Diagnostic Endoscopic Ultrasound: Technique, Current Status and Future Directions

( Tiing Leong Ang ),( Andrew Boon Eu Kwek ),( Lai Mun Wang ) 대한간학회 2018 Gut and Liver Vol.12 No.5

Endoscopic ultrasound (EUS) is now well established as an important tool in clinical practice. From purely diagnostic imaging, it has progressed to include tissue acquisition, which provided the basis for therapeutic procedures. Even as interventional EUS developed, there has been ongoing progress in EUS diagnostic capabilities due to improved imaging systems, better needles for tissue acquisition and development of enhanced imaging functions such as contrast harmonic EUS (CHEUS) and EUS elastography. EUS is well established for differentiation of subepithelial lesions, for T-staging of luminal gastrointestinal and pancreaticobiliary malignancies, for differentiation of benign pancreaticobiliary disorders and for diagnostic tissue acquisition, which can be achieved by EUS-guided fine needle aspiration or by EUS-guided fine needle biopsy using dedicated biopsy needles. This review briefly describes the technique of performing EUS and then discusses its clinical utility in terms of gastrointestinal cancer staging, the evaluation of pancreaticobiliary disorders and tissue acquisition. Enhanced imaging techniques such as CHEUS and EUS elastography are briefly reviewed. (Gut Liver 2018;12:483-496)

REVIEW : Screening Colonoscopy for Average Risk Individuals in Singapore

( Tiing Leong Ang ),( Kwong Ming Fock ) 대한장연구학회 2012 Intestinal Research Vol.10 No.3

In Singapore colorectal cancer (CRC) is the most common cancer for males, second most common cancer for females and most common cancer overall. A national CRC screening program for average risks individuals was started in July 2011, with the primary test modality being the faecal immunochemical test. Individuals may choose to undergo screening colonoscopy directly. Colonoscopy has two roles in CRC screening. It is performed either as a primary screening test or used to evaluate abnormal results from another screening test. Colonoscopy is a safe and effective procedure but potential risks exist. Local complications such as perforation and bleeding, cardiopulmonary events and even mortality may occur. Additionally there could be failed cecal intubation and missed lesions. It is imperative that prior to colonoscopy, there is a proper discussion of risks, benefits and alternatives. To provide quality assurance for colonoscopy in the CRC screening program, a set of quality indicators and criteria for endoscopists and endoscopy centres was established. The endoscopists must be qualified specialists with a lifetime experience of at least 500 colonoscopies and 50 polypectomies, and need to meet annual monitoring parameters that include at least 50 colonoscopies, >95% cecal intubation rate, >95% recovery rate of excised polyps, and withdrawal time of at least 6 minutes. In addition, complication rates must be within acceptable limits such as perforation rate of less than 0.1% and postpolypectomy bleeding rate less than 1%. (Intest Res 2012;10:219-228)

SR proteins regulate V6 exon splicing of CD44 pre-mRNA

( Tiing Jen Loh ),( Heegyum Moon ),( Ha Na Jang ),( Yongchao Liu ),( Namjeong Choi ),( Shengfu Shen ),( Darren Reece Williams ),( Da-woon Jung ),( Xuexiu Zheng ),( Haihong Shen ) 생화학분자생물학회(구 한국생화학분자생물학회) 2016 BMB Reports Vol.49 No.11

CD44 pre-mRNA includes 20 exons, of which exons 1-5 (C₁-C₅) and exons 16-20 (C₆-C₁₀) are constant exons, whereas exons 6-15 (V₁-V₁₀) are variant exons. V6-exon-containing isoforms have been known to be implicated in tumor cell invasion and metastasis. In the present study, we performed a SR protein screen for CD44 V₆ splicing using overexpression and lentivirus-mediated shRNA treatment. Using a CD44 V₆ minigene, we demonstrate that increased SRSF3 and SRSF4 expression do not affect V₆ splicing, but increased expression of SRSF1, SRSF6 and SRSF9 significantly inhibit V₆ splicing. In addition, using a constitutive exon-specific primer set, we could not detect alterations of CD44 splicing after SR protein-targeting shRNA treatment. However, using a V₆ specific primer, we identified that reduced SRSF2 expression significantly reduced the V6 isoform, but increased V_6-10 and V_6,8-10 isoforms. Our results indicate that SR proteins are important regulatory proteins for CD44 V₆ splicing. [BMB Reports 2016; 49(11): 612-616]

Helicobacter pylori Treatment Strategies in Singapore

( Tiing Leong Ang ),( Daphne Ang ) 대한간학회 2021 Gut and Liver Vol.15 No.1

The management of Helicobacter pylori infection in Singapore remains a clinical challenge. Similar to other regions, there has been an increase in antibiotic resistance rates through the years. Nonetheless, over the past two decades, clarithromycin-based triple therapy has continued to be used as the first line treatment option, with an eradication rate exceeding 90%, although the accepted treatment duration must now be lengthened from 1 to 2 weeks to maintain efficacy. Concomitant and sequential therapies did not demonstrate superiority over standard triple therapy. Current empiric second line treatment utilizes either bismuth-based quadruple therapy or levofloxacin-based triple therapy, but outcomes remain less than ideal. Identifying options to further improve treatment success rates is challenging. Strategies being considered include the use of potent acid suppressants, such as vonoprazan, and H. pylori culture and antibiotic susceptibility testing-guided therapy. (Gut Liver 2021;15:13-18)

SC35 promotes splicing of the C5-V6-C6 isoform of CD44 pre-mRNA

LOH, TIING JEN,MOON, HEEGYUM,CHO, SUNGHEE,JUNG, DA-WOON,HONG, SEONG-EUI,KIM, DO HAN,GREEN, MICHAEL R.,ZHENG, XUEXIU,ZHOU, JIANHUA,SHEN, HAIHONG D.A. Spandidos 2014 ONCOLOGY REPORTS Vol.31 No.1

<P>CD44 is a cell membrane glycoprotein that mediates the response of cells to their cellular microenvironment and regulates growth, survival, differentiation and motility. CD44 pre-mRNA contains 20 exons, 10 of which are alternatively spliced. Among the CD44 spliced variants, one of the V6 exon-containing isoforms, the V4–7 variant which contains variable exons 4, 5, 6 and 7, confers metastatic potential to non-metastatic cells. However, the splicing regulation of the V6 exon is not completely understood. SC35 is an arginine-serine rich protein that regulates alternative splicing of various pre-mRNAs. In the present study, we established a stable cell line which indicates inclusion or skipping of the V6 exon with the RFP or GFP signal. Using this stable cell line, we found that the V6 exon and flanking introns of CD44 pre-mRNA contained SC35 response elements that regulate V6 splicing. RT-PCR analyses of the endogenous CD44 splicing showed that SC35 promotes the production of the C5-V6-C6 isoform. shRNA knockdown of SC35 showed that reduced expression of SC35 decreased expression of the V6 exon-containing isoforms. Our results reveal a novel mechanism of CD44V6 splicing.</P>

CD44 alternative splicing and hnRNP A1 expression are associated with the metastasis of breast cancer.

Loh, Tiing Jen,Moon, Heegyum,Cho, Sunghee,Jang, Hana,Liu, Yong Chao,Tai, Hongmei,Jung, Da-Woon,Williams, Darren R,Kim, Hey-Ran,Shin, Myung-Geun,Liao, D Joshua,Zhou, Jianhua,Shi, Wei,Zheng, Xuexiu,Shen National Hellenic Research Foundation 2015 ONCOLOGY REPORTS Vol.34 No.3

<P>CD44 is a transmembrane receptor for hyaluronic acid. CD44 pre-mRNA contains 19?exons, 9?of which are alternatively spliced. Among the CD44 spliced variants, the v4-7 variant, one of the v6?exon-containing isoforms that contains variable exon?4, 5, 6 and?7, confers metastatic potential to non-metastatic cells. Splicing of CD44 and the function of CD44 isoforms are different in breast cancer cells. hnRNP?A1 is a ubiquitously expressed protein with an inhibitory function in pre-mRNA splicing. We showed that CD44v6 isoform, which includes all of the v6-containing mRNA isoforms, had the highest expression level in non-metatatic breast cancer cells (MCF7) when compared to the level in metastatic breast cancer cells (MDA-MB-231) and normal breast cells (MCF10A). Furthermore we showed that hnRNP?A1 knockdown regulated splicing of CD44 differently in breast cancer cells. We showed here that CD44 isoform expression is completely different in MDA-MB-231 cells than that in MCF7 and MCF10A cells, whereas MCF7 and MCF10A cells had a similar expression pattern of CD44 isoforms. RT-PCR analysis of CD44v6 showed that MCF7 and MCF10A cells predominantly expressed the c5v6v7v8v9v10c6 isoform. However, in addition to this isoform, MDA-MB-231 cells also expressed the c5v6v8v9v10c6 and c5v6c6 isoforms. We also found that knockdown of hnRNP?A1 significantly reduced the expression of c5v6v7v8v9v10c6 and c5v6v8v9v10c6, and promoted the expression of c5v6c6. hnRNP?A1 knockdown significantly induced cell death. In addition, hnRNP?A1 knockdown induced a decrease in cell invasion in the MDA-MB-231 cells. Our results indicate that the knockdown of hnRNP A1 has a specific function on the splicing of CD44 in breast cancer cells.</P>

CRISPR as a strong gene editing tool

( Shengfu Shen ),( Tiing Jen Loh ),( Hongling Shen ),( Xuexiu Zheng ),( Haihong Shen ) 생화학분자생물학회(구 한국생화학분자생물학회) 2017 BMB Reports Vol.50 No.1

Clustered regularly-interspaced short palindromic repeats (CRISPR) is a new and effective genetic editing tool. CRISPR was initially found in bacteria to protect it from virus invasions. In the first step, specific DNA strands of virus are identified by guide RNA that is composed of crRNA and tracrRNA. Then RNAse III is required for producing crRNA from pre-crRNA. In The second step, a crRNA:tracrRNA:Cas9 complex guides RNase III to cleave target DNA. After cleavage of DNA by CRISPR-Cas9, DNA can be fixed by Non- Homologous End Joining (NHEJ) and Homology Directed Repair (HDR). Whereas NHEJ is simple and random, HDR is much more complex and accurate. Gene editing by CRISPR is able to be applied to various biological field such as agriculture and treating genetic diseases in human. [BMB Reports 2017; 50(1): 20-24]

SRSF2 directly inhibits intron splicing to suppresses cassette exon inclusion

( Heegyum Moon ),( Sunghee Cho ),( Tiing Jen Loh ),( Ha Na Jang ),( Yongchao Liu ),( Namjeong Choi ),( Jagyeong Oh ),( Jiyeon Ha ),( Jianhua Zhou ),( Sungchan Cho ),( Dong-eun Kim ),( Michael B. Ye ) 생화학분자생물학회(구 한국생화학분자생물학회) 2017 BMB Reports Vol.50 No.8

SRSF2, a Serine-Arginine rich (SR) protein, is a splicing activator that mediates exon inclusion and exclusion events equally well. Here we show SRSF2 directly suppresses intron splicing to suppress cassette exon inclusion in SMN pre-mRNA. Through a serial mutagenesis, we demonstrate that a 10 nt RNA sequence surrounding the branch-point (BP), is important for SRSF2-mediated inhibition of cassette exon inclusion through directly interacting with SRSF2. We conclude that SRSF2 inhibits intron splicing to promote exon exclusion. [BMB Reports 2017; 50(8): 423-428]

Polypyrimidine tract binding protein inhibits IgM pre-mRNA splicing by diverting U2 snRNA base-pairing away from the branch point

Zheng, Xuexiu,Cho, Sunghee,Moon, Heegyum,Loh, Tiing Jen,Oh, Huyn Kyung,Green, Michael R.,Shen, Haihong Cold Spring Harbor Laboratory Press 2014 RNA Vol.20 No.4

<P>This study provides new insight into how the immunoglobulin splicing inhibitor sequence blocks splicing. Evidence indicates the polypyrimidine tract binding protein bound to the splicing inhibitor element prevents U2 snRNA from base-pairing to the branch point.</P>

Splicing inhibition of U2AF⁶⁵ leads to alternative exon skipping

Cho, Sunghee,Moon, Heegyum,Loh, Tiing Jen,Jang, Ha Na,Liu, Yongchao,Zhou, Jianhua,Ohn, Takbum,Zheng, Xuexiu,Shen, Haihong National Academy of Sciences 2015 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.112 No.32

<P><B>Significance</B></P><P>Transcription is a biological procedure in which DNA is transcribed to an RNA molecule. However, only fragments of this RNA are needed for protein synthesis. These fragments are exons that are interrupted by introns. Introns are removed by so-called RNA splicing process. Some exons could be alternatively included or excluded from the final RNA molecule. In this study, we have found that U2 snRNP auxiliary factor 65 kDa (U2AF<SUP>65</SUP>), a general splicing regulator, can significantly promote the exclusion of alternative exons. Strikingly, U2AF<SUP>65</SUP> suppresses flanking intron splicing of alternative exons, and even constitutive intron splicing. We deduce that the stimulatory effects of U2AF<SUP>65</SUP> on alternative exon exclusion are induced by the splicing inhibitory effects of U2AF<SUP>65</SUP>.</P><P>U2 snRNP auxiliary factor 65 kDa (U2AF<SUP>65</SUP>) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF<SUP>65</SUP> stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (<I>SMN</I>) pre-mRNA. A stronger 5′ splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF<SUP>65</SUP>. U2AF<SUP>65</SUP> overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF<SUP>65</SUP> inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF<SUP>65</SUP> effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF<SUP>65</SUP> even inhibits general splicing of adenovirus major late (Ad ML) or β-globin pre-mRNA. Thus, we conclude that U2AF<SUP>65</SUP> possesses a splicing Inhibitory function that leads to alternative exon skipping.</P>