Boqin Qiang

MicroRNA-21 down-regulates the expression of tumor suppressor PDCD4 in human glioblastoma cell T98G

MicroRNAs have been linked to different cancer-related processes. The microRNA miR-21 appears to function as an anti-apoptosis factor in glioblastomas. However, the functional target genes of miR-21 are largely unknown in glioblastomas. In this study, bioinformatics analysis was used to identify miR-21 target sites in various genes. Luciferase activity assay showed that a number of genes involved in apoptosis, PDCD4, MTAP, and SOX5, carry putative miR-21 binding sites. Expression of PDCD4 protein correlates inversely with expression of miR-21 in a number of human glioblastoma cell lines such as T98G, A172, U87, and U251. Inhibition of miR-21 increases endogenous levels of PDCD4 in cell line T98G and over-expression miR-21 inhibits PDCD4-dependent apoptosis. Together, these results indicate that miR-21 expression plays a key role in regulating cellular processes in glioblastomas and may serve as a target for effective therapies.

MicroRNA-16 targets amyloid precursor protein to potentially modulate Alzheimer's-associated pathogenesis in SAMP8 mice

Alzheimers disease (AD) is a progressive neurodegenerative disorder mainly characterized by amyloid-beta (Aβ) deposition and neurofibrillary tangles (NFTs). The abnormal enrichment of amyloid protein precursor (APP) leads to a high risk of AD. One of the plausible age-associated AD animal models, senescence-accelerated mouse prone 8 (SAMP8), have age-related learning and memory deficits. We found APP protein significantly increased in the hippocampus of aged SAMP8 mice. The 20 to 25 nucleotide (nt) tiny regulators, known as micro ribonucleic acids (miRNAs), have been found to play crucial roles in neurodegenerative diseases. Here, we examined the post-transcriptional regulation mechanism of APP mediated by micro ribonucleic acids and found that miR-16 was one of the post-transcriptional regulators of APP in SAMP8 mice. Overexpression of miR-16, both in vitro and in vivo, led to reduced APP protein expression. Furthermore, miR-16 and APP displayed complementary expression patterns in SAMP8 mice and BALb/c mice embryos. Taken together, these findings demonstrate that APP is a target of miR-16 and the abnormally low expression of miR-16 could potentially lead to APP protein accumulation in AD mice.

cAMP response element-binding protein promotes gliomagenesis by modulating the expression of oncogenic microRNA-23a

Gliomas are the most common and deadly type of primary brain tumor. In this study, we showed that cAMP response element-binding protein (CREB), a proto-oncogenic transcription factor that is overexpressed in gliomas, can promote gliomagenesis by modulating the expression of oncogenic microRNA-23a (mir-23a). First, we found that CREB is highly expressed in glioma tissues and cell lines. CREB is also essential for glioma cell growth and cell survival in vitro and is critical for gliomagenesis in vivo. Second, microRNA microarray, ChIP-chip, ChIP-quantitative PCR, and luciferase reporter assays showed that CREB directly binds to the regulatory sequences of mir-23a and enhance the expression of mir-23a. Moreover, mir-23a was confirmed as a functional downstream target of CREB in glioma cell growth and cell survival. Finally, using computational prediction followed by experimental confirmation, we identified PTEN, which is frequently silenced in gliomas, as a downstream target of mir-23a. Taken together, we propose that CREB promotes gliomagenesis and acts as a modulator of oncogenic mir-23a, which represses the tumor suppressor PTEN.

Disruption of Nectin-Like 1 Cell Adhesion Molecule Leads to Delayed Axonal Myelination in the CNS

Nectin-like 1 (Necl-1) is a neural-specific cell adhesion molecule that is expressed in both the CNS and PNS. Previous in vitro studies suggested that Necl-1 expression is essential for the axon-glial interaction and myelin sheath formation in the PNS. To investigate the in vivo role of Necl-1 in axonal myelination of the developing nervous system, we generated the Necl-1 mutant mice by replacing axons 2–5 with the LacZ reporter gene. Expression studies revealed that Necl-1 is exclusively expressed by neurons in the CNS. Disruption of Necl-1 resulted in developmental delay of axonal myelination in the optic nerve and spinal cord, suggesting that Necl-1 plays an important role in the initial axon-oligodendrocyte recognition and adhesion in CNS myelination.

RNA-binding protein PCBP2 modulates glioma growth by regulating FHL3

PCBP2 is a member of the poly(C)-binding protein (PCBP) family, which plays an important role in posttranscriptional and translational regulation by interacting with single-stranded poly(C) motifs in target mRNAs. Several PCBP family members have been reported to be involved in human malignancies. Here, we show that PCBP2 is upregulated in human glioma tissues and cell lines. Knockdown of PCBP2 inhibited glioma growth in vitro and in vivo through inhibition of cell-cycle progression and induction of caspase-3-mediated apoptosis. Thirty-five mRNAs were identified as putative PCBP2 targets/interactors using RIP-ChIP protein-RNA interaction arrays in a human glioma cell line, T98G. Four-and-a-half LIM domain 3 (FHL3) mRNA was downregulated in human gliomas and was identified as a PCBP2 target. Knockdown of PCBP2 enhanced the expression of FHL3 by stabilizing its mRNA. Overexpression of FHL3 attenuated cell growth and induced apoptosis. This study establishes a link between PCBP2 and FHL3 proteins and identifies a new pathway for regulating glioma progression.

The CREB-miR-9 Negative Feedback Minicircuitry Coordinates the Migration and Proliferation of Glioma Cells

Migration-proliferation dichotomy is a common mechanism in gliomagenesis; however, an understanding of the exact molecular mechanism of this “go or grow” phenomenon remains largely incomplete. In the present study, we first found that microRNA-9 (miR-9) is highly expressed in glioma cells. MiR-9 inhibited the proliferation and promoted the migration of glioma cells by directly targeting cyclic AMP response element-binding protein (CREB) and neurofibromin 1 (NF1), respectively. Our data also suggested a migration-inhibitory role for CREB through directly regulating the transcription of NF1. Furthermore, we found that the transcription of miR-9-1 is under CREBs control, forming a negative feedback minicircuitry. Taken together, miR-9 inhibits proliferation but promotes migration, whereas CREB plays a pro-proliferative and anti-migratory role, suggesting that the CREB-miR-9 negative feedback minicircuitry plays a critical role in the determination of “go or grow” in glioma cells.

D-Amino acids and D-Tyr-tRNATyr deacylase: stereospecificity of the translation machine revisited

Until 30 years ago, it had been considered that D‐amino acids were excluded from living systems except for D‐amino acids in the cell wall of microorganisms. However, D‐amino acids, in the form of free amino acids, peptides and proteins, were recently detected in various living organisms from bacteria to mammals. The extensive distribution of bio‐functional D‐amino acids challenges the current concept of protein synthesis: more attention should be paid to the stereospecificity of the translation machine. Besides aminoacyl‐tRNA synthetases, elongation factor Tu and some other mechanisms, D‐Tyr‐tRNATyr deacylases provide a novel checkpoint since they specifically recycle misaminoacylated D‐Tyr‐tRNATyr and some other D‐aminoacyl‐tRNAs. Their unique structure represents a new class of tRNA‐dependent hydrolase. These unexpected findings have far‐reaching implications for our understanding of protein synthesis and its origin.

Circadian gene Clock contributes to cell proliferation and migration of glioma and is directly regulated by tumor-suppressive miR-124

Although the roles of circadian Clock genes and microRNAs in tumorigenesis have been profoundly studied, mechanisms of cross‐talk between them in regulation of gliomagenesis are poorly understood. Here we show that the expression level of CLOCK is significantly increased in high‐grade human glioma tissues and glioblastoma cell lines. In contrast miR‐124 is attenuated in similar samples. Further studies show that Clock is a direct target of miR‐124, and either restoration of miR‐124 or silencing of CLOCK can reduce the activation of NF‐κB. In conclusion, we suggest that as a target of glioma suppressor miR‐124, CLOCK positively regulates glioma proliferation and migration by reinforcing NF‐κB activity.

Loss of NECL1, a novel tumor suppressor, can be restored in glioma by HDAC inhibitor-Trichostatin A through Sp1 binding site

Nectin‐like molecule 1 (NECL1)/CADM3/IGSF4B/TSLL1/SynCAM3 is a neural tissue‐specific immunoglobulin‐like cell–cell adhesion molecule downregulated at the mRNA level in 12 human glioma cell lines. Here we found that the expression of NECL1 was lost in six glioma cell lines and 15 primary glioma tissues at both RNA and protein levels. Re‐expression of NECL1 into glioma cell line U251 would repress cell proliferation in vitro by inducing cell cycle arrest. And also NECL1 could decrease the growth rate of tumors in nude mice in vivo. To further investigate the mechanism why NECL1 was silenced in glioma, the basic promoter region located at −271 to +81 in NECL1 genomic sequence was determined. DNA bisulfite sequencing was performed to study the methylation status of CpG islands in NECL1 promoter; however, no hypermethylated CpG site was found. Additionally, the activity of histone deacetylase (HDACs) in glioma was higher than that in normal brain tissues, and the expression of NECL1 in glioma cell lines could be reactivated by HDACs inhibitor‐Trichostatin A (TSA). So the loss of NECL1 in glioma was at least partly caused by histone deacetylation. Luciferase reporter assays, chromatin immunoprecipitation and co‐immunoprecipitation (co‐IP) assays indicated that Sp1 played an important role in this process by binding to either HDAC1 in untreated glioma cells or p300/CBP in TSA treated cells. Our finding suggests that NECL1 may act as a tumor suppressor in glioma and loss of it in glioma may be caused by histone deacetylation.

SARS-CoV nucleocapsid protein binds to hUbc9, a ubiquitin conjugating enzyme of the sumoylation system

SARS‐CoV is a newly identified coronavirus (CoV) that causes severe acute respiratory syndrome (SARS). The SARS‐CoV nucleocapsid (N) protein is an important structural and functional protein. To identify cellular proteins that interact with the SARS‐CoV N protein and to elucidate the possible involvement of N protein in SARS‐CoV pathogenesis, a human lymphocyte cDNA library was screened using a yeast two‐hybrid system assay. hUbc9, a ubiquitin conjugating enzyme of sumoylation system, was found to interact specifically with the N protein, implying the post‐translational sumoylation of the N protein. Mapping studies localized the critical N sequences for this interaction to amino acids 170–210, which includes the SR‐rich motif. However, the consensus motif of sumoylation GK62EE in the N protein is not responsible for binding to hUbc9. Mutations of hUbc9 at the enzyme active site C93A or C93S severely impair the interaction with the N protein. The two proteins were also shown to colocalize in the cytoplasm of the transfected 293T cells. This is the first report demonstrating the interaction of hUbc9 with a structural protein of plus‐strand RNA viruses, indicating a new drug target for SARS‐CoV. J. Med. Virol. 78:1365–1373, 2006.