Experiment Study Of Alkbh5 Regulating The Immuno Suppressive Function Of MDSCs In Tumor-Bearing Mice

ObjectiveN6-methyladenosine（m⁶A）is the most common chemical modification of eukaryotic mRNA.This study mainly studied the biological role of m⁶A demethylase Alkbh5 on the myeloid-derived suppressor cells（MDSCs）,and preliminarily explored its regulatory mechanism,providing a new experimental basis for immunotherapy against MDSCs.Methods（1）The mouse CT26 colon cancer transplanted tumor model was established by subcutaneous injection.MDSCs cells from mouse spleen were separated by immunomagnetic beads.The purity and liveness of MDSCs cells were detected by flow cytometry（FCM）.The levels of m⁶A in MDSCs of tumor-bearing mice were detected by colorimetry.The demethylase of m⁶A mainly contain ofα-ketoglutarate-dependent dioxygenase alk B homolog 5（α-Ketoglutarate-dependent dioxygenase alk B homolog 5,Alkbh5）,fat-mass and obesity-associated protein（FTO）.Quantitative real-time PCR（q RT-PCR）was used to detect the mRNA expression levels of Alkbh5 and FTO in MDSCs of tumor-bearing mice at different weeks.Western blot（WB）was used to detect the protein expression levels of Alkbh5 and FTO.（2）To explore the possible biological role of Alkbh5 in MDSCs.The RNA-sequencing was used to detect the differently expressed genes in MDSCs after Alkbh5 knockdown.The results showed that the mRNA expression of Arginase-1（Arg-1）and inducible nitric oxide synthase（i NOS）were significantly different in MDSCs,so we initially selected Arg-1 and i NOS as Alkbh5candidate target genes for further study.Mouse Alkbh5-specific si RNA（si Alkbh5）was transferred into MDSCs(MDSCs^{si Alkbh5}),or mouse Alkbh5-overexpressed lentivirus vector was transferred into MDSCs(MDSCs^Alkbh55).The mRNA expression changes of Arg-1 and NOS2 in MDSCs^{si Alkbh5} and MDSCs^Alkbh5 were detected by q RT-PCR,the activity of Arg-1 and the content of NO were detected by colorimetric method and Griess coupling method respectively.NCBI（https://www.ncbi.nlm.nih.gov/）was used to retrieve the mRNA sequence of Arg-1 and NOS2 of mice.Then input into SRAMP database（http://www.cuilab.cn/sramp/）to predict m⁶A modification site of Arg-1 mRNA and NOS2 mRNA.Alkbh5 binding mRNA was enriched by RIP,and q RT-PCR was used to detect the expression of Arg-1 and NOS2 of which.The mRNA m⁶A levels of Arg-1 and NOS2 in MDSCs^{si Alkbh5} and MDSCs^Alkbh5 cells were detected by Me RIP-PCR.Actinomycin D was used to block the mRNA synthesis in MDSCs cells,and q RT-PCR was used to detect the mRNA expression of Arg-1 in MDSCs^{si Alkbh5} and MDSCs^Alkbh5.The protein expression of Arg-1 in MDSCs^{si Alkbh5} and MDSCs^Alkbh5was detected by Western blot.（3）CD4⁺T cells was labeled with CFSE to establish CD4⁺T cells proliferation system in vitro,which was used to analyze the inhibitory ability of MDSCs on the proliferation of CD4⁺T cells.MDSCs of tumor bearing mice at different weeks were seperated and co-cultured with CD4⁺T cells,and the proliferation change of CD4⁺T cells was detected by FCM.The Alkbh5-specific si RNA or Alkbh5 overexpressed lentivirus vector was transferred into MDSCs,the inhibitory ability of MDSCs^{si Alkbh5} and MDSCs^Alkbh5 on CD4⁺T cell proliferation was detected by the same method.（4）MDSCs were transfected with the Alkbh5 specific si RNA or Alkbh5 overexpressed lentiviral vector,then 1x10⁶ MDSCs^{si Alkbh5} or 1x10⁶MDSCs^Alkbh5 mixed with 1x10⁶ CT26 tumor cells respectively for subcutaneous injection.The tumor growth and progression were observed every two days.After that,the mice were sacrificed at 23 days,the tumor tissue was stripped and weighed.The proportion of CD3⁺CD4⁺IFN-γ⁺T（Th1）cells and CD3⁺CD8⁺IFN-γ⁺T（CTL）cells in local lymph nodes,spleen and tumor tissues were detected by FCM.Results（1）The purity from spleen of tumor bearing mice was 94.40%and liveness of MDSCs cells was over 95%.The level of m⁶A in MDSCs of tumor-bearing mice at 3 weeks was twice as high as that of MDSCs of wild-type mice.And the level of m⁶A in MDSCs of tumor-bearing mice at 5weeks was twice as high as that of tumor-bearing mice at 3 weeks.Which indicating that the level of m⁶A in MDSCs of tumor-bearing mice was increased,and it further increased with the progression of tumor（P<0.05）.After detecting the expression of m⁶A demethylase Alkbh5 and fto in MDSCs.We find that the mRNA and protein expression of Alkbh5 in spleen MDSCs of tumor-bearing mice were significantly lower than those of wild-type mice（P<0.05）,and the expression of Alkbh5 in spleen MDSCs of tumor-bearing mice at 5 weeks was lower than that of MDSCs of tumor-bearing mice at 3 weeks（P<0.05）.The expression of m⁶A demethylase FTO was no significant difference.These results showed that the m⁶A level of spleen MDSCs of tumor-bearing mice was significantly increased with the tumor development,while the expression of Alkbh5 was down-regulated.（2）After Alkbh5 expression was knocked down in MDSCs,RNA sequencing results showed that159 genes showed significant differences in transcription levels,which contained 122 up-regulated genes and 37 down-regulated genes.We removed the genes which was extremely low expression and no potential m⁶A modification sites,combined with literature reading,screened out the MDSCs-related genes.Further analysis found that these genes were significantly enriched in the immunosuppressive function of MDSCs,followed by the chemotaxis and differentiation and development of MDSCs,suggesting that Alkbh5 may play an important role in the immunosuppressive function of MDSCs.Further analysis of the genes related to the immunosuppressive function of MDSC revealed that Arg-1,NOS2（i NOS）mRNA increased significantly,which is important for MDSCs exert immunosuppressive function as its directly suppressive molecule.Next,we further verified whether Alkbh5 could regulate the expression of Arg-1 and i NOS in MDSCs.After Alkbh5 in MDSCs was knocked down by Alkbh5 specific si RNA,the mRNA level of Arg-1 in MDSCs was 3.90 times higher than that in control cells（P<0.05）,NOS2 mRNA level was 5.30 times higher than that of control cells（P<0.05）.The activity of Arg-1 was 1.25 times higher than that of control cells（P<0.05）,and the content of NO was 1.19 times than that of control cells（P<0.05）.After overexpression of Alkbh5 in MDSCs,the mRNA level of Arg-1 decreased by 55.60%（P<0.05）,NOS2 mRNA level decreased by 50.00%（P<0.05）.The activity of Arg-1 in MDSCs^Alkbh5 cells decreased by 80.00%（P<0.001）,and the content of NO decreased by 66.00%（P<0.05）.These results showed that Alkbh5 could significantly inhibit the expression and activity of Arg-1 and i NOS of MDSCs in tumor-bearing mice.In order to further study whether Alkbh5 could regulate the m⁶A modification on mRNA of Arg-1and i NOS.The SRAMP database showed that there exist m⁶A modification sites on the mRNA of Arg-1 and NOS2.RIP-PCR detection system showed that Alkbh5 could directly bind to Arg-1mRNA and NOS2 mRNA in MDSCs.Me RIP-PCR showed that the level of m⁶A on Arg-1mRNA in MDSCs^{si Alkbh5} was 2.69 times higher than that in control cells（P<0.05）,and NOS2 had no change.The level of m⁶A on Arg-1 mRNA in MDSCs^Alkbh5 decreased by 63.60%compared with control cells（P<0.05）,while NOS2 had no change.These results indicated that Alkbh5could down-regulate the m⁶A modification level of Arg-1 mRNA,but had no direct regulation effect on the m⁶A modification of NOS2 mRNA.Further experiments are needed to find possible causes.m⁶A modification plays a regulatory role in the splicing,stability and metabolism of mRNA.To investigate the effect of m⁶A modification on the stability of Arg-1 mRNA.Alkbh5-specific si RNA or Alkbh5 overexpressed lentivirus vector was transfected into MDSCs respectively.The results showed that the degradation rate of Arg-1 mRNA in MDSCs^{si Alkbh5} was 40.00%slower than that in control cells.The degradation rate of Arg-1 mRNA in MDSCs^Alkbh5 was 42.00%faster than that in control cells,indicating that the stability of Arg-1 mRNA was decreased after overexpression of Alkbh5.Western blot analysis showed that the protein expression of Arg-1 in MDSCs^{si Alkbh5} increased significantly,and the protein expression of Arg-1 in MDSCs^Alkbh5decreased significantly.These results indicated that Alkbh5 could reduced Arg-1 mRNA stability and down-regulated its protein expression by down-regulating m⁶A on its mRNA.However,the molecular mechanism of Alkbh5 regulating i NOS expression has not been clarified,which will be discussed later.（3）Firstly,the immunosuppressive function of MDSCs in tumor bearing mice at different weeks was detected.The results showed that the inhibitory ability of MDSCs in tumor bearing mice at 5weeks was stronger than that of MDSCs at 3 weeks（P<0.05）,while the expression of Alkbh5 in MDSCs of tumor bearing mice at 5 weeks was lower significantly than that of MDSCs at 3weeks.Subsequently,Alkbh5-specific si RNA was transfected into MDSCs,and it was found that the ability of MDSCs^{si Alkbh5} inhibited the proliferation of CD4⁺T cells increased significantly（P<0.05）.MDSCs were transfected with the Alkbh5 overexpressed lentivirus vector,compared with control cells,the ability of MDSCs^Alkbh5 inhibited the proliferation of CD4⁺T cells decreased significantly（P<0.05）.These results indicated that Alkbh5 could down-regulate the immunosuppressive function of MDSCs by decreasing the expression of Arg-1 and i NOS.（4）Compared with the control group,tumor growth in MDSCs^{si Alkbh5} group was significantly faster than that in the control group,with increased volume and weight（P<0.05）.The proportion of CD3⁺CD4⁺IFN-γ⁺T（Th1）cells and CD3⁺CD8⁺IFN-γ⁺T（CTL）cells in spleen,local lymph nodes and tumor tissues of mice decreased significantly（P<0.05）.The tumor growth,volume and weight of MDSCs^Alkbh5 group were significantly reduced（P<0.05）,the proportion of CD3⁺CD4⁺IFN-γ⁺T（Th1）cells and CD3⁺CD8⁺IFN-γ⁺T（CTL）cells increased significantly（P<0.05）.These results indicated that Alkbh5 could delay tumor progression by down-regulating the immunosuppressive function of MDSCs.ConclusionsDuring the tumor-bearing development of mice,the expression of Alkbh5 in MDSCs cells was decreased,leading to the increased m⁶A modification level of Arg-1 mRNA,enhanced the Arg-1mRNA stability and its protein expression,thus enhancing the immunosuppressive function of MDSCs and promoting tumor growth.