[Effect of down-regulation of Id3 expression by microRNA on proliferation and apoptosis of A549 cells].

AIM: To investigate the effect of microRNA-mediated exogenous Id3 gene silencing on proliferation and apoptosis of human lung adenocarcinoma A549 cells in vitro. METHODS: A recombinant miRNA expression vector (pcDNA6.2-GW/EmGFPmiR-Id3, pcDNA/miRId3) which targets human Id3 gene was constructed. After 24 h of transfection, the transfection efficiency was monitored by inverted fluorescence microscopy.EGFP expression efficiency in A549 cells was analyzed by flow cytometry (FCM).Id3 expression vector pEGFP/Id3 and pcDNA/miRId3 were cotransfected into A549 cells by liposome-mediated method. After 24 h of transfection, the transfection efficiency was monitored by inverted fluorescence microscopy.Semi-quantitative RT-PCR and Western blot were used for identifying Id3 mRNA and protein expression respectively in A549 cells after transfection. Cell proliferation rate and apoptosis ratio were evaluated by MTT assay and Annexin V/7-ADD staining followed by FCM to observe the down-regulatory effect of Id3 expression by miRNA-mediated RNA interference (RNAi). RESULTS: pcDNA/miRId3 and pEGFP/Id3 were successfully transfected into A549 cells. RT-PCR and Western blot results showed that after 24 h of cotransfection of pEGFP/Id3 and pcDNA/miRId3 in A549 cells, the exogenous expression of Id3 both at mRNA and protein levels were significantly reduced compared with the pEGFP/Id3 group. MTT assay and Annexin V/7-AAD staining showed that after 24 h of transfection with pEGFP/Id3, the proliferation rates were significantly reduced and apoptotic cell ratios were significantly higher than those of pEGFP-transfected cells.Whereas there were not any significant differences in proliferation rates or apoptotic cell ratios between pcDNA/miRId3+pEGFP/Id3 cotransfected group and pEGFP or miRNA negative controls. CONCLUSION: Exogenous expression of Id3 in A549 cells could inhibit proliferation and induce apoptosis of A549 cells. Cotransfection of pcDNA/miRId3 and pEGFP/Id3 into A549 could reverse the Id3-induced proliferation inhibition and apoptosis. Construction and application of Id3-targeting miRNA expression vector may build some foundations for investigation the mechanisms of Id3-induced proliferation inhibition and apoptosis in A549 cells.

Upregulation of ID protein by growth and differentiation factor 5 (GDF5) through a smad-dependent and MAPK-independent pathway in HUVSMC.

GDF5 (growth and differentiation factor five), a member of the TGF-beta superfamily, binds specifically to BMPR1b, BMPR2 and ACTR2a receptors forming a heterodimeric complex, thereby inducing phosphorylation of smad1, 5, 8 and translocation to the nucleus. ID1 (inhibitor of differentiation or DNA binding) is essential for G1 to S phase transition inhibiting DNA binding thereby playing an important role in the control of differentiation, proliferation and angiogenesis. The objective of this study was, therefore, to characterize the signal transduction pathway of GDF5, especially the involvement of ID1, in human umbilical vein smooth muscle cells (HUVSMC). We observed the expression of BMPR1a, BMPR1b, BMPR2, ACTR2a, smad1, smad 5, ID1, ID2 and ID3 in HUVSMC. Application of GDF5 upregulated ID1 and ID3 expression by involvement of the smad signaling pathway. GDF5 caused phorsphorylation of smad1 followed by upregulation of ID1 and ID3. Co-incubation with anti-GDF5 prevented these effects. GDF5 significantly inhibited phosphorylation of p38 MAPK and induced phosphorylation of ERK. The specific inhibitor of p38 MAPK or ERK, SB203580 or U0126 did not induce ID protein expression. Smad1 siRNA transfection inhibited the upregulation of ID protein. GDF5 had chemotactic activity in HUVSMC; this effect was partly blocked by transfection of smad1 or ID1 siRNA. Our results indicate that GDF5 induces ID1 and ID3 in HUVSMC by a smad-dependent, MAPK-independent pathway. GDF5 binds to specific receptors, thereby inducing phosphorylation and translocation of smad1 to the nucleus where it is involved in the regulation of transcription. Since ID1 has been shown to be crucial for cell cycle control, we propose that GDF5 could be involved in the process of angiogenesis.

BMP-6 inhibits human bone marrow B lymphopoiesis--upregulation of Id1 and Id3.

OBJECTIVE: In mammals, factors produced by bone marrow (BM) stromal cells are instrumental in orchestrating the developmental process of B lymphocytes. Bone morphogenetic proteins (BMPs) are multifunctional cytokines previously found to regulate hematopoietic stem cells. In the present study, we have explored the role of BMP-6 in human B progenitor cells. MATERIALS AND METHODS: In vitro B lymphopoiesis of CD10(+) B progenitor cells from human BM was evaluated in the presence or absence of BMP-6 in short- or long-term coculture on MS-5 stromal cells, by tracking CFSE-labeled CD10(+) B progenitor cells or by quantification of CD19(+) cells. DNA synthesis in the pre-B cell line Nalm-6 was measured by (3)H-thymidine incorporation. BMP-6-induced phosphorylation of Smad1/5/8 was determined by Western blot analysis, whereas elevation of Id1-Id4 mRNA levels and basal BMP-6 mRNA levels were measured by real-time and conventional RT-PCR, respectively. RESULTS: By in vitro coculture of CD10(+) B progenitor cells or monoculture of Nalm-6 cells, we found that BMP-6 inhibited B lymphopoiesis by impeding cell proliferation. Furthermore, in CD10(+) B progenitors as well as in Nalm-6 cells, BMP-6 rapidly induced phosphorylation of Smad1/5/8, followed by an upregulation of Id1 and Id3 mRNA levels. Finally, we demonstrated that human bone marrow stromal cells express BMP-6 mRNA whereas B progenitor cells did not. CONCLUSIONS: We suggest that BMP-6, produced by the BM, may participate to fine-tune the balance between proliferation, apoptosis, and differentiation in human B progenitor cells during BM B lymphopoiesis.