Effect of HOXA10 gene silenced by shRNA on proliferation and apoptosis of U937cell line / 中国当代儿科杂志

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of lentivirus-mediated RNA interference targeting HOXA10 gene on the proliferation, apoptosis and morphology of leukemic cell line U937.</p><p><b>METHODS</b>Four different shRNA plasmids were designed and built to interfere with HOXA10 gene. The four interference plasmids were transfected into 293T cells with the HOXA10 over expression plasmid and then the RNAi efficiency of the four interference plasmids was determined by Western blot. The best one was chosen to transfect 293T cells with lentiviral helping plasmids to produce packaged lentivirus (lenti-shHOXA10). U937 cells were divided into interference group (lenti-shHOXA10), negative control group and untreated group. After infection with the packaged lentivirus, infection efficiency of lentivirus for U937 was detected by flow cytometry, and the expression of HOXA10 gene mRNA and protein was detected by real-time PCR and Western blot. Cell survival was determined by MTT assay. Apoptosis rate was detected by flow cytometry.</p><p><b>RESULTS</b>Lentiviral-shRNA vector of HOXA10 gene was successfully constructed. Compared with the negative control and untreated groups, mRNA level of HOXA10 decreased by (92.3±1.3)%, protein levels decreased by 91.1%, and the inhibition rate of U937 cells [(43.9±0.7)%] increased in the interference group (P<0.05). Wright's staining showed that the ratio of karyon to cytoplasm was reduced and mitotic phase was rare in the interference group. Apoptosis rate in the interference group [(27.1±1.4)%] was significantly higher than in the negative [(19.4±1.9)%] and untreated groups [(5.5±1.3)%] (P<0.05).</p><p><b>CONCLUSIONS</b>Lentivirus mediated RNAi can reduce the expression level of HOXA10, effectively inhibit proliferation and promote apoptosis of U937 cells. HOXA10 gene is expected to become a new target for the treatment of leukemia at gene level.</p>