The Regulatory Effects Of MicroRNA-29 In Organ Transplant Rejection And The Underlying Mechanisms

Organ transplantation,as a great breakthrough in medical science in the last century,has saved tens of thousands of lives,it has become an exclusively effective therapeutic means for patients with organ failure.Although the organ transplantation has made a huge progress since the invention of cyclosporine A in 1970 s.Due to the extreme mechanism of immunological rejection,which not only involves the acquired immunity,but also the innate immunity;not only related to cellular immunity,but also closely related to humoral immunity;moreover,different types of immunological reaction occur simultaneously or one after another,interweaved with each other,even to this day,our understanding of mechanism of occurrence of transplant rejection and tolerance and regulatory network is still very limited.In recent years,a lot of studies on microRNAs showed that,they have exerted crucial regulatory effects in terms of the development of immune cells,immune response,immune tolerance and other aspects of immunology.Although the studies on microRNAs concerning other aspects are abundant,the studies concerning transplantation immunity are few,most of the studies took the microRNAs as a diagnostic marker for transplant rejection;the studies on solid organ transplantation only involved a few microRNAs such as miR-155 and miR-17-92.Further study the regulatory mechanism of microRNAs in graft rejection can help us to further understand the mechanisms of graft rejection and graft tolerance and even the regulatory network.In the early stage,it was found by other researchers in our institute that,miR-29 can directly target the interferon-γ.The interferon γ was highly expressed in the serum of mouse after miR-29 was knocked down,and the Listeria monocytogenes infection can be resisted.However,during an allo-transplantation,we were surprised by the discovery that the graft survival time was significantly prolonged to over 2 weeks when miR-29 was knocked down.Generally speaking,as a cytokine of Th1 cell(one of the main cellular-mediated rejection effect cells),a high expression of IFN-γ is usually regarded as the sign of strengthened reactivity of Th1,the graft rejection is supposed to be strengthened.Why can the survival time of graft be significantly prolonged after miR-29 was knocked down? Whether or not other targets of miR-29 have played a key role in the graft rejection? Based on such hypothesis,we have studied the mechanism of regulation of transplant rejection by miR-29.PartⅠ: Significant suppression of acute transplant rejective reaction of allogene by low expression of miR-29Firstly,we used sponge technology to build the transgenic mice GS29 which miR-29 were knocked down.The GS29 mice were selected as a receptor,BALB/c mice as a donor,the modified Cuff technology was used to carry out a hetero-topic heart allo-transplantation model,the effects of miR-29 knockdown on transplant rejection were observed.It was found that,compared to the control group,GS29 group had a significantly prolonged survival time of grafts,meanwhile,it was markedly superior to the control group in terms of the thrombus formation of grafts and the swelling of grafts and peripheral immune organs.Meanwhile,a flow cytometer detection was used to detect the cell subsets of GS29 mice on day 5 after transplantation,which found that,in contrast to control group,the percentage of B cell in lymph nodes and spleen was evidently reduced;the percentage of T cell went up;the percentage of CD4+T evidently went down,the percentage of CD8+T cell evidently went up in GS29 mice.However,in contrast to T cells,both the percentage and the total number of B cells in the lymph nodes and spleen of GS29 mice were evidently lower than that of control mice.Meanwhile,we have analyzed the Treg subgroup in spleen and lymph nodes and Th1 subgroup in spleen.It was found that the percentage of Treg and Th1 subgroups of two groups had no significant difference.A histochemical detection was usd to find that,the immune cellular infiltration was evidently reduced in the GS29 group grafts.A further histochemical test found that,compared with the control group,the CD4,CD8 and CD138 stain in GS29 group grafts were evidently reduced.To sum up,it is indicated that,the significant suppression of allogeneic transplant rejective reaction by miR-29 knockdown might be associated with the humoral rejection.Part Ⅱ: effect of miR-29 on the AICD B cell to regulate the humoral immunityFor this part,we firstly detected the development and differentiation of B cells in the two group mice.It was found that,the development of pro-B into Pre-B and of immature B cell into mature B cell in GS29 mice had been blocked.And the differentiation of B2 cell subsets(follicle B cells)in in the spleen of GS29 mice were reduced.Furthermore,we have used the classical NP conjugated antigen and the method of intraperitoneal injection of TI-1 antigen NP-LPS and TD antigen NP-KLH to severally study the TI-1 and TD reaction of GS29 mice,of which the result showed that,the TD reaction of GS29 mice was weaker than that of control group.Furthermore,we have detected the ability of GS29 mice to generate DSA,and found that it also was weake than that of control group.The activation of B cell depended on the Tfh cells,afterwards,we used the flow cytometer to detect the germinal center B cells and Tfh cells in the spleen of GS29 mice after allo-transplantation.It was found that,after allo-transplantation,the germinal center B cells in the spleen of GS29 mice were significantly reduced,but the Tfh cells had no significant differences.Meanwhile,the histochemical result showed that,the expression of the two specific markers of AMR,IgG and C4 d,were both down-regulated in the GS29 group grafts,indicating that,the AMR of GS29 mice group was evidently lower than that of control group.It can thus be seen that,in the case of allo-transplantation,the inhibited effect of AMR in GS29 mice due to a decreased formation of germinal center.According to previous studies,activation induced cell death(AICD)played a key role in the germinal center formations of B cells.So we have sorted the CD43-na?ve B2 in the spleen,which was stimulated by anti-IgM in vitro for 10 hours,the AICD of B cell was detected by FCM.The experimental result showed that,compared with control group,the AICD of na?ve cell in GS29 mice was evidently increased,which was consistent with the result of histochemistry staining of TUNEL after allo-transplantation.Part Ⅲ: direct targeting of PTEN by miR-29 influences the regulation of B cell AICD by PI3K/AKT pathway.In previous experiment,we have confirmed that,in the na?ve B cell derived from the transgenic GS29 mice,miR-29 knockdown can promote the AICD of B cell.Due to the fact that the primary B cell has difficulty in surviving in vitro,making it difficult for the subsequent experiments to carry out.Thus,we have firstly selected the in-vitro model of AICD of B cell that has been generally recognized,namely,in-vitro stimulation of WEHI-231 cell by Anti-Mouse IgM to induce AICD.Then we found that transfection with microRNA-29a/b/c mimics can reduce the AICD of WEHI-231 cell;while transfection with microRNA-29a/b/c inhibitors can promote the AICD of WEHI-231 cell.These experiments again confirmed that,miR-29 can effectively regulate the AICD of B cell.Furthermore,we have conducted a GO enrichment analysis to more than 1000 targets of miR-29 provided by targetscan Website.It was found that,85 out of these targets were involved in the cell survival and growth processes.A kegg pathway enrichment analysis was conducted to study the 85 genes,4 pathways that have relatively high couts were chosen for vanny analysis,and two molecules that have an extensive influence were screened out: PTEN and PIK3 CA.Then,we have synthesized the reporter gene of PTEN,PIK3 CA and their mutants for dual-luciferase reporter assey,in order to verify the target.The results showed that,miR-29 can directly target PTEN and inhibit the activity of reporter gene of PTEN3 UTR.We have synthesized the plasmid and siRNA of PTEN subsequently,and further confirmed that,miR-29 can regulate AICD of WEHI-231 cell through PTEN.PTEN(phosphatase and tensin homolog deleted on chromosome ten),on the one hand,PTEN is star cancer suppressor gene,on the other hand,it has dual specificity phosphatase,enabling the dephosphorylation of PIP3(phosphatidylinositol-3,4,5-triphosphate,PIP3),and finally suppressing the PI3K/AKT pathway.While the AICD of B cell mainly involves three signals,PLCγ,Ras and PI3KPI3K/AKT.Therefore,we used western blot to detect the level of p-AKT in the WEHI-231 cell treated by antagomir-29a-3p,which was found to be consistent with what we have speculated,the level of p-AKT was lower than that of control group significantly.It was confirmed that,mir-29 influenced PI3K/AKT pathway by targeting PTEN.By a further study,we found that,the level of p-BAD was reduced evidently;so was the level of anti-apoptosis molecule Bcl-2 and Bcl-xl.The above results showed that,mir-29 inhibits PI3K/AKT pathway by targeting PTEN,leading to a decline in level of phosphorylation of BAD,and influencing the downstream expression of anti-apoptosis molecule Bcl-2 and Bcl-xl,finally promoting the AICD of WEHI-231 cell.Part Ⅳ: nucleic acid drug,antagomiR-29 can be used as a drug to intervene and treat the allograft rejection.antagomiR-29 is a cholesterol-modified inhibitor of miR-29,it can specifically inhibit the expression of miR-29 in vivo,and is usually used as a nucleic acid drug to intervene various disease models,to determine whether or not antagomiR-29 can be used as a drug to treat the allograft rejection,we commissioned Guangzhou Ruibo Biotech to synthesize antagomiR-29a-3p for intervention to allo-transplant rejecton by caudal vein injection.The experimental result showed that,the injection of antagomiR-29a-3p after allo-transplantation can significantly reduce allograft rejection,the survival time of transplant was longer than that of control group significantly.To conclude,mir-29 inhibits PI3K/AKT signal pathway by targeting PTEN,leading to a decline in level of AKT phosphorylation and influencing the downstream expression of anti-apoptosis molecule Bcl-2 and Bcl-xl,finally promoting the AICD of B cell.In the case of allo-transplantation,the B cells encounter the allogeneic antigen,owing to an enhanced function of AICD,leading to a decreased formation of germinal center,the plasmocytes that secrete the donor specific antibody are reduced significantly,and finally an inhibited effect of AMR is exerted.