Establishment of a bladder cancer cell line with toll-like receptor 2 gene knockdown and identification of its biological characteristics / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To establish a murine bladder cancer cell line with stable silencing of toll-like receptor 2 (TLR2) expression.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>