Donghai Li

Platelet-Secreted MicroRNA-223 Promotes Endothelial Cell Apoptosis Induced by Advanced Glycation End Products via Targeting the Insulin-like Growth Factor 1 Receptor

Platelets play a significant role in atherosclerosis, stroke, and asthma through active interaction with neutrophils, monocytes, and vascular endothelial cells. The mechanism underlying these intercellular interactions, however, is incompletely understood. In this study, we report that platelets can remotely modulate vascular endothelial cell apoptosis through releasing microRNA-223 (miR-223)–containing microvesicles (MVs). First, platelets expressed abundant miRNAs, and miR-223 had the highest level of expression. Platelet miR-223 and other miRNAs can be upregulated by the stimulation with thrombopoietin (TPO) or thrombin. Unlike leukocytes, platelets contained high levels of pre-miRNAs, and upregulation of mature platelet miRNAs by TPO was correlated with decreased pre-miRNAs. Second, under stimulation with TPO, platelets released a large amount of MVs, which also contain higher levels of miR-223. Elevation of miR-223 inside circulating platelet MVs (P-MVs) was also observed in plasma samples from patients with enteritis, hepatitis, nephritis, or atherosclerosis. Third, incubation of P-MVs with HUVECs, which had significantly lower levels of miR-223 than platelets, showed that P-MVs effectively delivered miR-223 into HUVECs. Finally, in HUVECs, exogenous platelet miR-223 decreased the level of insulin-like growth factor 1 receptor and thus promoted HUVEC apoptosis induced by advanced glycation end products. The proapoptotic effect of P-MVs on HUVECs was largely abolished by depleting cellular miR-223 using anti–miR-223 antisense oligonucleotide. In conclusion, our study presents the first evidence, to our knowledge, that platelet-released miR-223 promotes advanced glycation end product–induced vascular endothelial cell apoptosis via targeting insulin-like growth factor 1 receptor.

miR-203 Suppresses the Proliferation and Migration and Promotes the Apoptosis of Lung Cancer Cells by Targeting SRC

SRC, also known as proto-oncogene c-Src, is a non-receptor tyrosine kinase that plays an important role in cancer progression by promoting survival, angiogenesis, proliferation, and invasion pathways. In this study, we found that SRC protein levels were consistently upregulated in lung cancer tissues, but that SRC mRNA levels varied randomly, suggesting that a post-transcriptional mechanism was involved in SRC regulation. Because microRNAs (miRNAs) are powerful post-transcriptional regulators of gene expression, we used bioinformatic analyses to search for miRNAs that potentially target SRC. We identified specific targeting sites for miR-203 in the 3′-untranslated region (3′-UTR) of SRC. We then experimentally validated miR-203 as a direct regulator of SRC using cell transfection and luciferase assays and showed that miR-203 inhibited SRC expression and consequently triggered suppression of the SRC/Ras/ERK pathway. Finally, we demonstrated that the repression of SRC by miR-203 suppressed the proliferation and migration and promoted the apoptosis of lung cancer cells. In summary, this study provides the first clues regarding the role of miR-203 as a tumor suppressor in lung cancer cells through the inhibition of SRC translation.

Investigation of MicroRNA Expression in Human Serum During the Aging Process

BACKGROUND Although serum microRNAs (miRNAs) play essential roles in the diagnosis of various diseases, little is known about circulating miRNAs in the aging process. METHODS Solexa sequencing technology was used for an initial miRNA screening of serum samples pooled from 21 healthy Chinese subjects with an average age of 22 years, 10 subjects with an average age of 40 years, 10 subjects with an average age of 59 years, and 9 subjects with an average age of 70 years. Other serum samples were obtained from 123 normal people with approximately 31 samples in each age period. A stem-loop quantitative reverse transcription-PCR assay was conducted to confirm the concentrations of the miRNAs altered in the aging process. RESULTS Solexa sequencing demonstrated 10 markedly altered miRNAs in the aging process. Quantitative reverse transcription-PCR analysis identified five downregulated miRNAs (miR-29b, miR-106b, miR-130b, miR-142-5p, and miR-340) and three upregulated miRNAs (miR-92a, miR-222, and miR-375) with age. Their target genes, related diseases, molecular and cellular functions, and participated pathways were further analyzed. CONCLUSIONS The measurement of miRNAs in serum provides a novel, noninvasive approach for the identification of the aging process. Our bioinformatic analyses could form a useful knowledge base for the potential future development of novel therapeutic treatments.

Catalytic Mechanism Investigation of Lysine-Specific Demethylase 1 (LSD1): A Computational Study

Lysine-specific demethylase 1 (LSD1), the first identified histone demethylase, is a flavin-dependent amine oxidase which specifically demethylates mono- or dimethylated H3K4 and H3K9 via a redox process. It participates in a broad spectrum of biological processes and is of high importance in cell proliferation, adipogenesis, spermatogenesis, chromosome segregation and embryonic development. To date, as a potential drug target for discovering anti-tumor drugs, the medical significance of LSD1 has been greatly appreciated. However, the catalytic mechanism for the rate-limiting reductive half-reaction in demethylation remains controversial. By employing a combined computational approach including molecular modeling, molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations, the catalytic mechanism of dimethylated H3K4 demethylation by LSD1 was characterized in details. The three-dimensional (3D) model of the complex was composed of LSD1, CoREST, and histone substrate. A 30-ns MD simulation of the model highlights the pivotal role of the conserved Tyr761 and lysine-water-flavin motif in properly orienting flavin adenine dinucleotide (FAD) with respect to substrate. The synergy of the two factors effectively stabilizes the catalytic environment and facilitated the demethylation reaction. On the basis of the reasonable consistence between simulation results and available mutagenesis data, QM/MM strategy was further employed to probe the catalytic mechanism of the reductive half-reaction in demethylation. The characteristics of the demethylation pathway determined by the potential energy surface and charge distribution analysis indicates that this reaction belongs to the direct hydride transfer mechanism. Our study provides insights into the LSD1 mechanism of reductive half-reaction in demethylation and has important implications for the discovery of regulators against LSD1 enzymes.

Small Molecule Inhibitor of Myogenic microRNAs Leads to a Discovery of miR-221/222-myoD-myomiRs Regulatory Pathway

Myogenic microRNAs (myomiRs) that are specifically expressed in cardiac and skeletal muscle are highly relevant to myogenic development and diseases. Discovery and elucidation of unknown myomiRs-involved regulatory pathways in muscle cells are important, but challenging due to the lack of proper molecular tools. We report here a miR-221/222-myoD-myomiRs regulatory pathway revealed by using a small-molecule probe that selectively inhibits myomiRs including miR-1, miR-133a, and miR-206. The small-molecule inhibitor screened from luciferase assay systems was found to inhibit myomiRs and differentiation of C2C12 cells. Using the small molecule as a probe, we found that the transcriptional factor myoD, which is upstream of myomiRs, was further regulated by miR-221/222. This miR-221/222-myoD-myomiRs regulatory pathway was confirmed by over-expressing or knockdown miR-221/222 in muscle cells, which respectively led to the inhibition or enhancement of myoD protein expression and subsequent downregulation or upregulation of myomiR expression.

Helix unfolding/refolding characterizes the functional dynamics of Staphylococcus aureus CLP protease

Background: The molecular mechanism of ClpP dynamic switching between different conformations is poorly understood. Results: MD simulations describe the molecular pathway of the transition between three conformations of SaClpP. Conclusion: Helix unfolding/refolding characterizes the functional dynamics and mechanism of ClpP. Significance: This study provides molecular insights into the dynamics and mechanism of ClpP in general. The ATP-dependent Clp protease (ClpP) plays an essential role not only in the control of protein quality but also in the regulation of bacterial pathogen virulence, making it an attractive target for antibacterial treatment. We have previously determined the crystal structures of Staphylococcus aureus ClpP (SaClpP) in two different states, extended and compressed. To investigate the dynamic switching of ClpP between these states, we performed a series of molecular dynamics simulations. During the structural transition, the long and straight helix E in the extended SaClpP monomer underwent an unfolding/refolding process, resulting in a kinked helix very similar to that in the compressed monomer. As a stable intermediate in the molecular dynamics simulation, the compact state was suggested and subsequently identified in x-ray crystallographic experiment. Our combined studies also determined that Ala140 acted as a “hinge” during the transition between the extended and compressed states, and Glu137 was essential for stabilizing the compressed state. Overall, this study provides molecular insights into the dynamics and mechanism of the functional conformation changes of SaClpP. Given the highly conserved sequences of ClpP proteins among different species, these findings potentially reflect a switching mechanism for the dynamic process shared in the whole ClpP family in general and thus aid in better understand the principles of Clp protease assembly and function.

Salmonella enterica serovar enteritidis modulates intestinal epithelial miR-128 levels to decrease macrophage recruitment via macrophage colony-stimulating factor.

BACKGROUND The mechanism underlying the ability of virulent Salmonella organisms to escape clearance by macrophages is incompletely understood. Here, we report a novel mechanism by which Salmonella escapes macrophages. METHODS Microarray and quantitative real-time polymerase chain reaction analyses were used to screen key microRNAs regulating Salmonella-host cell interactions. Target gene was tested using luciferase reporter and Western blot assays. The role of microRNA 128 (miR-128) was assayed using intestinal epithelial cells and a mouse infection model. RESULTS The miR-128 level in human intestinal epithelial HT29 cells was strongly increased by infection with strain SE2472, and the elevation in miR-128 levels in mouse intestine and colon tissues correlated with the level of Salmonella infection in mice. Macrophage colony-stimulating factor (M-CSF) was identified as a target of miR-128, and increased miR-128 levels in epithelial cells due to infection with strain SE2472 significantly decreased the level of cell-secreted M-CSF, leading to impaired M-CSF-mediated macrophage recruitment. The secreted proteins from Salmonella were identified as possible effectors to induce miR-128 expression via the p53 signaling pathway. Moreover, intragastric delivery of anti-miR-128 antagomir into mice significantly increased M-CSF-mediated macrophage recruitment and suppressed Salmonella infection. CONCLUSIONS Salmonella can upregulate intestinal epithelial miR-128 expression, which, in turn, decreases levels of epithelial cell-secreted M-CSF and M-CSF-induced macrophage recruitment.

Major vault protein regulates class A scavenger receptor-mediated tumor necrosis factor-α synthesis and apoptosis in macrophages.

Background: Class A scavenger receptor (SR-A) influences the synthesis of pro-inflammatory mediators and apoptosis in macrophages. Results: Major vault protein (MVP) interacts with SR-A and modulates SR-A-caveolin-p38/JNK-mediated TNF-α production and apoptosis in macrophages. Conclusion: MVP may fine-tune SR-A activity in macrophages contributing to atherogenesis. Significance: Targeting MVP-SR-A complex in macrophages may yield viable solutions for the intervention of atherosclerosis. Atherosclerosis is considered a disease of chronic inflammation largely initiated and perpetuated by macrophage-dependent synthesis and release of pro-inflammatory mediators. Class A scavenger receptor (SR-A) expressed on macrophages plays a key role in this process. However, how SR-A-mediated pro-inflammatory response is modulated in macrophages remains ill defined. Here through immunoprecipitation coupled with mass spectrometry, we reported major vault protein (MVP) as a novel binding partner for SR-A. The interaction between SR-A and MVP was confirmed by immunofluorescence staining and chemical cross-linking assay. Treatment of macrophages with fucoidan, a SR-A ligand, led to a marked increase in TNF-α production, which was attenuated by MVP depletion. Further analysis revealed that SR-A stimulated TNF-α synthesis in macrophages via the caveolin- instead of clathrin-mediated endocytic pathway linked to p38 and JNK, but not ERK, signaling pathways. Importantly, fucoidan invoked an enrichment of MVP in lipid raft, a caveolin-reliant membrane structure, and enhanced the interaction among SR-A, caveolin, and MVP. Finally, we demonstrated that MVP elimination ameliorated SR-A-mediated apoptosis in macrophages. As such, MVP may fine-tune SR-A activity in macrophages which contributes to the development of atherosclerosis.

MicroRNA-196a/b Mitigate Renal Fibrosis by Targeting TGF-β Receptor 2

Organ-specific microRNAs have essential roles in maintaining normal organ function. However, the microRNA profile of the kidney and the role of microRNAs in modulating renal function remain undefined. We performed an unbiased assessment of the genome-wide microRNA expression profile in 14 mouse organs using Solexa deep sequencing and found that microRNA-196a (miR-196a) and miR-196b are selectively expressed in kidney, with 74.37% of mouse total miR-196a and 73.19% of mouse total miR-196b distributed in the kidneys. We confirmed the predominant expression of miR-196a/b in mouse and human kidney, particularly in the glomeruli and tubular epithelium, by quantitative RT-PCR and in situ hybridization assays. During unilateral ureteral obstruction (UUO)-induced mouse renal fibrosis, renal miR-196a/b levels rapidly decreased. Elevation of renal miR-196a/b expression by hydrodynamic-based delivery of a miR-196a/b-expressing plasmid before or shortly after UUO significantly downregulated profibrotic proteins, including collagen 1 and α-smooth muscle actin, and mitigated UUO-induced renal fibrosis. In contrast, depletion of renal miR-196a/b by miR-196a/b antagomirs substantially aggravated UUO-induced renal fibrosis. Mechanistic studies further identified transforming growth factor beta receptor II (TGFβR2) as a common target of miR-196a and miR-196b. Decreasing miR-196a/b expression in human HK2 cells strongly activated TGF-β-Smad signaling and cell fibrosis; whereas increasing miR-196a/b levels in mouse primary cultured tubular epithelial cells inhibited TGF-β-Smad signaling. In the UUO model, miR-196a/b silenced TGF-β-Smad signaling, decreased the expression of collagen 1 and α-smooth muscle actin, and attenuated renal fibrosis. Our findings suggest that elevating renal miR-196a/b levels may be a novel therapeutic strategy for treating renal fibrosis.

Human Cytomegalovirus miR-UL148D Facilitates Latent Viral Infection by Targeting Host Cell Immediate Early Response Gene 5

The mechanisms underlying human cytomegalovirus (HCMV) latency remain incompletely understood. Here, we showed that a HCMV-encoded miRNA, miR-UL148D, robustly accumulates during late stages of experimental latent HCMV infection in host cells and promotes HCMV latency by modulating the immediate early response gene 5 (IER5)-cell division cycle 25B (CDC25B) axis in host cells. miR-UL148D inhibited IER5 expression by directly targeting the three-prime untranslated region(3’UTR) of IER5 mRNA and thus rescued CDC25B expression during the establishment of viral latency. Infection with NR-1ΔmiR-UL148D, a derivative of the HCMV clinical strain NR-1 with a miR-UL148D knockout mutation, resulted in sustained induction of IER5 expression but decreased CDC25B expression in host cells. Mechanistically, we further showed that CDC25B plays an important role in suppressing HCMV IE1 and lytic gene transcription by activating cyclin-dependent kinase 1 (CDK-1). Both gain-of-function and lose-of-function assays demonstrated that miR-UL148D promotes HCMV latency by helping maintain CDC25B activity in host cells. These results provide a novel mechanism through which a HCMV miRNA regulates viral latency.