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Fulltext Differential expression of basal microRNAs' patterns in human dental pulp stem cells

Vasanthan P, Govindasamy V, Gnanasegaran N, Kunasekaran W, Musa S, Abu Kasim NH

J Cell Mol Med, 2015 Mar;19(3):566-80.
PMID: 25475098 DOI: 10.1111/jcmm.12381

MicroRNAs (miRNAs) are small non-coding RNAs that regulate translation of mRNA into protein and play a crucial role for almost all biological activities. However, the identification of miRNAs from mesenchymal stem cells (MSCs), especially from dental pulp, is poorly understood. In this study, dental pulp stem cells (DPSCs) were characterized in terms of their proliferation and differentiation capacity. Furthermore, 104 known mature miRNAs were profiled by using real-time PCR. Notably, we observed 19 up-regulated miRNAs and 29 significantly down-regulated miRNAs in DPSCs in comparison with bone marrow MSCs (BM-MSCs). The 19 up-regulated miRNAs were subjected to ingenuity analysis, which were composed into 25 functional networks. We have chosen top 2 functional networks, which comprised 10 miRNA (hsa-miR-516a-3p, hsa-miR-125b-1-3p, hsa-miR-221-5p, hsa-miR-7, hsa-miR-584-5p, hsa-miR-190a, hsa-miR-106a-5p, hsa-mir-376a-5p, hsa-mir-377-5p and hsa-let-7f-2-3p). Prediction of target mRNAs and associated biological pathways regulated by each of this miRNA was carried out. We paid special attention to hsa-miR-516a-3p and hsa-miR-7-5p as these miRNAs were highly expressed upon validation with qRT-PCR analysis. We further proceeded with loss-of-function analysis with these miRNAs and we observed that hsa-miR-516a-3p knockdown induced a significant increase in the expression of WNT5A. Likewise, the knockdown of hsa-miR-7-5p increased the expression of EGFR. Nevertheless, further validation revealed the role of WNT5A as an indirect target of hsa-miR-516a-3p. These results provide new insights into the dynamic role of miRNA expression in DPSCs. In conclusion, using miRNA signatures in human as a prediction tool will enable us to elucidate the biological processes occurring in DPSCs.

Matched MeSH terms: Gene Knockdown Techniques
Deregulation of hsa-miR-20b expression in TNF-α-induced premature senescence of human pulmonary microvascular endothelial cells

Wong PF, Jamal J, Tong KL, Khor ES, Yeap CE, Jong HL, et al.

Microvasc Res, 2017 11;114:26-33.
PMID: 28595801 DOI: 10.1016/j.mvr.2017.06.002

miRNAs are important regulators of cellular senescence yet the extent of their involvement remains to be investigated. We sought to identify miRNAs that are involved in cytokine-induced premature senescence (CIPS) in endothelial cells. CIPS was established in young human pulmonary microvascular endothelial cells (HMVEC-Ls) following treatment with a sublethal dose (20ng/ml) of tumor necrosis factor alpha (TNF-α) for 15days. In parallel, HMVEC-Ls were grown and routinely passaged until the onset of replicative senescence (RS). Differential expression analysis following miRNA microarray profiling revealed an overlapped of eight deregulated miRNAs in both the miRNA profiles of RS and TNF-α-induced premature senescence cells. Amongst the deregulated miRNAs were members of the miR 17-92 cluster which are known regulators of angiogenesis. The role of hsa-miR-20b in TNF-α-induced premature senescence, a paralog member of the miR 17-92 cluster, was further investigated. Biotin-labeled hsa-miR-20b captured the enriched transcripts of retinoblastoma-like 1 (RBL1), indicating that RBL1 is a target of hsa-miR-20b. Knockdown of hsa-miR-20b attenuated premature senescence in the TNF-α-treated HMVEC-Ls as evidenced by increased cell proliferation, increased RBL1 mRNA expression level but decreased protein expression of p16INK4a, a cellular senescence marker. These findings provide an early insight into the role of hsa-miR-20b in endothelial senescence.

Matched MeSH terms: Gene Knockdown Techniques
CTP synthase knockdown during early development distorts the nascent vertebral column and causes fluid retention in multiple tissues in zebrafish

Dzaki N, Wahab W, Azlan A, Azzam G

Biochem Biophys Res Commun, 2018 10 20;505(1):106-112.
PMID: 30241946 DOI: 10.1016/j.bbrc.2018.09.074

CTP Synthase (CTPS) is a metabolic enzyme that is recognized as a catalyst for nucleotide, phospholipid and sialoglycoprotein production. Though the structural characteristics and regulatory mechanisms of CTPS are well-understood, little is known regarding the extent of its involvement during the early developmental stages of vertebrates. Zebrafish carries two CTPS genes, annotated as ctps1a and ctps1b. Phylogenetic analyses show that both genes had diverged from homologues in the ancestral Actinopterygii, Oreochromis niloticus. Conservation of common CTPS-catalytic regions further establishes that both proteins are likely to be functionally similar to hsaCTPS. Here, we show that ctps1a is more critical throughout the initial period of embryonic development than ctps1b. The effects of concurrent partial knockdown are dependent on ctps1a vs ctps1b dosage ratios. When these are equally attenuated, abnormal phenotypes acquired prior to the pharyngula period disappear in hatchlings (48hpf); however, if either gene is more attenuated than the other, these only become more pronounced in advanced stages. Generally, disruption to normal ctps1a or ctps1b expression levels by morpholinos culminates in the distortion of the early spinal column as well as multiple-tissue oedema. Other effects include slower growth rates, increased mortality rates and impaired structural formation of the young fish's extremities. Embryos grown in DON, a glutamine-analogue drug and CTPS antagonist, also exhibit similar characteristics, thus strengthening the validity of the morpholino-induced phenotypes observed. Together, our results demonstrate the importance of CTPS for the development of zebrafish embryos, as well as a disparity in activity and overall importance amongst isozymes.

Matched MeSH terms: Gene Knockdown Techniques
Fulltext AP1S3 mutations are associated with pustular psoriasis and impaired Toll-like receptor 3 trafficking

Setta-Kaffetzi N, Simpson MA, Navarini AA, Patel VM, Lu HC, Allen MH, et al.

Am J Hum Genet, 2014 May 01;94(5):790-7.
PMID: 24791904 DOI: 10.1016/j.ajhg.2014.04.005

Adaptor protein complex 1 (AP-1) is an evolutionary conserved heterotetramer that promotes vesicular trafficking between the trans-Golgi network and the endosomes. The knockout of most murine AP-1 complex subunits is embryonically lethal, so the identification of human disease-associated alleles has the unique potential to deliver insights into gene function. Here, we report two founder mutations (c.11T>G [p.Phe4Cys] and c.97C>T [p.Arg33Trp]) in AP1S3, the gene encoding AP-1 complex subunit σ1C, in 15 unrelated individuals with a severe autoinflammatory skin disorder known as pustular psoriasis. Because the variants are predicted to destabilize the 3D structure of the AP-1 complex, we generated AP1S3-knockdown cell lines to investigate the consequences of AP-1 deficiency in skin keratinocytes. We found that AP1S3 silencing disrupted the endosomal translocation of the innate pattern-recognition receptor TLR-3 (Toll-like receptor 3) and resulted in a marked inhibition of downstream signaling. These findings identify pustular psoriasis as an autoinflammatory phenotype caused by defects in vesicular trafficking and demonstrate a requirement of AP-1 for Toll-like receptor homeostasis.

Matched MeSH terms: Gene Knockdown Techniques
Fulltext IFITM3 knockdown reduces the expression of CCND1 and CDK4 and suppresses the growth of oral squamous cell carcinoma cells

Gan CP, Sam KK, Yee PS, Zainal NS, Lee BKB, Abdul Rahman ZA, et al.

Cell Oncol (Dordr), 2019 Aug;42(4):477-490.
PMID: 30949979 DOI: 10.1007/s13402-019-00437-z

PURPOSE: Oral squamous cell carcinoma (OSCC) is a challenging disease to treat. Up to 50% of OSCC patients with advanced disease develop recurrences. Elucidation of key molecular mechanisms underlying OSCC development may provide opportunities to target specific genes and, thus, to improve patient survival. In this study, we examined the expression and functional role of interferon transmembrane protein 3 (IFITM3) in OSCC development.
METHODS: The expression of IFITM3 in OSCC and normal oral mucosal tissues was assessed by qRT-PCR and immunohistochemistry. The role of IFITM3 in driving OSCC cell proliferation and survival was examined using siRNA-mediated gene knockdown, and the role of IFITM3 in driving cell cycle regulators was examined using Western blotting.
RESULTS: We found that IFITM3 is overexpressed in more than 79% of primary OSCCs. We also found that IFITM3 knockdown led to impaired OSCC cell growth through inhibition of cell proliferation, induction of cell cycle arrest, senescence and apoptosis. In addition, we found that IFITM3 knockdown led to reduced expressions of CCND1 and CDK4 and reduced RB phosphorylation, leading to inhibition of OSCC cell growth. This information may be instrumental for the design of novel targeted therapeutic strategies.
CONCLUSIONS: From our data we conclude that IFITM3 is overexpressed in OSCC and may regulate the CCND1-CDK4/6-pRB axis to mediate OSCC cell growth.

Matched MeSH terms: Gene Knockdown Techniques*
Fulltext MicroRNA-362 negatively and positively regulates SMAD4 expression in TGF-β/SMAD signaling to suppress cell migration and invasion

Cheng HP, Huang CJ, Tsai ML, Ong HT, Cheong SK, Choo KB, et al.

Int J Med Sci, 2021;18(8):1798-1809.
PMID: 33746597 DOI: 10.7150/ijms.50871

Cell migration and invasion are modulated by epithelial-to-mesenchymal transition (EMT) and the reverse MET process. Despite the detection of microRNA-362 (miR-362, both the miR-362-5p and -3p species) in cancers, none of the identified miR-362 targets is a mesenchymal or epithelial factor to link miR-362 with EMT/MET and metastasis. Focusing on the TGF-β/SMAD signaling pathway in this work, luciferase assays and western blot data showed that miR-362 targeted and negatively regulated expression of SMAD4 and E-cadherin, but not SNAI1, which is regulated by SMAD4. However, miR-362 knockdown also down-regulated SMAD4 and SNAI1, but up-regulated E-cadherin expression. Wound-healing and transwell assays further showed that miR-362 knockdown suppressed cell migration and invasion, effects which were reversed by over-expressing SMAD4 or SNAI1, or by knocking down E-cadherin in the miR-362 knockdown cells. In orthotopic mice, miR-362 knockdown inhibited metastasis, and displayed the same SMAD4 and E-cadherin expression profiles in the tumors as in the in vitro studies. A scheme is proposed to integrate miR-362 negative regulation via SMAD4, and to explain miR-362 positive regulation of SMAD4 via miR-362 targeting of known SMAD4 suppressors, BRK and DACH1, which would have resulted in SMAD4 depletion and annulment of subsequent involvement in TGF-β signaling actions. Hence, miR-362 both negatively and positively regulates SMAD4 expression in TGF-β/SMAD signaling pathway to suppress cell motility and invasiveness and metastasis, and may explain the reported clinical association of anti-miR-362 with suppressed metastasis in various cancers. MiR-362 knockdown in miR-362-positive cancer cells may be used as a therapeutic strategy to suppress metastasis.

Matched MeSH terms: Gene Knockdown Techniques
Fulltext Network Analysis for the Identification of Differentially Expressed Hub Genes Using Myogenin Knock-down Muscle Satellite Cells

Malik A, Lee EJ, Jan AT, Ahmad S, Cho KH, Kim J, et al.

PLoS One, 2015;10(7):e0133597.
PMID: 26200109 DOI: 10.1371/journal.pone.0133597

Muscle, a multinucleate syncytium formed by the fusion of mononuclear myoblasts, arises from quiescent progenitors (satellite cells) via activation of muscle-specific transcription factors (MyoD, Myf5, myogenin: MYOG, and MRF4). Subsequent to a decline in Pax7, induction in the expression of MYOG is a hallmark of myoblasts that have entered the differentiation phase following cell cycle withdrawal. It is evident that MYOG function cannot be compensated by any other myogenic regulatory factors (MRFs). Despite a plethora of information available regarding MYOG, the mechanism by which MYOG regulates muscle cell differentiation has not yet been identified. Using an RNA-Seq approach, analysis of MYOG knock-down muscle satellite cells (MSCs) have shown that genes associated with cell cycle and division, DNA replication, and phosphate metabolism are differentially expressed. By constructing an interaction network of differentially expressed genes (DEGs) using GeneMANIA, cadherin-associated protein (CTNNA2) was identified as the main hub gene in the network with highest node degree. Four functional clusters (modules or communities) were identified in the network and the functional enrichment analysis revealed that genes included in these clusters significantly contribute to skeletal muscle development. To confirm this finding, in vitro studies revealed increased expression of CTNNA2 in MSCs on day 12 compared to day 10. Expression of CTNNA2 was decreased in MYOG knock-down cells. However, knocking down CTNNA2, which leads to increased expression of extracellular matrix (ECM) genes (type I collagen α1 and type I collagen α2) along with myostatin (MSTN), was not found significantly affecting the expression of MYOG in C2C12 cells. We therefore propose that MYOG exerts its regulatory effects by acting upstream of CTNNA2, which in turn regulates the differentiation of C2C12 cells via interaction with ECM genes. Taken together, these findings highlight a new mechanism by which MYOG interacts with CTNNA2 in order to promote myoblast differentiation.

Matched MeSH terms: Gene Knockdown Techniques*
Knockdown of c-MET induced apoptosis in ABCB1-overexpressed multidrug-resistance cancer cell lines

Hung TH, Li YH, Tseng CP, Lan YW, Hsu SC, Chen YH, et al.

Cancer Gene Ther, 2015 May;22(5):262-70.
PMID: 25908454 DOI: 10.1038/cgt.2015.15

Inappropriate c-MET signaling in cancer can enhance tumor cell proliferation, survival, motility, and invasion. Inhibition of c-MET signaling induces apoptosis in a variety of cancers. It has also been recognized as a novel anticancer therapy approach. Furthermore, reports have also indicated that constitutive expression of P-glycoprotein (ABCB1) is involved in the HGF/c-MET-related pathway of multidrug resistance ABCB1-positive human hepatocellular carcinoma cell lines. We previously reported that elevated expression levels of PKCδ and AP-1 downstream genes, and HGF receptor (c-MET) and ABCB1, in the drug-resistant MES-SA/Dx5 cells. Moreover, leukemia cell lines overexpressing ABCB1 have also been shown to be more resistant to the tyrosine kinase inhibitor imatinib mesylate. These findings suggest that chemoresistant cancer cells may also develop a similar mechanism against chemotherapy agents. To circumvent clinical complications arising from drug resistance during cancer therapy, the present study was designed to investigate apoptosis induction in ABCB1-overexpressed cancer cells using c-MET-targeted RNA interference technology in vitro and in vivo. The results showed that cell viability decreased and apoptosis rate increased in c-MET shRNA-transfected HGF/c-MET pathway-positive MES-SA/Dx5 and MCF-7/ADR2 cell lines in a dose-dependent manner. In vivo reduction of tumor volume in mice harboring c-MET shRNA-knockdown MES-SA/Dx5 cells was clearly demonstrated. Our study demonstrated that downregulation of c-MET by shRNA-induced apoptosis in a multidrug resistance cell line.

Matched MeSH terms: Gene Knockdown Techniques
Knockdown of ROS1 gene sensitizes breast tumor growth to doxorubicin in a syngeneic mouse model

Tiash S, Chua MJ, Chowdhury EH

Int J Oncol, 2016 Jun;48(6):2359-66.
PMID: 27035628 DOI: 10.3892/ijo.2016.3452

Treatment of breast cancer, the second leading cause of female deaths worldwide, with classical drugs is often accompanied by treatment failure and relapse of disease condition. Development of chemoresistance and drug toxicity compels compromising the drug concentration below the threshold level with the consequence of therapeutic inefficacy. Moreover, amplification and over-activation of proto-oncogenes in tumor cells make the treatment more challenging. The oncogene, ROS1 which is highly expressed in diverse types of cancers including breast carcinoma, functions as a survival protein aiding cancer progression. Thus we speculated that selective silencing of ROS1 gene by carrier-mediated delivery of siRNA might sensitize the cancer cells to the classical drugs at a relatively low concentration. In this investigation we showed that intracellular delivery of c-ROS1-targeting siRNA using pH-sensitive inorganic nanoparticles of carbonate apatite sensitizes mouse breast cancer cells (4T1) to doxorubicin, but not to cisplatin or paclitaxel, with the highest enhancement in chemosensitivity obtained at 40 nM of the drug concentration. Although intravenous administrations of ROS1-loaded nanoparticles reduced growth of the tumor, a further substantial effect on growth retardation was noted when the mice were treated with the siRNA- and Dox-bound particles, thus suggesting that silencing of ROS1 gene could sensitize the mouse breast cancer cells both in vitro and in vivo to doxorubicin as a result of synergistic effect of the gene knockdown and the drug action, eventually preventing activation of the survival pathway protein, AKT1. Our findings therefore provide valuable insight into the potential cross-talk between the pathways of ROS1 and doxorubicin for future development of effective therapeutics for breast cancer.

Matched MeSH terms: Gene Knockdown Techniques
Fulltext Gene knockout identification using an extension of Bees Hill Flux Balance Analysis

Choon YW, Mohamad MS, Deris S, Chong CK, Omatu S, Corchado JM

Biomed Res Int, 2015;2015:124537.
PMID: 25874200 DOI: 10.1155/2015/124537

Microbial strain optimisation for the overproduction of a desired phenotype has been a popular topic in recent years. Gene knockout is a genetic engineering technique that can modify the metabolism of microbial cells to obtain desirable phenotypes. Optimisation algorithms have been developed to identify the effects of gene knockout. However, the complexities of metabolic networks have made the process of identifying the effects of genetic modification on desirable phenotypes challenging. Furthermore, a vast number of reactions in cellular metabolism often lead to a combinatorial problem in obtaining optimal gene knockout. The computational time increases exponentially as the size of the problem increases. This work reports an extension of Bees Hill Flux Balance Analysis (BHFBA) to identify optimal gene knockouts to maximise the production yield of desired phenotypes while sustaining the growth rate. This proposed method functions by integrating OptKnock into BHFBA for validating the results automatically. The results show that the extension of BHFBA is suitable, reliable, and applicable in predicting gene knockout. Through several experiments conducted on Escherichia coli, Bacillus subtilis, and Clostridium thermocellum as model organisms, extension of BHFBA has shown better performance in terms of computational time, stability, growth rate, and production yield of desired phenotypes.

Matched MeSH terms: Gene Knockdown Techniques*
Fulltext Transcriptomic analysis of the role of RasGEF1B circular RNA in the TLR4/LPS pathway

Ng WL, Marinov GK, Chin YM, Lim YY, Ea CK

Sci Rep, 2017 09 25;7(1):12227.
PMID: 28947785 DOI: 10.1038/s41598-017-12550-w

Circular RNAs (circRNAs) have recently emerged as a large class of novel non-coding RNA species. However, the detailed functional significance of the vast majority of them remains to be elucidated. Most functional characterization studies targeting circRNAs have been limited to resting cells, leaving their role in dynamic cellular responses to stimuli largely unexplored. In this study, we focus on the LPS-induced cytoplasmic circRNA, mcircRasGEF1B, and combine targeted mcircRasGEF1B depletion with high-throughput transcriptomic analysis to gain insight into its function during the cellular response to LPS stimulation. We show that knockdown of mcircRasGEF1B results in altered expression of a wide array of genes. Pathway analysis revealed an overall enrichment of genes involved in cell cycle progression, mitotic division, active metabolism, and of particular interest, NF-κB, LPS signaling pathways, and macrophage activation. These findings expand the set of functionally characterized circRNAs and support the regulatory role of mcircRasGEF1B in immune response during macrophage activation and protection against microbial infections.

Matched MeSH terms: Gene Knockdown Techniques
Fulltext Assembly and activation of the Hippo signalome by FAT1 tumor suppressor

Martin D, Degese MS, Vitale-Cross L, Iglesias-Bartolome R, Valera JLC, Wang Z, et al.

Nat Commun, 2018 07 09;9(1):2372.
PMID: 29985391 DOI: 10.1038/s41467-018-04590-1

Dysregulation of the Hippo signaling pathway and the consequent YAP1 activation is a frequent event in human malignancies, yet the underlying molecular mechanisms are still poorly understood. A pancancer analysis of core Hippo kinases and their candidate regulating molecules revealed few alterations in the canonical Hippo pathway, but very frequent genetic alterations in the FAT family of atypical cadherins. By focusing on head and neck squamous cell carcinoma (HNSCC), which displays frequent FAT1 alterations (29.8%), we provide evidence that FAT1 functional loss results in YAP1 activation. Mechanistically, we found that FAT1 assembles a multimeric Hippo signaling complex (signalome), resulting in activation of core Hippo kinases by TAOKs and consequent YAP1 inactivation. We also show that unrestrained YAP1 acts as an oncogenic driver in HNSCC, and that targeting YAP1 may represent an attractive precision therapeutic option for cancers harboring genomic alterations in the FAT1 tumor suppressor genes.

Matched MeSH terms: Gene Knockdown Techniques
Fulltext FAT1 mutations cause a glomerulotubular nephropathy

Gee HY, Sadowski CE, Aggarwal PK, Porath JD, Yakulov TA, Schueler M, et al.

Nat Commun, 2016 Feb 24;7:10822.
PMID: 26905694 DOI: 10.1038/ncomms10822

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease (CKD). Here we show that recessive mutations in FAT1 cause a distinct renal disease entity in four families with a combination of SRNS, tubular ectasia, haematuria and facultative neurological involvement. Loss of FAT1 results in decreased cell adhesion and migration in fibroblasts and podocytes and the decreased migration is partially reversed by a RAC1/CDC42 activator. Podocyte-specific deletion of Fat1 in mice induces abnormal glomerular filtration barrier development, leading to podocyte foot process effacement. Knockdown of Fat1 in renal tubular cells reduces migration, decreases active RAC1 and CDC42, and induces defects in lumen formation. Knockdown of fat1 in zebrafish causes pronephric cysts, which is partially rescued by RAC1/CDC42 activators, confirming a role of the two small GTPases in the pathogenesis. These findings provide new insights into the pathogenesis of SRNS and tubulopathy, linking FAT1 and RAC1/CDC42 to podocyte and tubular cell function.

Matched MeSH terms: Gene Knockdown Techniques
Fulltext Regulation of nerve growth and patterning by cell surface protein disulphide isomerase

Cook GM, Sousa C, Schaeffer J, Wiles K, Jareonsettasin P, Kalyanasundaram A, et al.

Elife, 2020 05 28;9.
PMID: 32452761 DOI: 10.7554/eLife.54612

Contact repulsion of growing axons is an essential mechanism for spinal nerve patterning. In birds and mammals the embryonic somites generate a linear series of impenetrable barriers, forcing axon growth cones to traverse one half of each somite as they extend towards their body targets. This study shows that protein disulphide isomerase provides a key component of these barriers, mediating contact repulsion at the cell surface in chick half-somites. Repulsion is reduced both in vivo and in vitro by a range of methods that inhibit enzyme activity. The activity is critical in initiating a nitric oxide/S-nitrosylation-dependent signal transduction pathway that regulates the growth cone cytoskeleton. Rat forebrain grey matter extracts contain a similar activity, and the enzyme is expressed at the surface of cultured human astrocytic cells and rat cortical astrocytes. We suggest this system is co-opted in the brain to counteract and regulate aberrant nerve terminal growth.

Matched MeSH terms: Gene Knockdown Techniques
Fulltext Nutlin-3 sensitizes nasopharyngeal carcinoma cells to cisplatin-induced cytotoxicity

Voon YL, Ahmad M, Wong PF, Husaini R, Ng WT, Leong CO, et al.

Oncol Rep, 2015 Oct;34(4):1692-700.
PMID: 26252575 DOI: 10.3892/or.2015.4177

The small-molecule inhibitor of p53-Mdm2 interaction, Nutlin-3, is known to be effective against cancers expressing wild-type (wt) p53. p53 mutations are rare in nasopharyngeal carcinoma (NPC), hence targeting disruption of p53-Mdm2 interaction to reactivate p53 may offer a promising therapeutic strategy for NPC. In the present study, the effects of Nutlin-3 alone or in combination with cisplatin, a standard chemotherapeutic agent, were tested on C666-1 cells, an Epstein-Barr virus (EBV)-positive NPC cell line bearing wt p53. Treatment with Nutlin-3 activated the p53 pathway and sensitized NPC cells to the cytotoxic effects of cisplatin. The combined treatment also markedly suppressed soft agar colony growth formation and increased apoptosis of NPC cells. The effect of Nutlin-3 on NPC cells was inhibited by knockdown of p53, suggesting that its effect was p53-dependent. Extended treatment with increasing concentrations of Nutlin-3 did not result in emergence of p53 mutations in the C666-1 cells. Collectively, the present study revealed supportive evidence of the effectiveness of combining cisplatin and Nutlin-3 as a potential therapy against NPC.

Matched MeSH terms: Gene Knockdown Techniques
Fulltext The Immunomodulatory CEA Cell Adhesion Molecule 6 (CEACAM6/CD66c) Is a Protein Receptor for the Influenza a Virus

Rahman SK, Ansari MA, Gaur P, Ahmad I, Chakravarty C, Verma DK, et al.

Viruses, 2021 04 21;13(5).
PMID: 33919410 DOI: 10.3390/v13050726

To establish a productive infection in host cells, viruses often use one or multiple host membrane glycoproteins as their receptors. For Influenza A virus (IAV) such a glycoprotein receptor has not been described, to date. Here we show that IAV is using the host membrane glycoprotein CD66c as a receptor for entry into human epithelial lung cells. Neuraminidase (NA), a viral spike protein, binds to CD66c on the cell surface during IAV entry into the host cells. Lung cells overexpressing CD66c showed an increase in virus binding and subsequent entry into the cell. Upon comparison, CD66c demonstrated higher binding capacity than other membrane glycoproteins (EGFR and DC-SIGN) reported earlier to facilitate IAV entry into host cells. siRNA mediated knockdown of CD66c from lung cells inhibited virus binding on cell surface and entry into cells. Blocking CD66c by antibody on the cell surface resulted in decreased virus entry. We found that CD66c is a specific glycoprotein receptor for influenza A virus that did not affect entry of non-IAV RNA virus (Hepatitis C virus). Finally, IAV pre-incubated with recombinant CD66c protein when administered intranasally in mice showed decreased cytopathic effects in mice lungs. This publication is the first to report CD66c (Carcinoembryonic cell adhesion molecule 6 or CEACAM6) as a glycoprotein receptor for Influenza A virus.

Matched MeSH terms: Gene Knockdown Techniques
Fulltext Mechanisms of sex determination and transmission ratio distortion in Aedes aegypti

Hoang KP, Teo TM, Ho TX, Le VS

Parasit Vectors, 2016 Jan 28;9:49.
PMID: 26818000 DOI: 10.1186/s13071-016-1331-x

BACKGROUND: More effective mosquito control strategies are urgently required due to the increasing prevalence of insecticide resistance. The sterile insect technique (SIT) and the release of insects carrying a dominant lethal allele (RIDL) are two proposed methods for environmentally-friendly, species-targeted population control. These methods may be more suitable for developing countries if producers reduce the cost of rearing insects. The cost of control programs could be reduced by producing all-male mosquito populations to circumvent the isolation of females before release without reducing male mating competitiveness caused by transgenes.
RESULTS: An RNAi construct targeting the RNA recognition motif of the Aedes aegypti transformer-2 (tra-2) gene does not trigger female-to-male sex conversion as commonly observed among dipterous insects. Instead, homozygous insects show greater mortality among m-chromosome-bearing sperm and mm zygotes, yielding up to 100% males in the subsequent generations. The performance of transgenic males was not significantly different to wild-type males in narrow-cage competitive mating experiments.
CONCLUSION: Our data provide preliminary evidence that the knockdown of Ae. aegypti tra-2 gene expression causes segregation distortion acting at the level of gametic function, which is reinforced by sex-specific zygotic lethality. This finding could promote the development of new synthetic sex distorter systems for the production of genetic sexing mosquito strains.

Matched MeSH terms: Gene Knockdown Techniques
Fulltext Jerantinine A induces tumor-specific cell death through modulation of splicing factor 3b subunit 1 (SF3B1)

Chung FF, Tan PF, Raja VJ, Tan BS, Lim KH, Kam TS, et al.

Sci Rep, 2017 02 15;7:42504.
PMID: 28198434 DOI: 10.1038/srep42504

Precursor mRNA (pre-mRNA) splicing is catalyzed by a large ribonucleoprotein complex known as the spliceosome. Numerous studies have indicated that aberrant splicing patterns or mutations in spliceosome components, including the splicing factor 3b subunit 1 (SF3B1), are associated with hallmark cancer phenotypes. This has led to the identification and development of small molecules with spliceosome-modulating activity as potential anticancer agents. Jerantinine A (JA) is a novel indole alkaloid which displays potent anti-proliferative activities against human cancer cell lines by inhibiting tubulin polymerization and inducing G2/M cell cycle arrest. Using a combined pooled-genome wide shRNA library screen and global proteomic profiling, we showed that JA targets the spliceosome by up-regulating SF3B1 and SF3B3 protein in breast cancer cells. Notably, JA induced significant tumor-specific cell death and a significant increase in unspliced pre-mRNAs. In contrast, depletion of endogenous SF3B1 abrogated the apoptotic effects, but not the G2/M cell cycle arrest induced by JA. Further analyses showed that JA stabilizes endogenous SF3B1 protein in breast cancer cells and induced dissociation of the protein from the nucleosome complex. Together, these results demonstrate that JA exerts its antitumor activity by targeting SF3B1 and SF3B3 in addition to its reported targeting of tubulin polymerization.

Matched MeSH terms: Gene Knockdown Techniques
N-cadherin regulates beta-catenin signal and its misexpression perturbs commissural axon projection in the developing chicken spinal cord

Yang C, Li X, Wang C, Fu S, Li H, Guo Z, et al.

J Mol Histol, 2016 Dec;47(6):541-554.
PMID: 27650519

N-cadherin is a calcium-sensitive cell adhesion molecule that plays an important role in the formation of the neural circuit and the development of the nervous system. In the present study, we investigated the function of N-cadherin in cell-cell connection in vitro with HEK293T cells, and in commissural axon projections in the developing chicken spinal cord using in ovo electroporation. Cell-cell connections increased with N-cadherin overexpression in HEK293T cells, while cell contacts disappeared after co-transfection with an N-cadherin-shRNA plasmid. The knockdown of N-cadherin caused the accumulation of β-catenin in the nucleus, supporting the notion that N-cadherin regulates β-catenin signaling in vitro. Furthermore, N-cadherin misexpression perturbed commissural axon projections in the spinal cord. The overexpression of N-cadherin reduced the number of axons that projected alongside the contralateral margin of the floor plate, and formed intermediate longitudinal commissural axons. In contrast, the knockdown of N-cadherin perturbed commissural axon projections significantly, affecting the projections alongside the contralateral margin of the floor plate, but did not affect intermediate longitudinal commissural axons. Taken together, these findings suggest that N-cadherin regulates commissural axon projections in the developing chicken spinal cord.

Matched MeSH terms: Gene Knockdown Techniques
Targeted RNAi of the mitogen-activated protein kinase pathway genes in acute myeloid leukemia cells

M.R. Mohd Hafiz, M.Z. Mazatulikhma, F.A. Mohd Faiz, M.S. Mohamed Saifulaman

Sains Malaysiana, 2013;42:1131-1137.

In this study, RNA interference (RNAi) was carried out as an experimental technique to knockdown three mitogen-activated protein kinase (MAPK) pathway genes, raf-1, mekk1 and mlk3 in acute myeloid leukemia (AML) cells. Conventionally, RNAi knockdown experiments target a single gene for functional studies or therapeutic purposes. We wanted to explore the potential differences or similarities between targeting single targets or multiple target genes in a single application. We achieved knockdown of gene expression levels of between 40 and 60% for the RNAi experiments, with better knockdown observed in single target gene experiments in comparison with the multiple target gene experiment. Microarray analysis indicated that the transfection process had most likely induced the immune response from the cells in every RNAi treatment. This might indicate that when the MAPK signaling pathway is partially blocked, in tandem with the immune response, the cells will begin signaling for apoptosis leading to cellular death of the leukemic cells.

Matched MeSH terms: Gene Knockdown Techniques

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