| Hematopoietic stem cells (HSCs) are so far the best researched adult stem cells, giving rise to both myeloid and lymphoid progenitors. HSCs research is a promising field with potential applications that may bring revolutionary changes to human life. MicroRNAs, a group of newly-discovered non-coding small RNAs involved in organism development by regulating cell proliferation and differentiation , have been found to play a role in committed differentiation and self-renewal of stem cells. Studies of various stem cells indicate a close relationship between miRNAs and stem cell development. Research on stem cell-related miRNAs is becoming a heated field. However, to date, little is known about the HSCs-specific miRNAs expression profile and roles of miRNAs in HSCs biology. To research on HSCs-related miRNAs, an important starting point is to examine expression profiles of miRNAs in HSCs by high throughput screening.In this study, we developed a novel miRNA-specific microarray, screened a set of HSCs-related miRNAs, and further predicted their targets by bioinformatics. After verifying the potential miRNA targets by functional experiments, we analyzed the roles of the miRNAs in promoting HSCs development. Our results provide theoretical support and experimental basis for further research on mechanism of miRNAs regulating HSCs development and induction of committed differentiation of HSCs.Objectives1. To develop novel mammalian miRNA-specific microarray.2. To obtain HSCs development-related miRNAs by using microarray.3. To predict miRNAs targets by bioinformatic analysis and verify them by functional experiments.4. To investigate roles of miRNAs in HSCs development.Materials and Methods1. Fabrication of mammalian miRNA-specific microarray MiRNAs probes were designed based on the known mammalian miRNAs sequences and synthesized, and then microRNA microarrays were fabricated. 2. Screening for HSCs development-related miRNAsAfter isolating CD34+CD38- HSCs and CD34+ hematopoietic cells by FACS, we extracted total RNA. Then, 18-100nt small RNAs were purified. After being amplified and fluorescence–labled, the small RNAs were hybridized with microarrays to get miRNAs expression profiles of HSCs and CD34+ hematopoietic cells, respectively. As a result, HSCs- related miRNAs were then obtained by comparison with the other.3. Verifying the microarray results by Real time RT-PCR (Q-RT-PCR)Expression of six differentially expressed miRNAs was confirmed by Q-RT-PCR. Three miRNAs that have Q-RT-PCR results concordant with that of microarray experiments were selected for repeated Q-RT-PCR for 3 times to test reliability of Q-RT-PCR results.4. Bioinformatic analysis and verification of potential miRNAs targetsBioinformatic analysis was performed for chromosomal localization and conservation analysis of miR-129, miR-526b* and miR-520h. Three online softwares for bioinformatic analysis, i.e., picTar, miRanda 3.0 and targetscan3.1, were used for miRNAs target prediction.MiR-129 and miR-520h were selected for target verification.First, DNA sequence and its complementary sequence of the target sequence for miRNA binding were synthesized, with restriction enzyme sites for Hind III or Spe I dangling at the 5'end of one oligonucleotide, respectively. Then, the double-stranded insert sequence was formed by annealing of two oligonucleotides. Next, the insert sequence was ligased with the linear Luciferace reporter vector, pMIR-Luc, to construct a reconstitute vector, pMIR-Luc-miR. Verification by restriction enzymes digestion and sequencing were performed.The reconstitute vector, pMIR-Luc-miR, the control vector for normalization, pMIR-REPORT-β-gal (pMIR-β-gal), and Pre-miRNAs were co-transfected to HeLa cells (the test group).Seventy-two hours after transfection,β-Galactosidase and luciferase activities were determined. Three control groups were set up, namely, non-miRNA control group, Pre-miR-negative control group and Anti-miRNA control group. All transfection data were expressed as luciferase activity normalized byβ-galactosidase activity. By this means, the reliability of miRNAs targets was verified.5. Research on the role of miR-520h in promoting HSCs developmentMiR-520h, enriched in CD34+CD38- HSCs, was selected for function characterization in promoting HSCs development. We transfected miR-520h into FACS-isolated CD34+ hematopoietic cells. At 24hr after transfection, CFC assays were performed to test the colony-forming ability of transfected cells. At 72hr after transfection, FACS analysis was performed to test the proportion change of HSCs and CD34+ hematopoietic cells. Three control groups were set up, namely, non-miRNA control group, Pre-miR-129 negative control group and Anti-miRNA-520h control group. The role of miR-520h in promoting HSCs development was then analyzed.Results1. Development of novel mammalian miRNA-specific microarrayThe microarray consists of 4 subarrays (23×21). The distance between two neighbouring spots is 185μm, and the diameter of each spot is 130μm. Each miRNA probe has three replicate spots on a microarray. To ensure reliability of the microarray, various control probes were spotted on the microarray. Altogether, there are 1754 spots on the microarray, including probes for 588 genes, 469 of which are for miRNAs.2. Screening of HSCs development correlative miRNAsCompared with CD34+ hematopoietic cells, 31 miRNAs differentially expressed in HSCs were screened, including 22 miRNAs (including miR-129) that have an expression level 4 times lower in HSCs than in CD34+cells and 9 miRNAs (including miR-526b* and miR-520h) that have an expression level 4 times higher in HSCs. Four predicted miRNAs are also included in the 31 miRNAs.3. Verification of microarray results by Q-RT-PCRSix miRNAs differentially expressed in CD34+CD38- HSCs were selected for Q-RT-PCR verification. 3 of 6 real time RT-PCR data were concordant with the microarray data, i.e., CD34+CD38- HSCs-overexpressed hsa-miR-520h, hsa-miR-526B* and CD34+CD38- HSCs-underexpressed hsa-miR-129.4. Bioinformatic analysis and verification of potential miRNAs targets Three online softwares for bioinformatic analysis, i.e., picTar, miRanda 3.0 and targetscan3.1, were used for predicting targets of miR-129, miR-526b* and miR-520h, respectively. Our results demonstrated that①Thirty-three potential targets of miR-129,including EIF2C3, CAMTA1, SH3KBP1, TGIF2, ING3 and DLGAP2, exist reservedly in human, mice, dogs, chimps and chickens.And the potential targets include genes that are transcription factors, or related to miRNA processing and signal transduction.②miR-526b* only exists in human, with 771 potential targets, including ABCG2, EIF2C3, CAMK4 and CSF2RA, genes involved in signal transduction, transcription regulation, development of hematopoiesis, apoptosis regulation, maintaining of stem cell function and miRNAs maturation.③miR-520h have 226 potential targets. Among the 30 potential target genes reserved between human, mice and troglodyte, are ABCG2, ID 1(Inhibitor of DNA binding 1), SRP19 and SMAD6, regulatory proteins related to signal transduction, transcription and maintaining of stem cell function.Potential targets of miR-129,EIF2C3 and CAMTA1, and potential targets of miR-520h, ABCG2 and SMAD6 were selected for verification. Using luciferase reporter vector, pMIR-REPORT, we constructed four reconstitute vectors: pMIR-Luc-miR129-EIF2C3, pMIR-Luc-miR129-CAMTA1, pMIR-Luc-miR520h-ABCG2 and pMIR-Luc-miR520h-SMAD.The results of target verification experiments demonstrated that three control groups had similar Luc/β-gal values. In the test groups,①As for miR-129, luciferase activity for EIF2C3 and CAMTA1 decreased 41% and 47%, respectively, indicating that EIF2C3 and CAMTA1 are real targets for miR-129.②As for miR-520h, luciferase activity for ABCG2 and SMAD6 decreased 68% and 27%, respectively, indicating that ABCG2 is real target for miR-520h, and miR-520h is relatively weak at inhibiting SMAD6.5. Research on the role of miR-520h in promoting HSCs developmentCFC assays: In the three control groups, the numbers of different progenitor colonies and the total number of progenitor colonies are similar between the groups. In the no-miRNA control group, numbers of CFU-E, BFU-E, CFU-GM and CFU-GEMM are 28.50±4.32, 37.83±5.60, 57.67±5.16 and 1.50±0.55, respectively. And the total number of progenitor colonies is 145.50±14.35. In the test group, numbers of CFU-E, BFU-E, CFU-GM and CFU-GEMM are37.83±7.70, 46.5±4.85, 68.17±7.73 and 1.67±0.52, respectively. And the total number of progenitor colonies is174.33±17.82.Analysis of variance showed that the numbers of CFU-E, BFU-E, CFU-GM and the total number of progenitor colonies between the test group and the control group have significant difference(P<0.05,n=6).FACS analysis: In the three control groups, the proportion of CD34+ subpopulation and that of CD34+CD38- subpopulation are similar between the groups. In the no-miRNA control group, the proportion of CD34+ subpopulation and that of CD34+CD38- subpopulation are 22.91%±2.03 and 12.57%±0.81,respectively. In the test group, the proportion of CD34+ subpopulation and that of CD34+CD38- subpopulation are 30.43%±2.34 and 13.08%±1.08,respectively. Analysis of variance showed that in the test group, the proportion of CD34+ subpopulation was significantly higher (P<0.05,n=6). However, the proportion of CD34+CD38- subpopulation was no significance in comparison with the control group(P>0.05,n=6).The results show that miR-520h plays a role in promoting HSCs development and differentiation.Conclusion1. We successfully developed novel mammalian miRNA-specific microarray, indicating the feasiability of screening HSCs-related miRNAs by miRNAs microarray and providing technological platform for research on miRNAs expression profile of other adult stem cells.2. We obtained CD34+CD38- HSCs development-related miRNAs expression profile by miRNAs microarray , and partially verified the results by Q-RT-PCR, providing basis for research on miRNAs'involvement in mechanism of HSCs development and biological function.3. We verified several potential targets of miR-129 and miR-520h, showed that by regulating genes involved in miRNA processing, transcription regulation, signal transduction and stem cell function meitaining, including EIF2C3, AMTA1 and ABCG2, miRNAs play a role in HSCs development, and form a miRNAs gene network, cooperating or rivaling with each other.4. MiR-520h is obviously involved in promoting HSCs development and differentiation. Identification of miRNAs and gene networks specifying either stemness or commitment will not only be of major relevance for miRNAs-targeted strategy for promoting HSCs development, but also for developmental biology and the emerging fields of tissue engineering and regenerative medicine. |
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