[Establishment of a bladder cancer cell line with toll-like receptor 2 gene knockdown and identification of its biological characteristics].
OBJECTIVE
To establish a murine bladder cancer cell line with stable silencing of toll-like receptor 2 (TLR2) expression.


METHODS
Three different recombinant plasmids pcDNA 6.2-GW/EmGFPmiR-TLR2 were constructed and transfected into T739 cells via lipofectamine. The recombinant plasmid with the strongest interference effect was selected by RT-PCR and transfected into T739 cells. The Blasticidin-resistant cell clones were screened to obtain bladder cancer cell lines with TLR2 gene knockdown, and the biological characteristics of the stable cell lines were observed.


RESULTS
Sequencing showed that the target DNA fragment was correctly inserted into the vector. The recombinant plasmid with the strongest silencing effect (pcDNA 6.2-GW/EmGFPmiR- TLR2.949) was screened, and transfection of this plasmid in T739 cells resulted in a cell line with stable TLR2 gene silencing (T739-TLR2delta), in which the expression of TLR2 mRNA and receptor were downregulated by over 95% and 90%, respectively. Compared with the negative control cells, T739-TLR2delta cell line exhibited significant different cell proliferation index with extended cell population doubling time.


CONCLUSIONS
The recombinant plasmid pcDNATM6.2-GW/EmGFPmiR-TLR2 can effectively suppress the expression of TLR2 gene, and the established cell line with stable TLR-2 gene knockdown allows further functional study the TLR2 gene in T739 cells.
Wei
Jian-an
JA
Central Laboratory, Second Affiliated Hospital of Guangzhou University of TCM, Guangzhou 510120, China. andycarrie@163.com
Zeng
Xing
X
Han
Ling
L
Huang
Yu
Y
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002295:Carcinoma, Transitional Cell / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D005822:Genetic Vectors; D051379:Mice; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051195:Toll-Like Receptor 2 / Q000235:genetics*; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000473:pathology
D012333:RNA, Messenger; D034741:RNA, Small Interfering; C493486:Tlr2 protein, mouse; D051195:Toll-Like Receptor 2