Browsing by Author "Chen, Yu-Chia"

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Alternative splicing in human cancer cells is modulated by the amiloride derivative 3,5-diamino-6-chloro-N-(N-(2,6-dichlorobenzoyl)carbamimidoyl)pyrazine-2-carboxide
(1970-01-01)
Lee, Chien-Chin
;
Chang, Wen-Hsin
;
Chang, Ya-Sian
;
Yang, Jinn-Moon
;
Chang, Chih-Shiang
Alternative splicing (AS) is a process that enables the generation of multiple protein isoforms with different biological properties from a single mRNA. Cancer cells often use the maneuverability conferred by AS to produce proteins that contribute to growth and survival. In our previous studies, we identified that amiloride modulates AS in cancer cells. However, the effective concentration of amiloride required to modulate AS is too high for use in cancer treatment. In this study, we used computational algorithms to screen potential amiloride derivatives for their ability to regulate AS in cancer cells. We found that 3,5-diamino-6-chloro-N-(N-(2,6-dichlorobenzoyl)carbamimidoyl)pyrazine-2-carboxamide (BS008) can regulate AS of apoptotic gene transcripts, including HIPK3, SMAC, and BCL-X, at a lower concentration than amiloride. This splicing regulation involved various splicing factors, and it was accompanied by a change in the phosphorylation state of serine/arginine-rich proteins (SR proteins). RNA sequencing was performed to reveal that AS of many other apoptotic gene transcripts, such as AATF, ATM, AIFM1, NFKB1, and API5, was also modulated by BS008. In vivo experiments further indicated that treatment of tumor-bearing mice with BS008 resulted in a marked decrease in tumor size. BS008 also had inhibitory effects in vitro, either alone or in a synergistic combination with the cytotoxic chemotherapeutic agents sorafenib and nilotinib. BS008 enabled sorafenib dose reduction without compromising antitumor activity. These findings suggest that BS008 may possess therapeutic potential for cancer treatment.
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
;
Chang, Jan-Gowth
;
Jong, Yuh-Jyh
;
Liu, Ting-Yuan
;
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.
Muscle developmental defects in heterogeneous nuclear Ribonucleoprotein A1 knockout mice
(2017-01-01)
Liu, Ting-Yuan
;
Chen, Yu-Chia
;
Jong, Yuh-Jyh
;
Tsai, Huai-Jen
;
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.
Muscle developmental defects in heterogeneous nuclear Ribonucleoprotein A1 knockout mice
(2017-01)
Liu, Ting-Yuan
;
Chen, Yu-Chia
;
Jong, Yuh-Jyh
;
Tsai, Huai-Jen
;
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.
Securinine enhances SMN2 exon 7 inclusion in spinal muscular atrophy cells
(2017-04)
Chen, Yu-Chia
;
Chang, Jan-Gowth
;
Liu, Ting-Yuan
;
Jong, Yuh-Jyh
;
Cheng, Wei-Lin
Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by the degeneration of motor neurons in the spinal cord, leading to muscular atrophy. SMA is caused by deletions or mutations in the survival motor neuron gene (SMN1) on chromosome 5q13. A second copy of the SMN gene (SMN2) also exists on chromosome 5, and both genes can produce functional protein. However, due to alternative splicing of the exon 7, the majority of SMN protein produced by SMN2 is truncated and unable to compensate for the loss of SMN1. Increasing full-length SMN protein production by promoting the exon 7 inclusion in SMN2 mRNA or increasing SMN2 gene transcription could be a therapeutic approach for SMA. In this study, we screened for the compounds that enhance SMN2 exon 7 inclusion by using SMN2 minigene-luciferase reporter system. We found that securinine can increase luciferase activity, indicating that securinine promoted SMN2 exon 7 inclusion. In addition, securinine increased full-length SMN2 mRNA and SMN protein expression in SMA patient-derived lymphoid cell lines. To investigate the mechanism of securinine effect on SMN2 splicing, we compared the protein levels of relevant splicing factors between securinine-treated and untreated cells. We found that securinine downregulated hnRNP A1 and Sam68 and upregulated Tra2-b1 expression. However, securinine, unlike HDAC inhibitors, did not enhance tra2-b1 gene transcription, indicating a post-transcriptional mechanism for Tra2-b1 upregulation. Furthermore, we treated SMA-like mice with securinine by i.p. injection and found that securinine treatment increased SMN2 exon 7 inclusion and SMN protein expression in the brain and spinal cord. According to our results, securinine might have the potential to become a therapeutic drug for SMA disease. (C) 2017 Elsevier Masson SAS. All rights reserved.