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High Expression Level of Tra2-beta 1 Is Responsible for Increased SMN2 Exon 7 Inclusion in the Testis of SMA Mice
(2015-03-17)
Chen, Yu-Chia
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Chang, Jan-Gowth
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Jong, Yuh-Jyh
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Liu, Ting-Yuan
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Yuo, Chung-Yee
Spinal muscular atrophy (SMA) is an inherited neuromuscular disease caused by deletion or mutation of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene, which produces low level of SMN protein due to mRNA exon 7 exclusion. Previously, we found that the testis of SMA mice (smn(-/-) SMN2) expresses high level of SMN2 full-length mRNA, indicating a testis-specific mechanism for SMN2 exon 7 inclusion. To elucidate the underlying mechanism, we established primary cultures of testis cells from SMA mice and analyzed them for SMN2 exon 7 splicing. We found that primary testis cells after a 2-hour culture still expressed high level of SMN2 full-length mRNA, but the level decreased after longer cultures. We then compared the protein levels of relevant splicing factors, and found that the level of Tra2-beta 1 also decreased during testis cell culture, correlated with SMN2 full-length mRNA downregulation. In addition, the testis of SMA mice expressed the highest level of Tra2-beta 1 among the many tissues examined. Furthermore, overexpression of Tra2-beta 1, but not ASF/SF2, increased SMN2 minigene exon 7 inclusion in primary testis cells and spinal cord neurons, whereas knockdown of Tra2-beta 1 decreased SMN2 exon 7 inclusion in primary testis cells of SMA mice. Therefore, our results indicate that high expression level of Tra2-beta 1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice. This study also suggests that the expression level of Tra2-beta 1 may be a modifying factor of SMA disease and a potential target for SMA treatment.
Long noncoding RNA TUG1 is downregulated in non-small cell lung cancer and can regulate CELF1 on binding to PRC2
(2016-08-02)
Lin, Pei-Chin
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Huang, Hsien-Da
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Chang, Chun-Chi
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Chang, Ya-Sian
;
Yen, Ju-Chen
Background: Long noncoding RNAs (lncRNAs) play crucial roles in tumorigenesis, and lncRNA taurine-upregulated gene 1 (TUG1) has been proven to be associated with several human cancers. However, the mechanisms of TUG1-involved regulation remain largely unknown. Methods: We examined the expressions of TUG1 in a cohort of 89 patients with non-small cell lung cancer (NSCLC) to determine the association between TUG1 expression and clinical parameters. We used circular chromosome conformation capture (4C) coupled with next-generation sequencing to explore the genome regions that interact with TUG1 and the TUG1-mediated regulation. Results: TUG1 was significantly downregulated, and the TUG1 downregulation correlated with sex (p = 0.006), smoking status (p = 0.016), and tumor differentiation grade (p = 0.001). Knockdown of TUG1 significantly promoted the proliferation of NSCLC cells. According to the bioinformatic analysis result of TUG1 4C sequencing data, 83 candidate genes and their interaction regions were identified. Among these candidate genes, CUGBP and Elav-like family member 1 (CELF1) are potential targets of TUG1 in-trans regulation. To confirm the interaction between TUG1 and CELF1, relative expressions of CELF1 were examined in TUG1 knockdown H520 cells; results showed that CELF1 was significantly upregulated in TUG1 knockdown H520 cells. RNA immunoprecipitation was then performed to examine whether TUG1 RNA was bound to PRC2, a TUG1-involved regulation mechanism reported in previous studies. The results demonstrated that TUG1 RNA was bound to enhancer of zeste protein 2/embryonic ectoderm development (EZH2/EED), which is essential for PRC2. Finally, our designed ChIP assay revealed that the EZH2/EED was bound to the promotor region of CELF1 within 992 bp upstream of the transcript start site. Conclusion: TUG1 is downregulated in NSCLC. Using TUG1 4C sequencing and bioinformatic analysis, we found CELF1 to be a potential target of TUG1 RNA in in-trans regulation. Moreover, subsequent experiments showed that TUG1 RNA could bind to PRC2 in the promotor region of CELF1 and negatively regulate CELF1 expressions in H520 cells. Our results may facilitate developing new treatment modalities targeting TUG1/PRC2/CELF1 interactions in patients with NSCLC.
Muscle developmental defects in heterogeneous nuclear Ribonucleoprotein A1 knockout mice
(2017-01-01)
Liu, Ting-Yuan
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Chen, Yu-Chia
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Jong, Yuh-Jyh
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Tsai, Huai-Jen
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Lee, Chien-Chin
Heterogeneous ribonucleoprotein A1 (hnRNP A1) is crucial for regulating alternative splicing. Its integrated function within an organism has not, however, been identified. We generated hnRNP A1 knockout mice to study the role of hnRNPA1 in vivo. The knockout mice, hnRNP A1(-/-), showed embryonic lethality because of muscle developmental defects. The blood pressure and heart rate of the heterozygous mice were higher than those of the wild-type mice, indicating heart function defects. We performed mouse exon arrays to study the muscle development mechanism. The processes regulated by hnRNP A1 included cell adhesion and muscle contraction. The expression levels of muscle development-related genes in hnRNPA1(+/-) mice were significantly different from those in wild-type mice, as detected using qRT-PCR. We further confirmed the alternative splicing patterns of muscle development-related genes including mef2c, lrrfip1, usp28 and abcc9. Alternative mRNA isoforms of these genes were increased in hnRNP A1(+/-) mice compared with wild-type mice. Furthermore, we revealed that the functionally similar hnRNP A2/B1 did not compensate for the expression of hnRNP A1 in organisms. In summary, our study demonstrated that hnRNP A1 plays a critical and irreplaceable role in embryonic muscle development by regulating the expression and alternative splicing of muscle-related genes.
IncRNAMap: A map of putative regulatory functions in the long non-coding transcriptome
(2014-06-01)
Chan, Wen-Ling
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Huang, Hsien-Da
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Chang, Jan-Gowth
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生物科技學系
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生物資訊及系統生物研究所
Background: Recent studies have demonstrated the importance of long non-coding RNAs (IncRNAs) in chromatin remodeling, and in transcriptional and post-transcriptional regulation. However, only a few specific IncRNAs are well understood, whereas others are completely uncharacterized. To address this, there is a need for user-friendly platform to studying the putative regulatory functions of human IncRNAs. Description: IncRNAMap is an integrated and comprehensive database relating to exploration of the putative regulatory functions of human IncRNAs with two mechanisms of regulation, by encoding siRNAs and by acting as miRNA decoys. To investigate IncRNAs producing siRNAs that regulate protein-coding genes, IncRNAMap integrated small RNAs (sRNAs) that were supported by publicly available deep sequencing data from various sRNA libraries and constructed IncRNA-derived siRNA-target interactions. In addition, IncRNAMap demonstrated that IncRNAs can act as targets for miRNAs that would otherwise regulate protein-coding genes. Previously studies indicated that intergenic IncRNAs (lincRNAs) either positive or negative regulated neighboring genes, therefore, IncRNAMap surveyed neighboring genes within a 1 Mb distance from the genomic location of specific IncRNAs and provided the expression profiles of IncRNA and its neighboring genes. The gene expression profiles may supply the relationship between incRNA and its neighboring genes. Conclusions: lncRNAMap is a powerful user-friendly platform for the investigation of putative regulatory functions of human IncRNAs with producing siRNAs and acting as miRNA decoy. IncRNAMap is freely available on the web at http://IncRNAMap.mbc.nctu.edu.tw/., (C) 2014 Elsevier Ltd. All rights reserved.
Small Molecule Amiloride Modulates Oncogenic RNA Alternative Splicing to Devitalize Human Cancer Cells
(2011-06-09)
Chang, Jan-Gowth
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Yang, Den-Mei
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Chang, Wen-Hsin
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Chow, Lu-Ping
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Chan, Wen-Ling
Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could "normalize" the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of "normalized" oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pretreatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics.
Transcribed pseudogene psi PPM1K generates endogenous siRNA to suppress oncogenic cell growth in hepatocellular carcinoma
(2013-04-01)
Chan, Wen-Ling
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Yuo, Chung-Yee
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Yang, Wen-Kuang
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Hung, Shih-Ya
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Chang, Ya-Sian
Pseudogenes, especially those that are transcribed, may not be mere genomic fossils, but their biological significance remains unclear. Postulating that in the human genome, as in animal models, pseudogenes may function as gene regulators through generation of endo-siRNAs (esiRNAs), antisense RNAs or RNA decoys, we performed bioinformatic and subsequent experimental tests to explore esiRNA-mediated mechanisms of pseudogene involvement in oncogenesis. A genome-wide survey revealed a partial retrotranscript pseudogene psi PPM1K containing inverted repeats capable of folding into hairpin structures that can be processed into two esiRNAs; these esiRNAs potentially target many cellular genes, including NEK8. In 41 paired surgical specimens, we found significantly reduced expression of two predicted psi PPM1K-specific esiRNAs, and the cognate gene PPM1K, in hepatocellular carcinoma compared with matched non-tumour tissues, whereas the expression of target gene NEK8 was increased in tumours. Additionally, NEK8 and PPM1K were downregulated in stably transfected psi PPM1K-overexpressing cells, but not in cells transfected with an esiRNA1-deletion mutant of psi PPM1K. Furthermore, expression of NEK8 in psi PPM1K-transfected cells demonstrated that NEK8 can counteract the growth inhibitory effects of psi PPM1K. These findings indicate that a transcribed pseudogene can exert tumour-suppressor activity independent of its parental gene by generation of esiRNAs that regulate human cell growth.
16-Hydroxycleroda-3,13-dien-15,16-olide deregulates PI3K and Aurora B activities that involve in cancer cell apoptosis
(2011-07-11)
Lin, Yi-Hsiung
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Lee, Chien-Chih
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Chan, Wen-Li
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Chang, Wen-Hsin
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Wu, Yang-Chang
The PI3K-AKT pathway and Aurora kinase play essential roles in such cellular processes as cell survival, angiogenesis, and differentiation, and are usually expressed at maximum levels during cancer cell proliferation. The present study investigated the effect of the natural compound, 16-hydroxycleroda-3,13-dien-15,16-olide (PL3), on regulating the PI3K-AKT pathway and Aurora B, which led to cancer cell apoptosis. PL3 acts as a PI3K inhibitor by influencing cell survival, signaling transduction, and cell cycle progression. It was observed that PL3 targeted and induced dephosphorylation of the PI3K pathway, degradation of Aurora B and mitotic-related gene expressions, and sequentially shut down the cell cycle. This eventually resulted in cell death. As Aurora B was downregulated, spindle dysfunction and destruction of the G(2)/M phase checkpoint resulted in DNA-damaged cells undergoing apoptosis. Moreover. PL3 also resensitized T31 5I-mutated Bcr-ABL(+) BA/F3 cells to improve the cytotoxicity of Imatinib in Imatinib-resistant cell line. Taken together, PL3 can perturb the PI3K-AKT pathway and Aurora B resulting in gene silencing and cell cycle disturbance. It was demonstrated that PL3 acted like a novel small-molecule PI3K modulator, thereby potentially contributing to cancer chemotherapy and combination medication. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
pseudoMap: an innovative and comprehensive resource for identification of siRNA-mediated mechanisms in human transcribed pseudogenes
(2013-02-08)
Chan, Wen-Ling
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Yang, Wen-Kuang
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Huang, Hsien-Da
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Chang, Jan-Gowth
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生物科技學系
RNA interference (RNAi) is a gene silencing process within living cells, which is controlled by the RNA-induced silencing complex with a sequence-specific manner. In flies and mice, the pseudogene transcripts can be processed into short interfering RNAs (siRNAs) that regulate protein-coding genes through the RNAi pathway. Following these findings, we construct an innovative and comprehensive database to elucidate siRNA-mediated mechanism in human transcribed pseudogenes (TPGs). To investigate TPG producing siRNAs that regulate protein-coding genes, we mapped the TPGs to small RNAs (sRNAs) that were supported by publicly deep sequencing data from various sRNA libraries and constructed the TPG-derived siRNA-target interactions. In addition, we also presented that TPGs can act as a target for miRNAs that actually regulate the parental gene. To enable the systematic compilation and updating of these results and additional information, we have developed a database, pseudoMap, capturing various types of information, including sequence data, TPG and cognate annotation, deep sequencing data, RNA-folding structure, gene expression profiles, miRNA annotation and target prediction. As our knowledge, pseudoMap is the first database to demonstrate two mechanisms of human TPGs: encoding siRNAs and decoying miRNAs that target the parental gene. pseudoMap is freely accessible at http://pseudomap.mbc.nctu.edu.tw/. Database URL: http://pseudomap.mbc.nctu.edu.tw/
Characteristics and prevalence of KRAS, BRAF, and PIK3CA mutations in colorectal cancer by high-resolution melting analysis in Taiwanese population
(2012-10-09)
Hsieh, Li-Ling
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Er, Tze-Kiong
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Chen, Chih-Chieh
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Hsieh, Jan-Sing
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Chang, Jan-Gowth
Background: The identification of KRAS, BRAF, and PIK3CA mutations before the administration of anti-epidermal growth factor receptor therapy of colorectal cancer has become important The aim of the present study was to investigate the occurrence of KRAS, BRAF, and PIK3CA mutations in the Taiwanese population with colorectal cancer. This study was undertaken to identify BRAF and PIK3CA mutations in patients with colorectal cancer by high-resolution melting (HRM) analysis. HRM analysis is a new gene scan tool that quickly performs the PCR and identifies sequence alterations without requiring post-PCR treatment Methods: In the present study, DNAs were extracted from 182 cases of formalin-fixed, paraffin-embedded (FFPE) colorectal cancer samples for clinical KRAS mutational analysis by direct sequencing. All the samples were also tested for mutations within BRAF V600E and PIK3CA (exons 9 and 20) by HRM analysis. Results: The results were confirmed by direct sequencing. The frequency of BRAF and PIK3CA mutations is 1.1%, and 7.1%, respectively. Intriguingly, we found that nine patients (4.9%) with the KRAS mutation were coexistent with the PIK3CA mutation. Four patients (22%) without the KRAS mutation were existent with the PIK3CA mutation. Two patients (1.1%) without the KRAS mutation were existent with the BRAF mutation. Conclusions: In the current study, we suppose that HRM analysis is rapid, feasible, and powerful diagnostic tool for the detection of BRAF and PIK3CA mutations in a clinical setting. Additionally, our results indicated the prevalence of KRAS. BRAF, and PIK3C4 mutational status in the Taiwanese population. (C) 2012 Elsevier B.V. All rights reserved.
Computational analysis of a novel mutation in ETFDH gene highlights its long-range effects on the FAD-binding motif
(2011-10-21)
Er, Tze-Kiong
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Chen, Chih-Chieh
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Liu, Yen-Yi
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Chang, Hui-Chiu
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Chien, Yin-Hsiu
Background: Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is an autosomal recessive disease caused by the defects in the mitochondrial electron transfer system and the metabolism of fatty acids. Recently, mutations in electron transfer flavoprotein dehydrogenase (ETFDH) gene, encoding electron transfer flavoprotein: ubiquinone oxidoreductase (ETF:QO) have been reported to be the major causes of riboflavin-responsive MADD. To date, no studies have been performed to explore the functional impact of these mutations or their mechanism of disrupting enzyme activity. Results: High resolution melting (HRM) analysis and sequencing of the entire ETFDH gene revealed a novel mutation (p.Phe128Ser) and the hotspot mutation (p.Ala84Thr) from a patient with MADD. According to the predicted 3D structure of ETF: QO, the two mutations are located within the flavin adenine dinucleotide (FAD) binding domain; however, the two residues do not have direct interactions with the FAD ligand. Using molecular dynamics (MD) simulations and normal mode analysis (NMA), we found that the p. Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF: QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF: QO protein show highly correlated motions with the FAD-binding site. Conclusions: Based on the present findings, we conclude that the changes made to the amino acids in ETF: QO are likely to influence the FAD-binding stability.

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