Biomedical subjects

Shi-Lung Lin

Publications and source records attributed to Shi-Lung Lin.

At least 19 recordsLinked to original sources

PubMed2006

Hyaluronan stimulates transformation of androgen-independent prostate cancer.

Interaction between extracellular matrices and cancer cell receptors frequently alters signal transduction pathways, leading to malignant transformation and metastasis. Hyaluronan (HA) is a tumor promoter and enhancer in transformation of androgen-independent (AI) prostate cancer (CaP); however, the signal transduction pathway involved in this mechanism remains unclear. We report here that HA-mediated CD168, a receptor for HA-mediated motility, and its downstream signal molecules, including ROK1, Gab-1, PI3K*p110alpha and eIF4E3, accelerate the progression of AI rather than androgen-dependent CaP and enhance AI cell invasion and metastasis in human bone marrow endothelial layers. MicroRNA-based small hairpin RNA-mediated suppression of ROK1 can reverse the malignant role of CD168 signaling in human AI CaP PC3 and DU145 cells. This differential activation of ROK-PI3K signaling in AI CaP cells may provide clues to shed light on some mechanisms of cancer relapse after androgen ablation. These findings reveal a novel signal transduction mechanism for matrix-mediated cancer transformation and metastasis in hormone-refractory CaP.

Androgens↗

PubMed2006

Intronic microRNA (miRNA).

Nearly 97% of the human genome is composed of noncoding DNA, which varies from one species to another. Changes in these sequences often manifest themselves in clinical and circumstantial malfunction. Numerous genes in these non-protein-coding regions encode microRNAs, which are responsible for RNA-mediated gene silencing through RNA interference (RNAi)-like pathways. MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) with complete or partial complementarity, are useful for the design of new therapies against cancer polymorphisms and viral mutations. Currently, many varieties of miRNA are widely reported in plants, animals, and even microbes. Intron-derived microRNA (Id-miRNA) is a new class of miRNA derived from the processing of gene introns. The intronic miRNA requires type-II RNA polymerases (Pol-II) and spliceosomal components for their biogenesis. Several kinds of Id-miRNA have been identified in C elegans, mouse, and human cells; however, neither function nor application has been reported. Here, we show for the first time that intron-derived miRNAs are able to induce RNA interference in not only human and mouse cells, but in also zebrafish, chicken embryos, and adult mice, demonstrating the evolutionary preservation of intron-mediated gene silencing via functional miRNA in cell and in vivo. These findings suggest an intracellular miRNA-mediated gene regulatory system, fine-tuning the degradation of protein-coding messenger RNAs.

Journal Article↗

PubMed2006

Activin signaling and its role in regulation of cell proliferation, apoptosis, and carcinogenesis.

Activins, cytokine members of the transforming growth factor-beta superfamily, have various effects on many physiological processes, including cell proliferation, cell death, metabolism, homeostasis, differentiation, immune responses endocrine function, etc. Activins interact with two structurally related serine/threonine kinase receptors, type I and type II, and initiate downstream signaling via Smads to regulate gene expression. Understanding how activin signaling is controlled extracellularly and intracellularly would not only lead to more complete understanding of cell growth and apoptosis, but would also provide the basis for therapeutic strategies to treat cancer and other related diseases. This review focuses on the recent progress on activin-receptor interactions, regulations of activin signaling by ligand-binding proteins, receptor-binding proteins, and nucleocytoplasmic shuttling of Smad proteins.

Activin Receptors↗

PubMed2006

The microRNA: overview of the RNA gene that modulates gene functions.

MicroRNAs (miRNAs), widely distributed, small regulatory RNA genes, target both messenger RNA (mRNA) degradation and suppression of protein translation based on sequence complementarity between the miRNA and its targeted mRNA. Different names have been used to describe various types of miRNA. During evolution, RNA retroviruses or transgenes invaded the eukaryotic genome and inserted itself in the noncoding regions of DNA, conceivably acting as transposon-like jumping genes, providing defense from viral invasion and fine-tuning of gene expression as a secondary level of gene modulation in eukaryotes. When a transposon is inserted in the intron, it becomes an intronic miRNA, taking advantage of the protein synthesis machinery, i.e., mRNA transcription and splicing, as a means for processing and maturation. Recently, miRNAs have been found to play an important, but not life-threatening, role in embryonic development. They might play a pivotal role in diverse biological systems in various organisms, facilitating a quick response and accurate plotting of body physiology and structures. Based on these unique properties, manufactured intronic miRNAs have been developed for in vitro evaluation of gene function, in vivo gene therapy, and generation of transgenic animal models. The biogenesis and identification of miRNAs, potential applications, and future directions for research are presented in this chapter, hopefully providing a guideline for further miRNA and gene function studies.

Animals↗

PubMed2006

Gene silencing in vitro and in vivo using intronic microRNAs.

MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. Numerous miRNAs have been reported to induce RNA interference (RNAi), a posttranscriptional gene-silencing mechanism. Recent evidence also indicates that they are involved in the transcriptional regulation of genome activities. They were first discovered in Caenorhabditis elegans as native RNA fragments that modulate a wide range of genetic regulatory pathways during embryonic development, and are now recognized as small gene silencers transcribed from the noncoding regions of a genome. In humans, nearly 97% of the genome is noncoding DNA, which varies from one individual to another, and changes in these sequences are frequently noted to manifest clinical and circumstantial malfunction. Type 2 myotonic dystrophy and fragile X syndrome were found to be associated with miRNAs derived from introns. Intronic miRNA is a new class of miRNAs derived from the processing of nonproteincoding regions of gene transcripts. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of RNA polymerase (Pol)-II and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in C. elegans, mouse, and human cells; however, neither function nor application has been reported. Here, we show for the first time that intron-derived miRNA is not only able to induce RNAi in mammalian cells but also in fish, chicken embryos, and adult mice, demonstrating the evolutionary preservation of this gene regulation system in vivo. These miRNA-mediated animal models provide artificial means to reproduce the mechanisms of miRNA-induced disease in vivo and will shed further light on miRNA-related therapies.

Animals↗

PubMed2006

Isolation and identification of gene-specific microRNAs.

Prediction of microRNA (miRNA) candidates using computer programming has identified hundreds and hundreds of genomic hairpin sequences, of which, the functions remain to be determined. Because direct transfection of hairpin-like miRNA precursors (pre)-miRNAs in mammalian cells is not always sufficient to trigger effective RNA-induced gene-silencing complex (RISC) assembly, a key step for RNA interference (RNAi)-related gene silencing, we developed an intronic miRNA-expressing system to overcome this problem, and successfully increased the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. By insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, this intronic miRNA biogenesis system has been found to depend on a coupled interaction of nascent precursor messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA was transcribed by RNA type II polymerases, coexpressed with a primary gene transcript, and excised out of its encoding gene transcript by intracellular RNA splicing and processing mechanisms. Currently, some ribonuclease III endonucleases have been found to be involved in the processing of spliced introns and probably facilitating the intronic miRNA maturation. Using this miRNA-expressing system, we have shown for the first time that the intron-derived miRNAs were able to induce strong RNAi effects in not only human and mouse cells but also zebrafish, chicken embryos, and adult mice. Based on the strand complementarity between the designed miRNA and its target gene sequence, we have also developed a miRNA isolation protocol to purify and identify the mature miRNAs generated by the intronic miRNA-expressing system. Several intronic miRNA identities and structures are currently confirmed to be active in vitro and in vivo. According to this proof- of-principle method, we now have the knowledge to design pre-miRNA inserts that are more efficient and effective for the intronic miRNA-expressing system.

Animals↗

PubMed2006

Transgene-like animal models using intronic microRNAs.

Transgenic animal models are valuable tools for testing gene functions and drug mechanisms in vivo. They are also the best similitude of a human body for etiological and pathological research of diseases. All pharmaceutically developed drugs must be proven safe and effective in animals before approval by the Food and Drug Administration to be used in clinical trials. To this end, the transgenic animal models of human diseases serve as a front line for drug evaluation. However, there is currently no transgenic animal model for microRNA (miRNA) research. miRNAs, small single-stranded regulatory RNAs capable of silencing intracellular gene transcripts that contain either complete or partial complementarity to the miRNAs, are useful for the design and development of new therapies against cancer polymorphism and viral mutation. Recently, varieties of natural miRNAs have been found to be derived from hairpin-like RNA precursors in almost all eukaryotes, including yeast (Schizosaccharomyces pombe), plant (Arabidopsis), nematode (Caenorhabditis elegans), fly (Drosophila melanogaster), fish, mouse, and human, involving intracellular defense against viral infections and regulation of certain gene expressions during development. To facilitate the miRNA research in vivo, we have developed a state-of-the-art transgenic strategy for silencing specific genes in zebrafish, chicken, and mouse, using intronic miRNAs. By insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, we have found that mature miRNAs were successfully transcribed by RNA polymerase (Pol)-II, coexpressed with the encoding gene transcript, and excised out of the encoding gene transcript by natural RNA splicing and processing mechanisms. In conjunction with retroviral transfection systems, the hairpin-like pre-miRNA construct was further inserted into the intron of a cellular gene for tissue-specific expression regulated by the gene promoter. Because the retroviral vectors were randomly integrated into the genome of its host cell, the most effective transgenic animal can be selected and propagated to be a stable transgenic line for future research. Here, we have shown for the first time that transgene-like animal models were generated using the intronic miRNA-expressing system described previously, which has been proven to be useful for both miRNA research and in vivo evaluation of miRNA-associated target genes.

Animals↗

PubMed2006

MicroRNA protocols. Perspectives.

The discoveries of microRNAs (miRNAs), a class of noncoding RNAs that can regulate gene expression by translational repression, have opened a new avenue on gene modulation in mammalian cells. Conceivably, this highly effective method of modulation of gene expression would be exploited for numerous prospectives, including human, therapeutics. This chapter offers some perspective, with emphasis on areas that can be further developed.

Animals↗

PubMed2005

Transcriptional control of Shh/Ptc1 signaling in embryonic development.

In vivo profiling of signal-directed gene expression patterns is a major bottleneck in studying developmental biology. A signal molecule initiates its specific gene expression pattern through the activation of certain transcription factor (TF); however, tissue heterogeneity often masks this pattern due to intercellular complexity of other signal transduction pathways. To decipher the synergistic regulation of signal-directed gene expression in the tissue level, we report here a unique transcriptional responsive element (TRE) existing in the 5'-upstream promoter regions (5'-UPR) of the genes responding to the Shh/Ptc1 signal transduction pathway during feather placode development in chicken embryos. By locating the TRE homologue and its interactive TF, we were able to reveal the gene expression pattern of the Shh/Ptc1 signaling. We firstly demonstrated that homology profiling of the 5'-UPR of the genes, Gli1, TGF-beta2 and Msx2, responding to the Shh/Ptc1 signaling showed a more than 70% conserved region. Computer alignment of the consensus sequences in the conserved region revealed a 37-nucleotide TRE sequence, containing two regulatory elements homologous to human and mouse Gli-binding sites. Activation of this newly identified Shh/Ptc1-responsive TRE by active Smo signaling in chicken hepatoepithelial carcinoma cells elicited a strong synergistic expression of the Shh/Ptc1-downstream genes. Based on previous bioinformatics and the present experimental findings, we successfully established an in vivo signaling model for the Shh/Ptc1-directed embryonic feather morphogenesis.

Amino Acid Sequence↗

PubMed2005

Current perspectives in intronic micro RNAs (miRNAs).

MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. Numerous miRNAs have been reported to induce RNA interference (RNAi), a post-transcriptional gene silencing mechanism. Intronic miRNAs, derived from introns by RNA splicing and Dicer processing, can interfere with intracellular mRNAs to silence that gene expression. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of type II RNA polymerases (Pol-II) and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in Caenorhabditis elegans, mouse and human cells; however, neither their function nor application has been reported. To this day, the computer searching program for miRNA seldom include the intronic portion of protein-coding RNAs. The functional significance of artificially generated intronic miRNAs has been successfully ascertained in several biological systems such as zebrafishes, chicken embryos and adult mice, indicating the evolutionary preservation of this gene regulation system in vivo. Multiple miRNAs can be generated from the same cluster of introns; however, non-homologous miRNAs may have different targets and functions while homologous miRNA may be derived from different intronic clusters. Taken together, the model of intronic miRNA-mediated transgenic animals provides a tool to investigate the mechanism of miRNA-associated diseases in vivo and will shed light on miRNA-related therapies.

Animals↗

PubMed2005

MicroRNA: fine-tunes the function of genes in zebrafish.

MicroRNAs (miRNAs), 18-25 nt single-stranded RNAs, act as regulators in fine-tuning gene function. The absence of miRNAs is not life-threatening in early embryonic development of Dicer-knockout zebrafish and mice, which may account for genetic expression of various traits as a result of miRNA complexity in higher animals during natural evolution. The Pol-II-mediated intronic miRNA is a useful tool to validate the function of computer-predicated miRNAs in zebrafish and mice.

Animals↗

PubMed2005

Asymmetry of intronic pre-miRNA structures in functional RISC assembly.

The two oligonucleotide strands of a siRNA duplex are functionally asymmetric in assembling the RNAi effector, RNA-induced gene silencing complex (RISC). Based on this asymmetric RISC assembly model in vitro, formation of a microRNA (miRNA) and complementary miRNA (miRNA*) duplex was proposed to be an essential step for the assembly of miRNA-associated RISC (miRISC). We observed here that a strong structural bias exists in the selection of a mature miRNA strand for RISC assembly in zebrafish using an intronic miRNA-like vector to target EGFP mRNA for regulation. The position of the stemloop in a precursor miRNA (pre-miRNA) was involved in the determination of miRNA-miRNA* asymmetry of the pre-miRNA stemarm, leading to different miRNA maturation during miRISC assembly. These findings suggest that the miRISC assembly is likely different from the RISC assembly model of siRNA in zebrafish, providing the first in vivo evidence for asymmetric miRISC assembly.

Animals↗

PubMed2005

Intronic microRNAs.

MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular mRNAs that contain partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. MiRNA was originally discovered in the intergenic regions of the Caenorhabditis elegans genome as native RNA fragments that modulate a wide range of genetic regulatory pathways during animal development. However, neither RNA promoter nor polymerase responsible for miRNA biogenesis was determined. Recent findings of intron-derived miRNA in C. elegans, mouse, and human have inevitably led to an alternative pathway for miRNA biogenesis, which relies on the coupled interaction of Pol-II-mediated pre-mRNA transcription and intron excision, occurring in certain nuclear regions proximal to genomic perichromatin fibrils.

Gene Expression Regulation↗

PubMed2004

Intron-derived microRNAs--fine tuning of gene functions.

New evidence suggests that abundant small RNAs, microRNAs (miRNAs) act as regulatory vehicles to repress translation or cleave RNA transcripts, depending on their complementarity to the target gene, and result in modulation of gene expression. This review describes that the miRNAs derived from introns can suppress intracellular RNA homologues and regulate the gene function.

Animals↗

PubMed2004

Gene expression in precursor cells of prostate cancer associated with activin by combination of subtractive hybridization and microarray technologies.

Prostatic intraepithelial neoplasia (PIN) is considered the pre-malignant stage of prostate carcinoma, but little is known of its initiation and evolution. The identification of genes associated with these precursors of prostate cancer may elucidate the pathways of the early oncogenesis of this disease. Previously, we have reported that activin, a member of the TGFbeta superfamily, acted as an inhibitory growth factor in prostate cancer. We used laser capture microdissection, mRNA-library amplification (RNA-PCR), subtractive hybridization, and complementary DNA microarray to examine gene expression profiles in activin-positive PIN, compared with activin-negative PIN. Subtractive hybridization showed that 28 genes were differentially expressed (13 and 15 genes were up- and down-regulated, respectively). Microarray analysis identified 29 and 56 more genes (4 times) up- and down-regulated, respectively, suggesting that DNA microarray is a more effective method in screening gene profiles. We have validated the known genes identified by both subtractive hybridization and microarray technologies, using Northern blot analysis in the mRNA libraries generated from cells microdissected from pathological slides. We have successfully showed that at least 13 genes are involved in activin-associated PIN. The evaluation of candidate genes that emerge from these experiments provides a rational approach to investigate those genes significant in evolution from PIN to prostate carcinoma.

Activins↗

PubMed2004

Combinational therapy for potential HIV-1 eradication and vaccination.

Eradication of HIV would avert the pandemic high rates of deaths and save the healthcare resources in treatment and prevention costs, however, it is clear that this goal is not achievable with existing anti-HIV drugs due to HIV evolvement in great magnitude and diversity. Despite recent reports of short interfering RNA-directed silencing of HIV gene activities, there remain many challenges in overcoming the complications of HIV mutation in the management of clinical HIV-infected cells. Here we describe a combinational therapy of antiviral deoxynucleotidylated RNA hybrid transfection and interleukin-2 adjuvant treatment that was able to consistently silence 99% mutant type HIV-1 infections of T lymphocytes mediated through a novel post-transcriptional gene silencing mechanism. This combined therapy not only induced strong suppression on mixed type HIV-1 replication but also boosted the immunity and proliferation of normal T lymphocytes. The outgrowth of non-infected T cells resulted in the elimination of 76% HIV-infected cells 1 week after the combinational therapy. These findings promise to provide an immediate therapy in both acute and chronic HIV infections as well as a potential vaccination strategy for AIDS eradication.

AIDS Vaccines↗

PubMed2003

A novel RNA splicing-mediated gene silencing mechanism potential for genome evolution.

Over 90% of the human genome consists of non-protein-coding regions. Introns constitute most of the non-coding regions located in precursor messenger RNAs (pre-mRNAs). During pre-mRNA maturation, the introns are excised out of mRNA and thought to be completely digested prior to translation. If the introns were merely metabolic "leavings," why would the genome hold such a large amount of extraneous genetic materials? Here we show a novel posttranscriptional gene silencing system identified within mammalian introns. By packaging human spliceosome-recognition sites along with an exonic insert into an artificial intron, we observed that the splicing and processing of such an exon-containing intron in either sense or antisense conformation produced equivalent gene silencing effects, while a palindromic hairpin insert containing both sense and antisense strands resulted in synergistic effects. These findings may explain how cells respond to the presence of transgenic introns that are homologous to pre-existing exons during genomic evolution.

Animals↗

PubMed2003

Truncated Bcl-2, a potential pre-metastatic marker in prostate cancer.

A novel truncated form of Bcl-2, termed Bcl-2psi, was discovered in invasive prostate cancer cells, using laser capture microdissection, RNA-polymerase cycling reaction, and microarray analysis. The expression of Bcl-2psi increased prior to metastasis in higher-grade prostate cancer. The immunoreactive Bcl-2psi was specifically identified in higher-grade prostate cancer cells. These findings suggest that Bcl-2psi may be a potential pre-metastatic marker for detection, diagnosis, and therapy during the initiation of metastasis in prostate cancer.

Base Sequence↗