The microRNA-21-PDCD4 axis prevents type 1 diabetes by blocking pancreatic beta cell death

1、The microRNA-21-PDCD4 axis prevents type 1 diabetes by blocking pancreatic beta cell deathQingguo Ruan, Ting Wang, Vasumathi Kameswaran, , and Youhai H. ChenDepartment of Pathology and Laboratory of Medicine, University of Pennsylvania School of MedicineINTRODUCTION1、miRNA：microRNAa.small noncoding

2、RNAs, binding to the 3 untranslated region(3UTR) of mRNAs and affecting their stability and/or translationb.miRNA-21，microRNA家族中的一个亚型，具有自主转录单位， up-regulated in many cancers2、PDCD4：programmed cell death protein 4a.tumor suppressorb.a translational and transcriptional regulator of gene expression3、NF-

3、B： nuclear factor-Ba.transcription factorb.Whereas NF-B prevents apoptosis in most cell types, it can both promote and inhibit islet cell death. Its related to the nature of the death-inducing conditions involved.RESULTS AND DISCUSSION1、PDCD4 Deficiency in Nonhematopoietic Cells Reduces the Suscepti

4、bility to Type 1 Diabetes. 2、Increased Th1, Th2, and Th17 Cytokine Gene Expression in Pdcd4/T Cells. 3、PDCD4 Deficiency Prevents Islet Cell Death, Whereas PDCD4Overexpression Induces It. 4、The NF-BmiR-21PDCD4 Axis That Controls Islet Cell Death.RESULTS AND DISCUSSION1、PDCD4 Deficiency in Nonhematopo

5、ietic Cells Reduces the Susceptibility to Type 1 Diabetes. (1)To determine the potential roles of PDCD4 in the pathogenesis of type 1 diabetes, Pdcd4-deficient nonobese diabetic (NOD) mice age-matched wild-type (WT) littermatesFig. S1A. (flow cytometry) The percentages of cells expressing CD4, CD8,

6、B220, and CD11c in the spleen and hymus of Pdcd4/ and WT mice were similar. Fig. S1B. (flow cytometry) We also found that there was no significant difference in the expression of the T-cell activation markers CD44, CD62L, and CD25 between Pdcd4/ and WT cells .Fig. S2A. (flow cytometry) We found that

7、 Pdcd4 deficiency did not affect anti-CD3 or LPS-induced apoptosis of splenocytes. Splenocyte proliferation was also similar between WT and Pdcd4/ NOD cellsFig. S2B. Splenocyte proliferation was also similar between WT and Pdcd4/ NOD cellsFig. 1 A and B. Pdcd4/ NOD mice were significantly resistant

8、to both spontaneous and CY-induced diabetes (2)To determine the effect of PDCD4 deficiency on T1D cyclophosphamide (CY)-induced diabetes in Pdcd4/ NOD mice. Age- and sex-matched wild-type (WT) littermatesFig. S3 A and B. (histochemical) Insulitis was less severe in Pdcd4/ than in WT mice.these resul

9、ts indicate that PDCD4 plays a crucial role in accelerating T1D.(3)To determine which cells(hematopoietic or nonhematopoietic cells) express PDCD4, which plays a role in T1D. Fig.1 C. Results are accumulated diabetes incidence and are representative of two independent experiments. We found that PDCD

10、4 deficiency in hematopoietic cells significantly exacerbated the diabetes, whereas PDCD4 deficiency in nonhematopoietic cells alleviated it. injecting WT bone marrow cells into WT mice. injecting WT bone marrow cells into Pdcd4/ mice. injecting Pdcd4/ bone marrow cells into WT mice.Eight weeks late

11、r, mice were injected with low-dose STZ to induce diabetes.Fig.1D. Similarly, following adoptive transfer of WT T cells into WT NOD.scid or PDCD4-deficient NOD.scid mice, the spontaneous T1D was significant decreased in PDCD4-deficient NOD.scid recipient mice. injecting WT bone marrow cells into WT

12、NOD scid . injecting WT bone marrow cells into Pdcd4/ NOD scid.Eight weeks later, mice were injected with low-dose STZ to induce diabetes.Taken together, these results indicate that PDCD4 expressed by hematopoietic cells plays an opposite role of that of nonhematopoietic cells in T1D.2、Increased Th1

13、, Th2, and Th17 Cytokine Gene Expression in Pdcd4/T Cells. Fig.2. (A)We found that, upon stimulation with anti-CD3, Pdcd4/ NOD splenocytes produced significantly more Th17 (IL-17A), Th1 (IL-2 and IFN-), and Th2-type cytokines (IL-4) . (B)IL-17A and IFN- were also significantly increased in Pdcd4/ T-

14、cell cultures stimulated with murine GAD65(1)To determine the potential effect of PDCD4 deficiency in T cellsFig.S4 . (B)We found that, upon stimulation with anti-CD3, Pdcd4/ NOD pancreatic lymph node cells, and islet infiltrating T cells produced significantly more Th17 (IL-17A), Th1 (IL-2 and IFN-

15、), and Th2-type cytokines (IL-4). (A)IL-17A and IFN- were also significantly increased in Pdcd4/ T-cell cultures stimulated with murine GAD65Fig.S5.(B)We found that, upon stimulation with anti-CD3, Pdcd4/ NOD islet infiltrating T cells produced significantly more Th17 (IL-17A), Th1 (IL-2 and IFN-),

16、and Th2-type cytokines (IL-4). (A)IL-17A and IFN- were also significantly increased in Pdcd4/ T-cell cultures stimulated with murine GAD65Fig.S6. In both pancreatic lymph nodes and islets, the frequency of CD4+CD44+ T cells was increased, whereas that of CD4+CD25+ T cells unchanged in PDCD4-deficien

17、ct mice compared with WT mice . Fig.S7. We found that the production of proinflammatory cytokines (IL-6, IL-12, and TNF-) by myeloid cells following stimulation with LPS was not signifi cantly different between WT and Pdcd4/ groups.These results indicate that PDCD4 may selectively regulate Th cell d

18、ifferentiation. (2)To test the possibility of that PDCD4 selectively regulate Th cell differentiation.Fig.S8.(flow cytometry) PDCD4 deficiency did not significantly affect the differentiation of Th1, Th2, Th17, or Treg cells.Fig.9A. The frequency of Tregs was not altered in PDCD4-deficient mice . Th

19、ese data indicates that the increased Th1, Th2, and Th17 responses in PDCD4-deficient cells is likely related to the PDCD4 effect on gene expression, not cell differentiation.3、PDCD4 Deficiency Prevents Islet Cell Death, Whereas PDCD4Overexpression Induces It. (1)To examine the impact of PDCD4 defic

20、iency on the death and survival of pancreatic islet cells. Fig. 3A. Compared with control animals, the mean pancreatic islet size in 12-wk-old Pdcd4/ NOD mice were significantly increased .(2)To determine the effect of PDCD4 deficiency on streptozococin-induced killing of cells in vivoFig. 3B. Two d

21、ays afte the STZ treatment, 100% of WT mice developed diabetes. B contrast, the incidence of the disease was reduced to 30% in the Pdcd4/ group(3)To examine the effect of PDCD4 deficiency and overexpression in a model of cytokine-induced death of islet cells in vitro. Fig. 3C.Compared with WT islet

22、cells, PDCD4-deficient cells were significantly less sensitive to apoptosis.Fig. 3D. Conversely, PDCD4 overexpression in TC-6 cells increased their susceptibility to cytokine-induced apoptosisFig. 3E. Consistent with these results, the mRNA levels of proapopotic genes (Bad, Bax, and Bid) were signif

23、icantly decreased in PDCD4-deficient islet cells, whereas that of antiapoptotic gene Bclxl was increased .4、The NF-BmiR-21PDCD4 Axis That Controls Islet Cell Death.(1)To test the relationship between miR-21 and PDCD4 levels in pancreatic islets of NOD mice.Fig. 4A .(real-time RT-PCR) In islets of 6-

24、 and 12-wk-old animals, the levels of miR-21 were significantly higher than that of 3-wk-old mice , the level of PDCD4 was significantly decreased in the islets of older animals. Fig. 4B .(real-time RT-PCR) The expression of miR-21 increased,whereas that of PDCD4 decreased.Fig. S9B. The infiltrating T cells in the islets of 3-wk-old and 12-wk-old NOD mice accounted for only 1.1 and 9% of all cells, respectively.Fig. S9C.(real-time RT-PCR) We found that the expression of PDCD4 was significantly dec