis,and inducing the secretion of IFNβ and IL-6.Complete knockout of Adar1 deprives the tumorigenicity of mouse tumor cells in vivo,which suggests that ADAR1 functions as an important tumor-promoter;(2)Different tumor cells have different sensitivities to ADAR1 loss.Only the combination of ADAR1 deficiency and interferon stimulation can inhibit the growth of Adar1 KO-insensitive tumor cells.However,both in Adar1 KO-sensitive and KO-insensitive tumor cells,loss of ADAR1 inhibits tumor growth in vivo and in vitro through activating the interferon-related signaling pathways,including the JAK-STAT pathway and the downstream PKR-sensing pathway;(3)The difference in the sensitivity of tumor cells to Adar1 deletion is derived from the difference in the intracellular expression levels of PKR and other ISGs.Therefore,the expression levels of ISGs can be used as a biomarker to judge whether tumors are sensitive to ADAR1 deficiency;(4)The immune environment is involved in mediating the deprivation of tumor formation by Adar1 deletion and Adar1 KO tumor cells can function as a prophylactic tumor vaccine in mice.Altogether this study indicated that ADAR1 mediates the immune tolerance of tumors in vivo by inhibiting the activation of the intracellular interferon-related signaling pathways,thus functions as a tumor promoter.Moreover,tumors with high expression levels of ISGs are sensitive to ADAR1 deficiency,providing a new basis and ideas for therapeutic interventions target ADAR1 in clinical. |
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