Displaying publications 1 - 20 of 28 in total

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  1. 3'-UTR variations and G6PD deficiency
    Amini F, Ismail E
    J Hum Genet, 2013 Apr;58(4):189-94.
    PMID: 23389243 DOI: 10.1038/jhg.2012.155
    The combination of two silent mutations, c.1311C>T in exon 11 and IVS11 T93C (glucose-6-phosphate dehydrogenase (G6PD) 1311T/93C), with unknown mechanism, have been reported in G6PD-deficient individuals in Asian populations including Malaysian aboriginal group, Negrito. Here, we report the screening of G6PD gene in 103 Negrito volunteers using denaturing high-performance liquid chromatography (dHPLC) and direct sequencing. A total of 48 individuals (46.6%) were G6PD deficient, 83.3% of these carried G6PD 1311T/93C with enzyme activity ranging from 1.8 to 4.8 U gHb(-1). Three novel single-nucleotide polymorphisms (SNPs), rs112950723, rs111485003 and rs1050757, were found in the G6PD 3'-untranslated region (UTR). Strong association was observed between haplotype 1311T/93C and rs1050757G, which is located inside the 35 bp AG-rich region. In silico analysis revealed that the transition of A to G at position rs1050757 makes significant changes in the G6PD mRNA secondary structure. Moreover, putative micro (mi)RNA target sites were identified in 3'-UTR of G6PD gene, two of these in the region encompassing rs1050757. It could be speculated that rs1050757 have a potential functional effect on the downregulation of mRNA and consequently G6PD deficiency either by affecting mRNA stability and translation or mirRNA regulation process. This is the first report of biochemical association of an SNP in 3'-UTR of G6PD gene and the possible role of mRNA secondary structure.
    Matched MeSH terms: 3' Untranslated Regions*
  2. Functional characterization of two variants in the 3'-untranslated region (UTR) of transcription factor 4 gene and their association with schizophrenia in sib-pairs from multiplex families
    Mohamed ZI, Tee SF, Chow TJ, Loh SY, Yong HS, Bakar AKA, et al.
    Asian J Psychiatr, 2019 Feb;40:76-81.
    PMID: 30771755 DOI: 10.1016/j.ajp.2019.02.001
    Transcription factor 4 (TCF4) gene plays an important role in nervous system development and it always associated with the risk of schizophrenia. Since miRNAs regulate targetgenes by binding to 3'UTRs of target mRNAs, the functional variants located in 3'UTR of TCF4 are highly suggested to affect the gene expressions in schizophrenia. To test the hypothesis regarding the effects of the variants located in 3'UTR of TCF4, we conducted an in silico analysis to identify the functional variants and their predicted functions. In this study, we sequenced the 3'UTR of TCF4 in 13 multiplex schizophrenia families and 14 control families. We found two functional variants carried by three unrelated patients. We determined that the C allele of rs1272363 and the TC insert of rs373174214 might suppress post- transcriptional expression. Secondly, we cloned the region that flanked these two variants into a dual luciferase reporter system and compared the luciferase activities between the pmirGLO-TCF4 (control), pmirGLO-TCF4-rs373174214 and pmirGLO-TCF4-rs1273263. Both pmirGLO-TCF4-rs373174214 and pmirGLO-TCF4-rs1273263 caused lower reporter gene activities, as compared to the control. However, only the C allele of rs1272363 reduced the luciferase activity significantly (p = 0.0231). Our results suggested that rs1273263 is a potential regulator of TCF4 expression, and might be associated with schizophrenia.
    Matched MeSH terms: 3' Untranslated Regions/genetics*
  3. Cellular proteins bind to the 3' and 5' untranslated regions of dengue 2 virus genome
    Ong CC, Lam SK, AbuBakar S
    Malays J Pathol, 1998 Jun;20(1):11-7.
    PMID: 10879258
    In vitro generated cloned full length dengue 2 virus untranslated regions (UTRs) were used in RNA gel mobility shift assays to examine cellular factors binding to the virus genomes. Cellular factors in lysates of Vero (monkey) and C6/36 (mosquito) cells bound specifically and non-specifically to the dengue 2 virus 3' UTR. Non-specific interaction with the 5' UTR, resulting in formation of at least 4 band shift complexes was noted with lysate of the C6/36 cells only. Pre-treating the cell lysates with proteinase K affected binding of cellular factors to the dengue 2 virus UTRs, suggesting that the cellular factors were proteins. These findings suggest that cellular proteins could interact with specific sites on the dengue virus genomes.
    Matched MeSH terms: 3' Untranslated Regions/genetics
  4. Association of functional polymorphisms in 3'-untranslated regions of COMT, DISC1, and DTNBP1 with schizophrenia: a meta-analysis
    Mohamed ZI, Tee SF, Tang PY
    Psychiatr Genet, 2018 12;28(6):110-119.
    PMID: 30252773 DOI: 10.1097/YPG.0000000000000210
    INTRODUCTION: In recent years, various studies have accumulated evidence of the involvement of single nucleotide polymorphisms (SNPs) in introns and exons in schizophrenia. The association of functional SNPs in the 3'-untranslated regions with schizophrenia has been explored in a number of studies, but the results are inconclusive because of limited meta-analyses. To systematically analyze the association between SNPs in 3'-untranslated regions and schizophrenia, we conducted a meta-analysis by combining all available studies on schizophrenia candidate genes.

    MATERIALS AND METHODS: We searched candidate genes from the schizophrenia database and performed a comprehensive meta-analysis using all the available data up to August 2017. The association between susceptible SNPs and schizophrenia was assessed by the pooled odds ratio with 95% confidence interval using fixed-effect and random-effect models.

    RESULTS: A total of 21 studies including 8291 cases and 9638 controls were used for meta-analysis. Three investigated SNPs were rs165599, rs3737597, and rs1047631 of COMT, DISC1, and DTNBP1, respectively. Our results suggested that rs3737597 showed a significant association with schizophrenia in Europeans (odds ratio: 1.584, P: 0.002, 95% confidence interval: 1.176-2.134) under a random-effect framework.

    CONCLUSION: This meta-analysis indicated that rs3737597 of DISC1 was significantly associated with schizophrenia in Europeans, and it can be suggested as an ethnic-specific risk genetic factor.

    Matched MeSH terms: 3' Untranslated Regions/genetics
  5. Fulltext Descriptive distribution and phylogenetic analysis of feline infectious peritonitis virus isolates of Malaysia
    Sharif S, Arshad SS, Hair-Bejo M, Omar AR, Zeenathul NA, Fong LS, et al.
    Acta Vet Scand, 2010 Jan 06;52(1):1.
    PMID: 20053278 DOI: 10.1186/1751-0147-52-1
    The descriptive distribution and phylogeny of feline coronaviruses (FCoVs) were studied in cats suspected of having feline infectious peritonitis (FIP) in Malaysia. Ascitic fluids and/or biopsy samples were subjected to a reverse transcription polymerase chain reaction (RT-PCR) targeted for a conserved region of 3'untranslated region (3'UTR) of the FCoV genome. Eighty nine percent of the sampled animals were positive for the presence of FCoV. Among the FCoV positive cats, 80% of cats were males and 64% were below 2 years of age. The FCoV positive cases included 56% domestic short hair (DSH), 40% Persian, and 4% Siamese cats. The nucleotide sequences of 10 selected amplified products from FIP cases were determined. The sequence comparison revealed that the field isolates had 96% homology with a few point mutations. The extent of homology decreased to 93% when compared with reference strains. The overall branching pattern of phylogenetic tree showed two distinct clusters, where all Malaysian isolates fall into one main genetic cluster. These findings provided the first genetic information of FCoV in Malaysia.
    Matched MeSH terms: 3' Untranslated Regions/genetics
  6. Fulltext Lack of association between RANTES-28, SDF-1 gene polymorphisms and systemic lupus erythematosus in the Malaysian population
    Lian LH, Kee BP, Ng HL, Chua KH
    Genet. Mol. Res., 2011;10(4):2841-50.
    PMID: 22095608 DOI: 10.4238/2011.November.17.2
    Regulated on activation, normal T-cell expressed and secreted (RANTES) and stromal cell-derived factor 1 (SDF-1) are members of the CC- and CXC-chemokine families, respectively. Both genes have been postulated to be involved in the pathogenesis of systemic lupus erythematosus (SLE). We analyzed position 28 of the RANTES gene promoter region, as well as the SNP observed in the 3' UTR of the SDF-1 gene at position 801, in 130 patients presenting SLE at the Malaya University Medical Centre. Screening of 130 healthy volunteer controls using RFLP was also performed. RANTES-28 polymorphism analysis showed no significant (P = 0.3520) relationship, even though homozygous C/C was more frequent in SLE patients (OR = 1.4183) and heterozygous C/G was more frequent in healthy controls (OR = 0.7051). There were no significant (P = 0.2650) associations between A/A (OR = 0.783), G/G (OR = 1.5914) and G/A (OR = 0.8289) genotypes in the SDF-1 gene polymorphism with SLE. We conclude that there is no significant association of RANTES-28 and SDF-1 gene polymorphisms and occurrence of SLE in Malaysia.
    Matched MeSH terms: 3' Untranslated Regions/genetics
  7. Evolutionary relationship of the L- and M-class genome segments of bat-borne fusogenic orthoreoviruses in Malaysia and Australia
    Voon K, Chua KB, Yu M, Crameri G, Barr JA, Malik Y, et al.
    J Gen Virol, 2011 Dec;92(Pt 12):2930-2936.
    PMID: 21849518 DOI: 10.1099/vir.0.033498-0
    We previously described three new Malaysian orthoreoviruses designated Pulau virus, Melaka virus and Kampar virus. Melaka and Kampar viruses were shown to cause respiratory disease in humans. These viruses, together with Nelson Bay virus, isolated from Australian bats, are tentatively classified as different strains within the species Pteropine orthoreovirus (PRV), formerly known as Nelson Bay orthoreovirus, based on the small (S) genome segments. Here we report the sequences of the large (L) and medium (M) segments, thus completing the whole-genome characterization of the four PRVs. All L and M segments were highly conserved in size and sequence. Conserved functional motifs previously identified in other orthoreovirus gene products were also found in the deduced proteins encoded by the cognate segments of these viruses. Detailed sequence analysis identified two genetic lineages divided into the Australian and Malaysian PRVs, and potential genetic reassortment among the M and S segments of the three Malaysian viruses.
    Matched MeSH terms: 3' Untranslated Regions
  8. Kernel-specific cDNA clones encoding three different isoforms of seed storage protein glutelin from oil palm Elaeis guineensis
    Cha TS, Habib Shah F
    Plant Sci, 2001 Apr;160(5):913-923.
    PMID: 11297788
    The mRNA differential display method was used to identify and isolate cDNAs corresponding to transcripts that accumulate during the period of lipid synthesis, 12-20 weeks after anthesis (WAA) in the kernel of Elaeis guineensis, var. Tenera. We successfully isolated two cDNA clones, KT7 (312 bp) and KT8 (266 bp). Interestingly, both clones show 79% nucleotide sequence identity to each other. This suggests that both clones encode the isoforms of the same protein. We screened the kernel (15 WAA) cDNA library and isolated the clone pKT7 (587 bp) using KT7 as probe, and isolated another isoform with KT8 probe, which designated as pKT9 (900 bp). Clone pKT9 has 93% nucleotide identity to KT8 and only 46% to pKT7 in their 3'-untranslated region. All three clones displayed significant amino acid sequence identity to seed storage protein glutelin from monocotyledon and globulin from dicotyledon plants. The coding sequence of KT8 (106 bp) shows 76 and 97% identity to pKT9 and pKT7, respectively. Therefore, we suggest that clones KT8 and pKT7 are members of the same subfamily (A), while pKT9 belongs to another subfamily (B) of glutelin multigene families. Southern analysis shows that there are at least four members for the subfamily B. Northern analysis shows that these three members of the glutelin family are co-ordinately expressed and developmentally regulated during the development of the kernel. The transcripts begin to accumulate at 12 WAA, increase in 15 WAA and show a significant reduction at 17 WAA.
    Matched MeSH terms: 3' Untranslated Regions
  9. Fulltext Systematic identification and characterization of Aedes aegypti long noncoding RNAs (lncRNAs)
    Azlan A, Obeidat SM, Yunus MA, Azzam G
    Sci Rep, 2019 08 21;9(1):12147.
    PMID: 31434910 DOI: 10.1038/s41598-019-47506-9
    Long noncoding RNAs (lncRNAs) play diverse roles in biological processes. Aedes aegypti (Ae. aegypti), a blood-sucking mosquito, is the principal vector responsible for replication and transmission of arboviruses including dengue, Zika, and Chikungunya virus. Systematic identification and developmental characterisation of Ae. aegypti lncRNAs are still limited. We performed genome-wide identification of lncRNAs, followed by developmental profiling of lncRNA in Ae. aegypti. We identified a total of 4,689 novel lncRNA transcripts, of which 2,064, 2,076, and 549 were intergenic, intronic, and antisense respectively. Ae. aegypti lncRNAs share many characteristics with other species including low expression, low GC content, short in length, and low conservation. Besides, the expression of Ae. aegypti lncRNAs tend to be correlated with neighbouring and antisense protein-coding genes. A subset of lncRNAs shows evidence of maternal inheritance; hence, suggesting potential role of lncRNAs in early-stage embryos. Additionally, lncRNAs show higher tendency to be expressed in developmental and temporal specific manner. The results from this study provide foundation for future investigation on the function of Ae. aegypti lncRNAs.
    Matched MeSH terms: 3' Untranslated Regions
  10. Identification of AKT1 3'UTR variants in two Indian schizophrenia patients with poor executive functioning
    Chow TJ, Tee SF, Loh SY, Yong HS, Abu Bakar AK, Song SL, et al.
    Asian J Psychiatr, 2018 Aug;36:17-18.
    PMID: 29864676 DOI: 10.1016/j.ajp.2018.05.025
    Matched MeSH terms: 3' Untranslated Regions
  11. Fulltext Cloning and Expression of Iranian Turkmen-thoroughbred Horse Follicle Stimulating Hormone in Pichia pastoris
    Elyasi Gorji Z, Amiri-Yekta A, Gourabi H, Hassani S, Fatemi N, Zerehdaran S, et al.
    Iran J Biotechnol, 2015 Jun;13(2):10-17.
    PMID: 28959285 DOI: 10.15171/ijb.1004
    BACKGROUND: Follicle stimulating hormone (FSH) plays an essential role in reproductive physiology and follicular development.

    OBJECTIVE: A new variant of the equine fsh (efsh) gene was cloned, sequenced, and expressed in Pichia pastoris (P. pastoris) GS115 yeast expression system.

    MATERIALS AND METHODS: The full-length cDNAs of the efshα and efshβ chains were amplified by reverse transcription polymerase chain reaction (RT-PCR) using the total RNA isolated from an Iranian Turkmen-thoroughbred horse's anterior pituitary gland. The amplified efsh chains were cloned into the pPIC9 vector and transferred into P. pastoris. The secretion of recombined eFSH using P. pastoris expression system was confirmed by Western blotting and immunoprecipitation (IP) methods.

    RESULTS: The DNA sequence of the efshβ chain accession number JX861871, predicted two putative differential nucleotide arrays, both of which are located in the 3'UTR. Western blotting showed a molecular mass of 13 and 18 kDa for eFSHα and eFSHβ subunits, respectively. The expression of desired protein was confirmed by protein G immunoprecipitation kit.

    CONCLUSIONS: eFSH successfully expressed in P. pastoris. These findings lay a foundation to improve ovulation and embryo recovery rates as well as the efficiency of total embryo-transfer process in mares.

    Matched MeSH terms: 3' Untranslated Regions
  12. Fulltext Oncogenic Role of miR-200c-3p in High-Grade Serous Ovarian Cancer Progression via Targeting the 3'-Untranslated Region of DLC1
    Ankasha SJ, Shafiee MN, Abdul Wahab N, Raja Ali RA, Mokhtar NM
    Int J Environ Res Public Health, 2021 05 27;18(11).
    PMID: 34071861 DOI: 10.3390/ijerph18115741
    High-grade serous ovarian cancer (HGSC) is the most common ovarian cancer with highly metastatic properties. A small non-coding RNA, microRNA (miRNA) was discovered to be a major regulator in many types of cancers through binding at the 3'-untranslated region (3'UTR), leading to degradation of the mRNA. In this study, we sought to investigate the underlying mechanisms involved in the dysregulation of miR-200c-3p in HGSC progression and metastasis. We identified the upregulation of miR-200c-3p expression in different stages of HGSC clinical samples and the downregulation of the tumor suppressor gene, Deleted in Liver Cancer 1 (DLC1), expression. Over expression of miR-200c-3p in HGSC cell lines downregulated DLC1 but upregulated the epithelial marker, E-cadherin (CDH1). Based on in silico analysis, two putative binding sites were found within the 3'UTR of DLC1, and we confirmed the direct binding of miR-200c-3p to the target binding motif at position 1488-1495 bp of 3'UTR of DLC1 by luciferase reporter assay in a SKOV3 cell line co-transfected with vectors and miR-200c-3p mimic. These data showed that miR-200c-3p regulated the progression of HGSC by regulating DLC1 expression post-transcription and can be considered as a promising target for therapeutic purposes.
    Matched MeSH terms: 3' Untranslated Regions
  13. Fulltext Detection of FZD4, LRP5 and TSPAN12 Genes Variants in Malay Premature Babies with Retinopathy of Prematurity
    Mohd Khair SZN, Ismail AS, Embong Z, Mohamed Yusoff AA
    J Ophthalmic Vis Res, 2019 5 23;14(2):171-178.
    PMID: 31114654 DOI: 10.4103/jovr.jovr_210_17
    Purpose: To determine the mutational analyses of familial exudative vitreoretinopathy (FEVR)-causing genes in Malay patients with retinopathy of prematurity (ROP) to obtain preliminary data for gene alterations in the Malay community.

    Methods: A comparative cross-sectional study involving 86 Malay premature babies (ROP = 41 and non-ROP = 45) was performed from September 2012 to December 2014. Mutation analyses in (FEVR)-causing genes (NDP, FZD4, LRP5, and TSPAN12) were performed using DNA from premature babies using polymerase chain reaction (PCR) and direct sequencing. Sequencing results were confirmed with PCR-Restriction Fragment Length Polymorphism (RFLP).

    Results: We found variants of FZD4, LRP5, and TSPAN12 in this study. One patient from each group showed a non-synonymous alteration in FZD4, c.502C>T (p.P168S). A synonymous variant of LRP5 [c.3357G>A (p.V1119V)] was found in 30 ROP and 28 non-ROP patients. Two variants of TSPAN12, c.765G>T (p.P255P) and c.*39C>T (3'UTR), were also recorded (29 and 21 in ROP, 33 and 26 in non-ROP, respectively). Gestational age and birth weight were found to be significantly associated with ROP (P value < 0.001 and 0.001, respectively).

    Conclusion: Analysis of data obtained from the ROP Malay population will enhance our understanding of these FEVR-causing gene variants. The c.3357G>A (p.V1119V) variant of LRP5, and c.765G>T (p.P255P) and c.*39C>T variants of TSPAN12 could be common polymorphisms in the Malay ethnic group; however, this requires further elucidation. Future studies using larger groups and higher numbers of advanced cases are necessary to evaluate the relationship between FEVR-causing gene variants and the risk of ROP susceptibility in Malaysian infants.

    Matched MeSH terms: 3' Untranslated Regions
  14. Fulltext Molecular Characterization and Gene Expression of Glutathione Peroxidase 1 in Tor tambroides Exposed to Temperature Stress
    Do TD, Thi Mai N, Duy Khoa TN, Abol-Munafi AB, Liew HJ, Kim CB, et al.
    Evol Bioinform Online, 2019;15:1176934319853580.
    PMID: 31236006 DOI: 10.1177/1176934319853580
    Temperature is an abiotic factor that affects various biological and physiological processes in fish. Temperature stress is known to increase the production of reactive oxygen species (ROS) that subsequently cause oxidative stress. Fish is known to evolve a system of antioxidant enzymes to reduce ROS toxicology. Glutathione peroxidase (GPx) family consists of key enzymes that protect fish from oxidative stress. In this study, full-length GPx1 cDNA (GenBank accession no. KY984468) of Tor tambroides was cloned and characterized by rapid amplification of cDNA ends (RACE). The 899-base-pair (bp) GPx1 cDNA includes a 576-bp open reading frame encoding for 191 amino acids, plus 28 bp of 5'-untranslated region (UTR) and 295 bp of 3'-UTR. Homology analysis revealed that GPx1 of T tambroides (Tor-GPx1) shared high similarity with GPx1 sequences of other fish species. The phylogenetic construction based on the amino acid sequence showed that Tor-GPx1 formed a clade with GPx1 sequences of various fish species. Real-time polymerase chain reaction (PCR) was performed to assess the levels of GPx1 gene expression in the liver and muscle of T tambroides under thermal stress. The results indicated that GPx1 gene expression was down-regulated under decreased temperature. However, there was no significant difference between GPx1 gene expression in fish exposed to high temperature and control. Our study provides the first data regarding GPx gene expression in T tambroides under thermal stress.
    Matched MeSH terms: 3' Untranslated Regions
  15. Study of the CTLA-4 gene polymorphisms in systemic lupus erythematosus (SLE) samples from Malaysia
    Chua KH, Puah SM, Chew CH, Tan SY, Lian LH
    Ann Hum Biol, 2010 Apr;37(2):274-80.
    PMID: 19951233 DOI: 10.3109/03014460903325185
    In this study, we investigated the polymorphisms of the exon 1 (+49A/G), promoter sites (-1722T/C, -1661A/G, -318C/T), and 3'-untranslated region (3'-UTR) (+6230 A/G) of the CTLA-4 gene in systemic lupus erythematosus (SLE) affected patients. Polymerase chain reaction-restriction fragment length polymorphism was used to determine genotypes of these five markers in 130 SLE patients and 130 healthy controls. Of the five tested polymorphisms, there was no statistical significant difference between the genotypic and allelic frequencies of patients with SLE and controls. Hence, we propose that the CTLA-4 gene does not play a major role in the genetic susceptibility to the development of SLE in the Malaysian population.
    Matched MeSH terms: 3' Untranslated Regions
  16. MicroRNA-367-3p induces apoptosis and suppresses migration of MCF-7 cells by downregulating the expression of human choline kinase α
    Raikundalia S, Sa'Dom SAFM, Few LL, Too WCS
    Oncol Lett, 2021 Mar;21(3):183.
    PMID: 33574922 DOI: 10.3892/ol.2021.12444
    Choline kinase (ChK) catalyzes the first step in the CDP-choline pathway for the synthesis of phosphatidylcholine. The α isoform of this enzyme is overexpressed in various types of cancer and its inhibition or downregulation has been applied as an anticancer strategy. In spite of increasing attention being paid to ChK expression, as well as its activity and inhibition in cancer, there are only limited studies available on the regulation of ChK, including its regulation by microRNAs (miRNAs/miRs). The dysregulation of gene expression by miRNAs is a common cause for carcinogenesis. In the present study, miR-367-3p was predicted to target the 3'-untranslated region (UTR) of the ChK α (chka) mRNA transcript. The binding of miR-367-3p to the 3'-UTR of chka was validated by a luciferase assay. The effects of the miR-367-3p mimic on chka gene and protein expression levels were determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. miR-367-3p significantly downregulated the expression of chka to ~60% of the negative control. Cells transfected with miR-367-3p exhibited higher levels of apoptosis and a lower cell migration compared with the control. To the best of our knowledge, the present study provided the first experimental evidence of the regulation of chka expression by miR-367-3p. The pro-apoptotic and suppressive effects of miR-367-3p on cell migration were similar to the anticancer effects resulting from the inhibition of ChK enzyme activity or the knockdown of chka gene expression by small interfering RNA. Therefore, these findings may potentially lead to the use of miR-367-3p in anticancer strategies that target ChK.
    Matched MeSH terms: 3' Untranslated Regions
  17. Fulltext Emergence of Japanese encephalitis virus genotype V in the Republic of Korea
    Takhampunya R, Kim HC, Tippayachai B, Kengluecha A, Klein TA, Lee WJ, et al.
    Virol J, 2011;8:449.
    PMID: 21943222 DOI: 10.1186/1743-422X-8-449
    Japanese encephalitis virus (JEV) genotype V reemerged in Asia (China) in 2009 after a 57-year hiatus from the continent, thereby emphasizing a need to increase regional surveillance efforts. Genotypic characterization was performed on 19 JEV-positive mosquito pools (18 pools of Culex tritaeniorhynchus and 1 pool of Cx. bitaeniorhynchus) from a total of 64 positive pools collected from geographically different locations throughout the Republic of Korea (ROK) during 2008 and 2010.
    Matched MeSH terms: 3' Untranslated Regions/genetics
  18. Cyp2a5 Promoter-based Gene Reporter Assay: A Novel Design of Cell-based Bioassay for Toxicity Prediction
    Abu-Bakar A, Hu H, Lang MA
    Basic Clin Pharmacol Toxicol, 2018 Sep;123 Suppl 5:72-80.
    PMID: 29788535 DOI: 10.1111/bcpt.13046
    The murine cytochrome P450 2a5 (Cyp2a5) gene is regulated by complex interactions of various stress-activated transcription factors (TFs). Elevated Cyp2a5 transcription under chemical-induced stress conditions is achieved by interplay between the various TFs - including as aryl hydrocarbon receptor (AhR) and nuclear factor (erythroid-derived 2)-like 2 wild-type (Nrf2) - at the 'stress-responding' cluster of response elements on the Cyp2a5 promoter, as well as through mRNA stabilization mediated by interaction of the stress-activated heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with the 3'-UTR of the CYP2A5 mRNA. We designed a unique toxicity pathway-based reporter assay to include regulatory regions from both the 5' and the 3' untranslated regions of Cyp2a5 in a luciferase reporter plasmid to reflect in vivo responses to chemical insult. Human breast cancer MCF-7 cells were stably transfected with pGL4.38-Cyp2a5_Wt3k (wild-type) or mutant - pGL4.38-Cyp2a5_StREMut and pGL4.38-Cyp2a5_XREMut - reporter gene to monitor chemical-induced cellular response mediated by AhR and Nrf2 signalling. The recombinant cells were treated with representative of AhR agonist, polycyclic aromatic hydrocarbons, brominated flame retardant, fluorosurfactant, aromatic organic compound and metal, to determine the sensitivity of the Cyp2a5 promoter-based gene reporter assays to chemical insults by measuring the LC50 and EC50 of the respective chemicals. The three assays are sensitive to sublethal cellular responses of chemicals, which is an ideal feature for toxicity pathway-based bioassay for toxicity prediction. The wild-type reporter responded well to chemicals that activate crosstalk between the AhR and Nrf2, whilst the mutant reporters effectively gauge cellular response driven by either Nrf2/StRE or AhR/XRE signalling. Thus, the three gene reporter assays could be used tandemly to determine the predominant toxicity pathway of a given compound.
    Matched MeSH terms: 3' Untranslated Regions/genetics
  19. Characterization of Malaysian velogenic NDV strain AF2240-I genomic sequence: a comparative study
    Murulitharan K, Yusoff K, Omar AR, Molouki A
    Virus Genes, 2013 Jun;46(3):431-40.
    PMID: 23306943 DOI: 10.1007/s11262-012-0874-y
    Newcastle disease virus (NDV) strain AF2240 is a viscerotropic velogenic strain that is used as a vaccine challenge virus in Malaysia. The identification of the full length genome will be a crucial platform for further studies of this isolate. In this study, we fully sequenced the genome of a derivative of this strain named AF2240-I. The 15,192 nt long genome contains a 55-nt leader sequence at the 3' whereas the trailer region consists of 114 nt at the 5'. The intergenic sequences between the NP-P, P-M, M-F, F-HN, and HN-L genes comprise 1, 1, 1, 31, and 47 nt, respectively. The acknowledged cleavage site of fusion protein showed amino acid sequence of 112-R-R-Q-K-R-F-117, which corresponds to those of virulent NDV strains. Phylogenetic analysis of the whole virus genome shows that the strain AF2240-I belongs to genotype VIII and is more closely related to velogenic strains QH1, QH4, Fontana, Largo, and Italienas compared to other strains of NDV. Differences are noticed in the hemagglutinin-neuraminidase (HN) and matrix (M) gene between AF2240 and its derivative AF2240-I. This is the first report of a complete genome sequence of an NDV strain isolated in Malaysia.
    Matched MeSH terms: 3' Untranslated Regions
  20. miR-608 regulates apoptosis in human lung adenocarcinoma via regulation of AKT2
    Othman N, Nagoor NH
    Int J Oncol, 2017 Dec;51(6):1757-1764.
    PMID: 29075783 DOI: 10.3892/ijo.2017.4174
    Lung cancer remains a major health problem with a low 5-year survival rate of patients. Recent studies have shown that dysregulation of microRNAs (miRNAs) are prevalent in lung cancer and these aberrations play a significant role in the progression of tumour progression. In the present study, bioinformatics analyses was employed to predict potential miR-608 targets, which are associated with signaling pathways involved in cancer. Luciferase reporter assay identified AKT2 as a novel target of miR-608, and suppression of its protein levels was validated through western blot analysis. Zebrafish embryos were microinjected with cells transfected with miR-608 to elucidate the role of miR-608 in vivo, and immunostained with antibodies to detect activated caspase-3. We present the first evidence that miR-608 behaves as a tumour suppressor in A549 and SK-LU-1 cells through the regulation of AKT2, suggesting that selective targeting of AKT2 via miR-608 may be developed as a potential therapeutic strategy for miRNA-based non-small cell lung cancer (NSCLC) therapy.
    Matched MeSH terms: 3' Untranslated Regions
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