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Fulltext Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants: Application of a points-based ACMG/AMP approach

Thomassen M, Mesman RLS, Hansen TVO, Menendez M, Rossing M, Esteban-Sánchez A, et al.

Hum Mutat, 2022 Dec;43(12):1921-1944.
PMID: 35979650 DOI: 10.1002/humu.24449

Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength.

Matched MeSH terms: RNA, Messenger/genetics
Fulltext Differential proteome analysis of chikungunya virus infection on host cells

Thio CL, Yusof R, Abdul-Rahman PS, Karsani SA

PLoS One, 2013;8(4):e61444.
PMID: 23593481 DOI: 10.1371/journal.pone.0061444

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach.

Matched MeSH terms: RNA, Messenger/genetics
Expression profiles of defence related cDNAs in oil palm (Elaeis guineensis Jacq.) inoculated with mycorrhizae and Trichoderma harzianum Rifai T32

Tan YC, Wong MY, Ho CL

Plant Physiol Biochem, 2015 Nov;96:296-300.
PMID: 26322853 DOI: 10.1016/j.plaphy.2015.08.014

Basal stem rot is one of the major diseases of oil palm (Elaies guineensis Jacq.) caused by pathogenic Ganoderma species. Trichoderma and mycorrhizae were proposed to be able to reduce the disease severity. However, their roles in improving oil palm defence system by possibly inducing defence-related genes in the host are not well characterized. To better understand that, transcript profiles of eleven putative defence-related cDNAs in the roots of oil palm inoculated with Trichoderma harzianum T32 and mycorrhizae at different time points were studied. Transcripts encoding putative Bowman-Birk protease inhibitor (EgBBI2) and defensin (EgDFS) increased more than 2 fold in mycorrhizae-treated roots at 6 weeks post inoculation (wpi) compared to those in controls. Transcripts encoding putative dehydrin (EgDHN), glycine-rich RNA binding protein (EgGRRBP), isoflavone reductase (EgIFR), type 2 ribosome inactivating protein (EgT2RIP), and EgDFS increased in the oil palm roots treated with T. harzianum at 6 and/or 12 wpi compared to those in the controls. Some of these genes were also expressed in oil palm roots treated with Ganoderma boninense. This study provides an insight of some defence-related genes induced by Trichoderma and mycorrhizae, and their roles as potential agents to boost the plant defence system.

Matched MeSH terms: RNA, Messenger/genetics
Deep parallel sequencing reveals conserved and novel miRNAs in gill and hepatopancreas of giant freshwater prawn

Tan TT, Chen M, Harikrishna JA, Khairuddin N, Mohd Shamsudin MI, Zhang G, et al.

Fish Shellfish Immunol, 2013 Oct;35(4):1061-9.
PMID: 23816854 DOI: 10.1016/j.fsi.2013.06.017

MicroRNAs (miRNAs) are ~20-22 nucleotides, non protein-coding RNA regulatory genes that post-transcriptionally regulate many protein-coding genes, influencing critical biological and metabolic processes. While the number of known microRNA is increasing, there is currently no published data for miRNA from giant freshwater prawns, Macrobrachium rosenbergii (M. rosenbergii), a commercially cultured and economically important food species. In this study, we identified novel miRNAs in the gill and hepatopancreas of M. rosenbergii. Through a deep parallel sequencing analysis and an in silico data analysis approach, 327 miRNA families were identified from small RNA libraries with reference to both the de novo transcriptome of M. rosenbergii obtained from RNA-Seq and to miRBase (Release 18.0, November 2012). Based on the identified mature miRNA and recovered precursor sequences that form appropriate hairpin structures, three conserved miRNA (miR125, miR750, miR993) and 27 novel miRNA candidates encoding messenger-like non-coding RNA were identified. miR-125, miR-750, G-m0002/H-m0009, G-m0005, G-m0008/H-m0016, G-m0011/H-m0027 and G-m0015 were selected for experimental validation with stem-loop quantitative RT-PCR and were found to be coherent with the expression profile of deep sequencing data as evaluated with Pearson's correlation coefficient (r = 0.835178 for miRNA in gill, r = 0.724131 for miRNA in hepatopancreas). Using a combinatorial approach of pathway enrichment analysis and inverse expression relationship of miRNA and mRNA, four co-expressed novel miRNA candidates (G-m0005, G-m0008/H-m0016, G-m0011/H-m0027, and G-m0015) were found to be associated with energy metabolism. In addition, the expression of the three novel miRNA candidates (G-m0005, G-m0008/H-m0016, and G-m0011/H-m0027) were also found to be significantly reduced at 9 and 24 h post infection in M. rosenbergii challenged with infectious hypodermal and hematopoietic necrosis virus, suggesting a functional role of these miRNAs in crustacean immune defense.

Matched MeSH terms: RNA, Messenger/genetics
Elucidating the roles of miR-372 in cell proliferation and apoptosis of nasopharyngeal carcinoma TW01 cells

Tan JK, Tan EL, Gan SY

Exp Oncol, 2014 Sep;36(3):170-3.
PMID: 25265349

Deregulation of microRNA has been associated with cancer progression and the modification of cancer phenotypes could be achieved by targeting microRNA expression. This study aimed to determine the effects of miR-372 on cell progression and gene expression in nasopharyngeal carcinoma cell line, TW01.

Matched MeSH terms: RNA, Messenger/genetics
Fulltext Neonatal feed restriction modulates circulating levels of corticosterone and expression of glucocorticoid receptor and heat shock protein 70 in aged Japanese quail exposed to acute heat stress

Soleimani AF, Zulkifli I, Omar AR, Raha AR

Poult Sci, 2011 Jul;90(7):1427-34.
PMID: 21673157 DOI: 10.3382/ps.2011-01403

This study aimed to determine the effect of neonatal feed restriction on plasma corticosterone concentration (CORT), hippocampal glucocorticoid receptor (GR) expression, and heat shock protein (Hsp) 70 expression in aged male Japanese quail subjected to acute heat stress. Equal numbers of chicks were subjected to either ad libitum feeding (AL) or 60% feed restriction on d 4, 5, and 6 (FR). At 21 (young) and 270 (aged) d of age, birds were exposed to 43 ± 1°C for 1 h. Blood and hippocampus samples were collected to determine CORT and Hsp 70 and GR expressions before heat stress and following 1 h of heat stress, 1 h of post-heat stress recovery, and 2 h of post-heat stress recovery. With the use of real-time PCR and enzyme immunoassay, we examined the hippocampal expression of GR and Hsp 70 and CORT. The GR expression of the young birds increased following heat stress and remained consistent throughout the period of recovery. Conversely, no significant changes were noted on GR expression of aged birds. Although both young and aged birds had similar CORT before and during heat stress, the latter exhibited greater values following 1 and 2 h of recovery. Within the young group, feeding regimens had no significant effect on Hsp 70 expression. However, neonatal feed restriction improved Hsp 70 expression in aged birds. Neonatal feed restriction, compared with the AL group, resulted in higher CORT on d 21 but the converse was noted on d 270. Neonatal feed restriction appears to set a robust reactive hypothalamo-pituitary-adrenal response allowing the development of adaptive, healthy, and resilient phenotypes in aged quail as measured by a higher hippocampal Hsp 70 expression along with lower CORT.

Matched MeSH terms: RNA, Messenger/genetics
Fulltext Invasion and Metastasis Suppression by Anti-Neonatal Nav1.5 Antibodies in Breast Cancer

Sharudin NA, Murtadha Noor Din AH, Azahar II, Mohd Azlan M, Yaacob NS, Sarmiento ME, et al.

Asian Pac J Cancer Prev, 2022 Sep 01;23(9):2953-2964.
PMID: 36172657 DOI: 10.31557/APJCP.2022.23.9.2953

BACKGROUND: Detectable neonatal Nav1.5 (nNav1.5) expression in tumour breast tissue positive for lymph node metastasis and triple-negative subtype serves as a valid tumour-associated antigen to target and prevent breast cancer invasion and metastasis. Therapeutic antibodies against tumour antigens have become the predominant class of new drugs in cancer therapy because of their fewer adverse effects and high specificity.
OBJECTIVE: This study was designed to investigate the therapeutic and anti-metastatic potential of the two newly obtained anti-nNav1.5 antibodies, polyclonal anti-nNav1.5 (pAb-nNav1.5) and monoclonal anti-nNav1.5 (mAb-nNav1.5), on breast cancer invasion and metastasis.
METHODS: MDA-MB-231 and 4T1 cells were used as in vitro models to study the effect of pAb-nNav1.5 (59.2 µg/ml) and mAb-nNav1.5 (10 µg/ml) (24 hours treatment) on cell invasion. 4T1-induced mammary tumours in BALB/c female mice were used as an in vivo model to study the effect of a single dose of intravenous pAb-nNav1.5 (1 mg/ml) and mAb-nNav1.5 (1 mg/ml) on the occurrence of metastasis. Real-time PCR and immunofluorescence staining were conducted to assess the effect of antibody treatment on nNav1.5 mRNA and protein expression, respectively. The animals' body weight, organs, lesions, and tumour mass were also measured and compared.
RESULTS: pAb-nNav1.5 and mAb-nNav1.5 treatments effectively suppressed the invasion of MDA-MB-231 and 4T1 cells in the 3D spheroid invasion assay. Both antibodies significantly reduced nNav1.5 gene and protein expression in these cell lines. Treatment with pAb-nNav1.5 and mAb-nNav1.5 successfully reduced mammary tumour tissue size and mass and prevented lesions in vital organs of the mammary tumour animal model whilst maintaining the animal's healthy weight. mRNA expression of nNav1.5 in mammary tumour tissues was only reduced by mAb-nNav1.5.
CONCLUSION: Overall, this work verifies the uniqueness of targeting nNav1.5 in breast cancer invasion and metastasis prevention, but more importantly, humanised versions of mAb-nNav1.5 may be valuable passive immunotherapeutic agents to target nNav1.5 in breast cancer.

Matched MeSH terms: RNA, Messenger/genetics
Fulltext Reference gene validation for gene expression normalization in canine osteosarcoma: a geNorm algorithm approach

Selvarajah GT, Bonestroo FAS, Timmermans Sprang EPM, Kirpensteijn J, Mol JA

BMC Vet Res, 2017 Nov 25;13(1):354.
PMID: 29178874 DOI: 10.1186/s12917-017-1281-3

BACKGROUND: Quantitative PCR (qPCR) is a common method for quantifying mRNA expression. Given the heterogeneity present in tumor tissues, it is crucial to normalize target mRNA expression data using appropriate reference genes that are stably expressed under a variety of pathological and experimental conditions. No studies have validated specific reference genes in canine osteosarcoma (OS). Previous gene expression studies involving canine OS have used one or two reference genes to normalize gene expression. This study aimed to validate a panel of reference genes commonly used for normalization of canine OS gene expression data using the geNorm algorithm. qPCR analysis of nine canine reference genes was performed on 40 snap-frozen primary OS tumors and seven cell lines.
RESULTS: Tumors with a variety of clinical and pathological characteristics were selected. Gene expression stability and the optimal number of reference genes for gene expression normalization were calculated. RPS5 and HNRNPH were highly stable among OS cell lines, while RPS5 and RPS19 were the best combination for primary tumors. Pairwise variation analysis recommended four and two reference genes for optimal normalization of the expression data of canine OS tumors and cell lines, respectively.
CONCLUSIONS: Appropriate combinations of reference genes are recommended to normalize mRNA levels in canine OS tumors and cell lines to facilitate standardized and reliable quantification of target gene expression, which is essential for investigating key genes involved in canine OS metastasis and for comparative biomarker discovery.

Matched MeSH terms: RNA, Messenger/genetics
Palm tocotrienols inhibit proliferation of murine mammary cancer cells and induce expression of interleukin-24 mRNA

Selvaduray KR, Radhakrishnan AK, Kutty MK, Nesaretnam K

J Interferon Cytokine Res, 2010 Dec;30(12):909-16.
PMID: 21121862 DOI: 10.1089/jir.2010.0021

Several mechanisms have been postulated for the anticancer effects of tocotrienols. In this study, for the first time, the anticancer effect of tocotrienols is linked to increased expression of interleukin-24 (IL-24) mRNA, a cytokine reported to have antitumor effects in many cancer models. Tocotrienol isomers (α-T3, γ-T3, and δ-T3) and tocotrienol-rich fraction (TRF) inhibited the growth of the 4T1 murine mammary cancer cells (P γ-T3 > TRF > α-T3 > α-T, which was similar to their antiproliferative effects. The IL-24 mRNA levels in tumor tissues of BALB/c mice supplemented with TRF increased 2-fold when compared with control mice. Increased levels of IL-24 have been associated with inhibition of tumor growth and angiogenesis. Treatment of 4T1 cells with TRF and δ-T3 significantly decreased IL-8 and vascular endothelial growth factor mRNA levels. Hence, we report that tocotrienols have potent antiangiogenic and antitumor effects that is associated with increased levels of IL-24 mRNA.

Matched MeSH terms: RNA, Messenger/genetics
Testosterone Induces Increase in Aquaporin (AQP)-1, 5, and 7 Expressions in the Uteri of Ovariectomized Rats

Salleh N, Mokhtar HM, Kassim NM, Giribabu N

J. Membr. Biol., 2015 Dec;248(6):1097-105.
PMID: 26198330 DOI: 10.1007/s00232-015-9823-8

Testosterone has been reported to cause a decrease in uterine fluid volume in which this could involve the aquaporins (AQPs). This study aimed to investigate effect of testosterone on uterine AQP-1, 5, and 7 expressions in order to explain the reported reduction in uterine fluid volume under testosterone influence. Ovariectomized adult female rats received peanut oil, testosterone (1 mg/kg/day), estrogen (0.2 µg/kg/day), or combined estrogen plus testosterone for three consecutive days. Other groups received 3 days estrogen followed by 2 days either peanut oil or testosterone with or without flutamide or finasteride. A day after last injection, uteri were harvested, and the levels of AQP-1, 5, and 7 messenger RNA (mRNA) in uterine tissue homogenates were analyzed by real-time PCR (qPCR). Distributions of AQP-1, 5, and 7 proteins in uterus were observed by immunofluorescence. Levels of AQP-1 mRNA were elevated in rats receiving either estrogen or testosterone-only treatment; however, levels of AQP-5 and 7 mRNAs were elevated in rats receiving testosterone-only treatment. In rats pre-treated with estrogen, testosterone treatment resulted in higher AQP-1, 5, and 7 mRNA levels compared to vehicle treatment. Testosterone effects were antagonized by flutamide but not finasteride. Immunofluorescence study showed that AQP-1 was highly distributed in uterine lumenal epithelium following estrogen or testosterone-only treatment. However, AQP-5 and 7 distributions were high in uterine lumenal epithelium following testosterone-only treatment. Testosterone-induced up-regulation of AQP-1, 5, and 7 expressions in uterus could explain the observed reduction in uterine fluid volume as reported under this condition.

Matched MeSH terms: RNA, Messenger/genetics
Fulltext Immunogenicity mechanism of mRNA vaccines and their limitations in promoting adaptive protection against SARS-CoV-2

Salleh MZ, Norazmi MN, Deris ZZ

PeerJ, 2022;10:e13083.
PMID: 35287350 DOI: 10.7717/peerj.13083

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) in late 2019, hundreds of millions of people have been infected worldwide. There have been unprecedented efforts in acquiring effective vaccines to confer protection against the disease. mRNA vaccines have emerged as promising alternatives to conventional vaccines due to their high potency with the capacity for rapid development and low manufacturing costs. In this review, we summarize the currently available vaccines against SARS-CoV-2 in development, with the focus on the concepts of mRNA vaccines, their antigen selection, delivery and optimization to increase the immunostimulatory capability of mRNA as well as its stability and translatability. We also discuss the host immune responses to the SARS-CoV-2 infection and expound in detail, the adaptive immune response upon immunization with mRNA vaccines, in which high levels of spike-specific IgG and neutralizing antibodies were detected after two-dose vaccination. mRNA vaccines have been shown to induce a robust CD8+T cell response, with a balanced CD4+ TH1/TH2 response. We further discuss the challenges and limitations of COVID-19 mRNA vaccines, where newly emerging variants of SARS-CoV-2 may render currently deployed vaccines less effective. Imbalanced and inappropriate inflammatory responses, resulting from hyper-activation of pro-inflammatory cytokines, which may lead to vaccine-associated enhanced respiratory disease (VAERD) and rare cases of myocarditis and pericarditis also are discussed.

Matched MeSH terms: RNA, Messenger/genetics
Fulltext Effects of conjugated linoleic acid, fish oil and soybean oil on PPARs (α & γ) mRNA expression in broiler chickens and their relation to body fat deposits

Royan M, Meng GY, Othman F, Sazili AQ, Navidshad B

Int J Mol Sci, 2011;12(12):8581-95.
PMID: 22272093 DOI: 10.3390/ijms12128581

An experiment was conducted on broiler chickens to study the effects of different dietary fats (Conjugated linoleic acid (CLA), fish oil, soybean oil, or their mixtures, as well as palm oil, as a more saturated fat), with a as fed dose of 7% for single fat and 3.5 + 3.5% for the mixtures, on Peroxisome Proliferator-Activated Receptors (PPARs) gene expression and its relation with body fat deposits. The CLA used in this experiment was CLA LUTA60 which contained 60% CLA, so 7% and 3.5% dietary inclusions of CLA LUTA60 were equal to 4.2% and 2.1% CLA, respectively. Higher abdominal fat pad was found in broiler chickens fed with a diet containing palm oil compared to chickens in the other experimental groups (P ≤ 0.05). The diets containing CLA resulted in an increased fat deposition in the liver of broiler chickens (P ≤ 0.05). The only exception was related to the birds fed with diets containing palm oil or fish oil + soybean oil, where contents of liver fat were compared to the CLA + fish oil treatment. PPARγ gene in adipose tissue of chickens fed with palm oil diet was up-regulated compared to other treatments (P ≤ 0.001), whereas no significant differences were found in adipose PPARγ gene expression between chickens fed with diets containing CLA, fish oil, soybean oil or the mixture of these fats. On the other hand, the PPARα gene expression in liver tissue was up-regulated in response to the dietary fish oil inclusion and the differences were also significant for both fish oil and CLA + fish oil diets compared to the diets with palm oil, soybean oil or CLA as the only oil source (P ≤ 0.001). In conclusion, the results of present study showed that there was a relationship between the adipose PPARγ gene up-regulation and abdominal fat pad deposition for birds fed with palm oil diet, while no deference was detected in n-3 and n-6 fatty acids, as well as CLA on PPARγ down regulation in comparison to a more saturated fat. When used on its own, fish oil was found to be a more effective fat in up-regulating hepatic PPARα gene expression and this effect was related to a less fat deposition in liver tissue. A negative correlation coefficient (-0.3) between PPARα relative gene expression and liver tissue fat content confirm the anti-lipogenic effect of PPARα, however, the change in these parameters was not completely parallel.

Matched MeSH terms: RNA, Messenger/genetics
The role of specific Smad linker region phosphorylation in TGF-β mediated expression of glycosaminoglycan synthesizing enzymes in vascular smooth muscle

Rostam MA, Kamato D, Piva TJ, Zheng W, Little PJ, Osman N

Cell Signal, 2016 08;28(8):956-66.
PMID: 27153775 DOI: 10.1016/j.cellsig.2016.05.002

Hyperelongation of glycosaminoglycan chains on proteoglycans facilitates increased lipoprotein binding in the blood vessel wall and the development of atherosclerosis. Increased mRNA expression of glycosaminoglycan chain synthesizing enzymes in vivo is associated with the development of atherosclerosis. In human vascular smooth muscle, transforming growth factor-β (TGF-β) regulates glycosaminoglycan chain hyperelongation via ERK and p38 as well as Smad2 linker region (Smad2L) phosphorylation. In this study, we identified the involvement of TGF-β receptor, intracellular serine/threonine kinases and specific residues on transcription factor Smad2L that regulate glycosaminoglycan synthesizing enzymes. Of six glycosaminoglycan synthesizing enzymes, xylosyltransferase-1, chondroitin sulfate synthase-1, and chondroitin sulfotransferase-1 were regulated by TGF-β. In addition ERK, p38, PI3K and CDK were found to differentially regulate mRNA expression of each enzyme. Four individual residues in the TGF-β receptor mediator Smad2L can be phosphorylated by these kinases and in turn regulate the synthesis and activity of glycosaminoglycan synthesizing enzymes. Smad2L Thr220 was phosphorylated by CDKs and Smad2L Ser250 by ERK. p38 selectively signalled via Smad2L Ser245. Phosphorylation of Smad2L serine residues induced glycosaminoglycan synthesizing enzymes associated with glycosaminoglycan chain elongation. Phosphorylation of Smad2L Thr220 was associated with XT-1 enzyme regulation, a critical enzyme in chain initiation. These findings provide a deeper understanding of the complex signalling pathways that contribute to glycosaminoglycan chain modification that could be targeted using pharmacological agents to inhibit the development of atherosclerosis.

Matched MeSH terms: RNA, Messenger/genetics
Isolation and characterization of differentially expressed transcripts from the suspension cells of oil palm (Elaeis guineensis Jacq.) in response to different concentration of auxins

Roowi SH, Ho CL, Alwee SS, Abdullah MO, Napis S

Mol Biotechnol, 2010 Sep;46(1):1-19.
PMID: 20390382 DOI: 10.1007/s12033-010-9262-9

Oil palm suspension cultures were initiated by transferring the gel-like friable embryogenic tissue onto liquid medium supplemented with auxins. In this study, transcripts that were differentially expressed in oil palm suspension cells cultured at different auxin concentrations were examined using suppression subtractive hybridization. Total RNA was first isolated from oil palm suspension cells proliferated in liquid medium with different hormone concentrations for 6 months. Four different hormone combinations: T1 (0.1 mg/l 2,4-D and 1.0 mg/l NAA), T2 (0.4 mg/l 2,4-D and 1.0 mg/l NAA), T3 (1.0 mg/l NAA), and T4 (0.4 mg/l 2,4-D) were used for the treatments. The first and second subtractions were performed using samples T1 and T2 in forward and reverse order. The other two subtractions were forward and reverse subtractions of T3 and T4, respectively. Reverse northern analyses showed that 14.13% of these clones were preferentially expressed in T1, 13.70% in T2, 14.75% in T3, and 15.70% in T4. Among the 294 cDNA clones that were sequenced, 61 contigs (assembled from 165 sequences) and 129 singletons were obtained. Among the 61 contigs, 10 contigs consist of sequences from treatment T1, 8 contigs were from treatment T2, 10 contigs were contains sequences of treatment T3 and 13 contigs contains sequences of treatment T4. Northern analyses of five transcripts that were shown to be differentially expressed in the oil palm suspension cells by reverse northern analysis revealed that transcripts 16A1 (a putative lignostilbene-alpha,beta-dioxygenase, EgLSD) and 16H12 (a putative ethylene responsive 6, EgER6) were differentially expressed in oil palm suspension cells treated with different levels of auxin.

Matched MeSH terms: RNA, Messenger/genetics
Fulltext Mapping a Circular RNA-microRNA-mRNA-Signaling Regulatory Axis That Modulates Stemness Properties of Cancer Stem Cell Populations in Colorectal Cancer Spheroid Cells

Rengganaten V, Huang CJ, Tsai PH, Wang ML, Yang YP, Lan YT, et al.

Int J Mol Sci, 2020 Oct 23;21(21).
PMID: 33114016 DOI: 10.3390/ijms21217864

Spheroidal cancer cell cultures have been used to enrich cancer stem cells (CSC), which are thought to contribute to important clinical features of tumors. This study aimed to map the regulatory networks driven by circular RNAs (circRNAs) in CSC-enriched colorectal cancer (CRC) spheroid cells. The spheroid cells established from two CRC cell lines acquired stemness properties in pluripotency gene expression and multi-lineage differentiation capacity. Genome-wide sequencing identified 1503 and 636 circRNAs specific to the CRC parental and spheroid cells, respectively. In the CRC spheroids, algorithmic analyses unveiled a core network of mRNAs involved in modulating stemness-associated signaling pathways, driven by a circRNA-microRNA (miRNA)-mRNA axis. The two major circRNAs, hsa_circ_0066631 and hsa_circ_0082096, in this network were significantly up-regulated in expression levels in the spheroid cells. The two circRNAs were predicted to target and were experimentally shown to down-regulate miR-140-3p, miR-224, miR-382, miR-548c-3p and miR-579, confirming circRNA sponging of the targeted miRNAs. Furthermore, the affected miRNAs were demonstrated to inhibit degradation of six mRNA targets, viz. ACVR1C/ALK7, FZD3, IL6ST/GP130, SKIL/SNON, SMAD2 and WNT5, in the CRC spheroid cells. These mRNAs encode proteins that are reported to variously regulate the GP130/Stat, Activin/Nodal, TGF-β/SMAD or Wnt/β-catenin signaling pathways in controlling various aspects of CSC stemness. Using the CRC spheroid cell model, the novel circRNA-miRNA-mRNA axis mapped in this work forms the foundation for the elucidation of the molecular mechanisms of the complex cellular and biochemical processes that determine CSC stemness properties of cancer cells, and possibly for designing therapeutic strategies for CRC treatment by targeting CSC.

Matched MeSH terms: RNA, Messenger/genetics*
Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel

Ravanfar SA, Aziz MA, Saud HM, Abdullah JO

Curr Genet, 2015 Nov;61(4):653-63.
PMID: 25986972 DOI: 10.1007/s00294-015-0494-x

An efficient system for shoot regeneration and Agrobacterium tumefaciens-mediated transformation of Brassica oleracea cv. Green Marvel cultivar is described. This study focuses on developing shoot regeneration from hypocotyl explants of broccoli cv. Green Marvel using thidiazuron (TDZ), zeatin, and kinetin, the optimization of factors affecting Agrobacterium-mediated transformation of the hypocotyl explants with heat-resistant cDNA, followed by the confirmation of transgenicity of the regenerants. High shoot regeneration was observed in 0.05-0.1 mg dm(-3) TDZ. TDZ at 0.1 mg dm(-3) produced among the highest percentage of shoot regeneration (96.67 %) and mean number of shoot formation (6.17). The highest percentage (13.33 %) and mean number (0.17) of putative transformant production were on hypocotyl explants subjected to preculture on shoot regeneration medium (SRM) with 200 µM acetosyringone. On optimization of bacterial density and inoculation time, the highest percentage and mean number of putative transformant production were on hypocotyl explants inoculated with a bacterial dilution of 1:5 for 30 min. Polymerase chain reaction (PCR) assay indicated a transformation efficiency of 8.33 %. The luciferase assay showed stable integration of the Arabidopsis thaliana HSP101 (AtHSP101) cDNA in the transgenic broccoli regenerants. Three out of five transgenic lines confirmed through PCR showed positive hybridization bands of the AtHSP101 cDNA through Southern blot analysis. The presence of AtHSP101 transcripts in the three transgenic broccoli lines indicated by reverse transcription-PCR (RT-PCR) confirmed the expression of the gene. In conclusion, an improved regeneration system has been established from hypocotyl explants of broccoli followed by successful transformation with AtHSP101 for resistance to high temperature.

Matched MeSH terms: RNA, Messenger/genetics
A B-myb--DREAM complex is not critical to regulate the G2/M genes in HPV-transformed cell lines

Rashid NN, Yusof R, Watson RJ

Anticancer Res, 2014 Nov;34(11):6557-63.
PMID: 25368258

It is well-established that HPV E7 proteins, encoded by human papillomavirus (HPV) genes, frequently associated with cervical cancers bind avidly to the retinoblastoma (RB) family of pocket proteins and disrupt their association with members of the E2F transcription factor family. Our previous study showed that the repressive p130-dimerization partner, RB-like, E2F and multi-vulval class (DREAM) complex was disrupted by HPV16 E7 proteins in order to maintain the viral replication in CaSki cells. However, we would like to address whether the activator B-myb-DREAM complex is critical in regulating the replication and mitosis phase since our previous study showed increased B-myb-DREAM expression in HPV-transformed cell lines when compared to control cells.

Matched MeSH terms: RNA, Messenger/genetics
Effects of a high-salt diet on MAP and expression levels of renal ENaCs and aquaporins in SHR

Ramachandran CD, Gholami K, Lam SK, Hoe SZ

Exp Biol Med (Maywood), 2023 Oct;248(20):1768-1779.
PMID: 37828834 DOI: 10.1177/15353702231198085

An increase in blood pressure by a high-salt (HS) diet may change the expression levels of renal epithelial sodium channels (ENaCs) and aquaporins (AQPs). Spontaneously hypertensive rats (SHRs) and Wistar Kyoto (WKY) rats were exposed to HS and regular-salt (RS) diets for 6 weeks. Mean arterial pressure (MAP) and plasma atrial natriuretic peptide (ANP), angiotensin II (Ang II), aldosterone, and arginine vasopressin (AVP) levels were determined. Expression of mRNA levels of ENaCs and AQPs were quantified by real-time PCR. The MAP was higher in SHRs on the HS diet. Plasma Ang II and aldosterone levels were low while plasma ANP level was high in both strains of rats. Renal expression of mRNA levels of α-, β-, and γ-ENaCs was lowered in SHRs on the HS diet. Meanwhile, renal AQP1, AQP2, and AQP7 mRNA expression levels were lowered in both strains of rats on the HS diet. Suppression of mRNA expression levels of ENaC and AQP subunits suggests that the high-salt-induced increase in the MAP of SHR may not be solely due to renal sodium and water retention.

Matched MeSH terms: RNA, Messenger/genetics
A variant e13a3 BCR-ABL1 fusion transcript in refractory adult B-cell acute lymphoblastic leukemia achieving complete remission with CAR-Tcell therapy

Phan CL, Tan SN, Tan SM, Kadir SSSA, Ramli NLM, Lim TO, et al.

Cancer Genet, 2021 01;250-251:20-24.
PMID: 33220656 DOI: 10.1016/j.cancergen.2020.11.003

Acute lymphoblastic leukemia (ALL) cases with e13a3 fusion transcripts are extremely rare. We report a 24-year-old male with Ph-positive (Ph+) ALL with an aberrant e13a3 fusion transcript treated with CD19-specific chimeric antigen receptor T-cell (CAR-T) therapy. He developed refractory disease post-chemotherapy induction, andreceived allogeneic hematopoietic stem cell transplantation (allo-HSCT) after salvage with imatinib in combination with chemotherapy regimen. Unfortunately, the patient relapsed after +90 days post-transplant. He was consented to CAR-T therapy trial and achieved complete remission, highlighting the efficacy of CAR-T treatment in relapsed-refractory B-ALL irrespective of the underlying genetic drivers in leukemia cells .

Matched MeSH terms: RNA, Messenger/genetics*
Fulltext Evaluation of the immunogenicity of an mRNA vectored Nipah virus vaccine candidate in pigs

Pedrera M, McLean RK, Medfai L, Thakur N, Todd S, Marsh G, et al.

Front Immunol, 2024;15:1384417.
PMID: 38726013 DOI: 10.3389/fimmu.2024.1384417

Nipah virus (NiV) poses a significant threat to human and livestock populations across South and Southeast Asia. Vaccines are required to reduce the risk and impact of spillover infection events. Pigs can act as an intermediate amplifying host for NiV and, separately, provide a preclinical model for evaluating human vaccine candidate immunogenicity. The aim of this study was therefore to evaluate the immunogenicity of an mRNA vectored NiV vaccine candidate in pigs. Pigs were immunized twice with 100 μg nucleoside-modified mRNA vaccine encoding soluble G glycoprotein from the Malaysia strain of NiV, formulated in lipid nanoparticles. Potent antigen-binding and virus neutralizing antibodies were detected in serum following the booster immunization. Antibody responses effectively neutralized both the Malaysia and Bangladesh strains of NiV but showed limited neutralization of the related (about 80% amino acid sequence identity for G) Hendra virus. Antibodies were also capable of neutralizing NiV glycoprotein mediated cell-cell fusion. NiV G-specific T cell cytokine responses were also measurable following the booster immunization with evidence for induction of both CD4 and CD8 T cell responses. These data support the further evaluation of mRNA vectored NiV G as a vaccine for both pigs and humans.

Matched MeSH terms: RNA, Messenger/genetics

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