Construction and characterization of a eukaryotic expression vector for small interfering RNA targeting the NEDD9 gene
- Authors:
- Jing-Xia Chang
- Hua-Qi Wang
- Guo-Qiang Zhao
- He-Ying Chu
- Guo-Jun Zhang
View Affiliations
Affiliations: Department of Respiratory Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, P.R. China, Department of Microorganisms and Immunization, Preclinical Medicine, Zhengzhou University, Zhengzhou, Henan, P.R. China
- Published online on: September 20, 2012 https://doi.org/10.3892/ijmm.2012.1137
-
Pages:
1343-1348
Metrics: Total
Views: 0 (Spandidos Publications: | PMC Statistics: )
Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )
This article is mentioned in:
Abstract
The aim of this study was to construct a eukaryotic expression vector for a small interfering RNA (siRNA) targeting the neural precursor cell expressed, developmentally downregulated 9 (NEDD9) gene, and to investigate the effects of RNA interference (RNAi) on NEDD9 expression in human lung adenocarcinoma A549 cells. We used the siRNA design and analysis software to determine the target oligonucleotides according to the sequence of NEDD9 mRNA available in GenBank. Four siRNA sequences were obtained, and the corresponding cDNAs were synthesized and inserted into the pRNAT-CMV3.2 plasmid to construct the recombinant plasmids. These were transformed into the E. coli strain DH5α. The plasmids, after identification by PCR and DNA sequencing, were transfected into the A549 cell line via the liposome method. NEDD9 mRNA and protein in the cells were determined by fluorescence quantitative RT-PCR (FQ-PCR) and western blotting, respectively. The pRNAT-CMV3.2-transfected plasmid was used as a control. Four recombinant plasmids were identified by PCR and sequence analysis, which contained the correct insertion of the designed sequences in the plasmids. FQ-PCR and western blotting showed substantially decreased mRNA and protein expression of the NEDD9 gene in the transfected cells, compared with the control group. In conclusion, the recombinant plasmids expressing the siRNA targeting the NEDD9 gene were successfully constructed, and the siRNA expression vectors inhibited the expression of NEDD9 in A549 cells.
View References
|
1.
|
SF LawJ EstojakB WangHuman enhancer of
filamentation 1 (HEF1/NEDD9/CAS-L), a novel p130Cas-like docking
protein, associates with FAK, and induces pseudohyphal growth in
yeastMol Cell Biol163327333719968668148
|
|
2.
|
M MinegishiK TachibanaT SatoStructure and
function of Cas-L, a 105-kD Crk-associated substrate-related
protein that is involved in beta-1 integrin-mediated signaling in
lymphocytesJ Exp
Med18413651375199610.1084/jem.184.4.13658879209
|
|
3.
|
M SinghL CowellS SeoMolecular basis for
HEF1/NEDD9/Cas-L action as a multifunctional co-ordinator of
invasion, apoptosis and cell cycleCell Biochem
Biophys485472200710.1007/s12013-007-0036-317703068
|
|
4.
|
AJ MinnGP GuptaPM SiegelGenes that mediate
breast cancer metastasis to
lungNature436518524200510.1038/nature0379916049480
|
|
5.
|
M NatarajanJE StewartEA GolemisHEF1 is a
necessary and specific downstream effector of FAK that promotes the
migration of glioblastoma
cellsOncogene2517211732200610.1038/sj.onc.120919916288224
|
|
6.
|
M KimJD GansC NogueiraComparative
oncogenomics identifies NEDD9 as a melanoma metastasis
geneCell12512691281200610.1016/j.cell.2006.06.00816814714
|
|
7.
|
A JemalF BrayMM CenterGlobal cancer
statisticsCA Cancer J Clin616990201110.3322/caac.20107
|
|
8.
|
JX ChangF GaoGJ ZhangExpression and
clinical significance of NEDD9 in lung tissuesMed
OncolMarch242012(Epub ahead of
print)10.1007/s12032-012-0213-02012
|
|
9.
|
SF LawYZ ZhangAJ Klein-SzantoCell
cycle-regulated processing of HEF1 to multiple protein forms
differentially targeted to multiple subcellular compartmentsMol
Cell Biol18354035511998
|
|
10.
|
M RyuT KinoshitaM KonishiSegmental
resection of the duodenum including the papilla of Vater for focal
cancer in adenomaHepatogastroenterology4383583819968884299
|
|
11.
|
JA LowellRL RossiJL MunsonPrimary
adenocarcinoma of third and fourth portions of duodenumArch
Surg127557560199210.1001/archsurg.1992.014200500810101349472
|
|
12.
|
IG KaklamanosOF BatheD FranceschiExtent of
resection in the management of duodenal adenocarcinomaAm J
Surg1793741200010.1016/S0002-9610(99)00269-X10737576
|
|
13.
|
EN PugachevaEA GolemisThe focal adhesion
scaffolding protein HEF1 regulates activation of the Aurora-A and
Nek2 kinases at the centrosomeNat Cell
Biol7937946200510.1038/ncb130916184168
|
|
14.
|
D DadkeM JarnikEN PugachevaDeregulation of
HEF1 impairs M-phase progression by disrupting the RhoA activation
cycleMol Biol Cell1712041217200610.1091/mbc.E05-03-023716394104
|
|
15.
|
S SeoT AsaiT SaitoCrk-associated substrate
lymphocyte type is required for lymphocyte trafficking and marginal
zone B cell maintenanceJ
Immunol17534923501200510.4049/jimmunol.175.6.349216148091
|
|
16.
|
JQ YinY WanRNA-mediated gene regulation
system: Now and the futureInt J Mol Med10355365200212239579
|
|
17.
|
YH WangS LiuG ZhangKnockdown of c-Myc
expression by RNAi inhibits MCF-7 breast tumor cells growth in
vitro and in vivoBreast Cancer
Res7220228200510.1186/bcr97515743499
|
