miRNAs play important roles in many biological processes, including erythropoiesis. Although several miRNAs regulate erythroid differentiation, how the key erythroid regulator, GATA-1, directly orchestrates differentiation through miRNA pathways remains unclear. In this study, we identified miR-23a as a key regulator of erythropoiesis, which was upregulated both during erythroid differentiation and in GATA-1 gain-of-function experiments, as determined by miRNA expression profile analysis. In primary human CD34+ hematopoietic progenitor cells, miR-23a increased in a GATA-1-dependent manner during erythroid differentiation. Gain- or loss-of-function analysis of miR-23a in mice or zebrafish demonstrated that it was essential for normal morphology in terminally differentiated erythroid cells. Furthermore, a protein tyrosine phosphatase, SHP2, was identified as a downstream target of miR-23a that mediated its regulation of erythropoiesis. Taken together, our data identify a key GATA-1–miRNA axis in erythroid differentiation.

cThe developmental stage-specific expression of the human ␤-like globin genes has been studied for decades, and many transcriptional factors as well as other important cis elements have been identified. However, little is known about the microRNAs that potentially regulate ␤-like globin gene expression directly or indirectly during erythropoiesis. In this study, we show that microRNA 23a (miR-23a) and miR-27a promote ␤-like globin gene expression in K562 cells and primary erythroid cells through targeting of the transcription factors KLF3 and SP1. Intriguingly, miR-23a and miR-27a further enhance the transcription of ␤-like globin genes through repression of KLF3 and SP1 binding to the ␤-like globin gene locus during erythroid differentiation. Moreover, KLF3 can bind to the promoter of the miR-23aϳ27aϳ24-2 cluster and suppress this microRNA cluster expression. Hence, a positive feedback loop comprised of KLF3 and miR-23a promotes the expression of ␤-like globin genes and the miR23aϳ27aϳ24-2 cluster during erythropoiesis.

During early development of the human embryo, chromosomal imbalance and instability may cause spontaneous miscarriages. In this study, we observe aberrant chromosome numbers in nearly half of spontaneous miscarriage embryo samples, most of which show abnormalities in karotype. We also detect significantly reduced expression of two important mitotic checkpoint proteins, Mad2 and Bub1. To further investigate the role of Bub1 and Mad2 in chromosome mis-segregation, in embryogenesis, and in errors leading to spontaneous miscarriages, we used RNA interference technology to knockdown Bub1 and Mad2 genes. We examined the effect of reduced expression of Mad2 and Bub1 on chromosome number, cell proliferation and cell cycle progression. Significant suppression of cell proliferation and increased abnormal chromosome numbers were detected. M phase arrest was observed in cultured villus cell lines with depleted Mad2 or Bub1 mRNA by RNAi technique. The results from the in vitro RNAi-mediated silencing model may provide an explanation for the observations in clinical samples of spontaneous miscarriages. Thus, our findings strongly suggest that the loss of spindle assembly checkpoint proteins, such as Bub1 and Mad2, may cause spontaneous miscarriages.

Bub1 is a critical component of the spindle assembly checkpoint (SAC) and closely linked to cell proliferation and differentiation. We previously found that spontaneous abortion embryos contained a low level of Bub1 protein but normal mRNA level, while the knockdown of Bub1 leads to abnormal numerical chromosomes in embryonic cells. Here, we investigated the mechanism through which governs the post-transcriptional regulation of Bub1 protein expression level. We first conducted bioinformatics analysis and identified eight putative miRNAs that may target Bub1. Luciferase reporter assay confirmed that miR-450a-3p can directly regulate Bub1 by binding to the 3′-untranslated region of Bub1 mRNA. We found that the overexpression of miR-450a-3p in mouse embryonic fibroblast (MEF) cells down-regulated Bub1 protein level, repressed cell proliferation, increased apoptosis and restricted most cells in G1 phase of the cell cycle. Furthermore, when the fertilized eggs were microinjected with miR-450a-3p mimics, the cleavage of zygotes was effectively suppressed. Our results strongly suggest that an abnormally decreased Bub1 level regulated by miRNAs may be implicated in the pathogenesis of spontaneous miscarriage. Therefore, the blockade of miR-450a-3p may be explored as a novel therapeutic strategy for preventing spontaneous miscarriages.

Esophageal cancer is a common malignancy with a high mortality rate. The lack of effective chemotherapy and a means to overcome drug resistance leads to the predictable failure of esophageal cancer treatment. Mitotic checkpoint proteins play a critical role in regulating the cell cycle and proliferation. Abnormal expression of the mitotic checkpoint protein BubR1 has been reported in several types of cancers. In this study, we investigated the role of BubR1 in conferring resistance of esophageal cancer cells to anti-microtubule drugs. Using quantitative real-time PCR analysis on 50 samples of paired esophageal squamous cell cancer (ESC) tissues and adjacent non-cancerous tissues, we found that 72% (36 of 50) of the analyzed ESC samples exhibited high expression levels of BubR1, which was also confirmed in ESC cell lines. ESC cells with high levels of BubR1 were less sensitive to the anti-microtubule drugs paclitaxel and nocodazole. Recombinant adenovirus-mediated enforced expression of BubR1 in relatively sensitive ESC cell lines resulted in increased resistance to paclitaxel. Conversely, RNAi-mediated knockdown of BubR1 restored ESC cell sensitivity to paclitaxel. Cell cycle analysis indicated that the sub-G1 population increased in the ESC cells with reduced BubR1 levels. Taken together, our results suggest that upregulation of BubR1 expression may be associated with ESC resistance to paclitaxel treatment. Thus, BubR1 may serve as a potential chemosensitizing target to overcome chemoresistance.