The Role Of Homeodomain Protein DLX4 In Apoptosis And Invasion Of Choriocarcinoma Cells

Choriocarcinoma rises from embryo trophoblast cells, but malignant trophoblast cells lose the characteristics of normal villus, invade uterine muscles and metastasize other organs, which causes damage, even leads to death. Choriocarcinoma has a high malignancy. Despite extensive cytogenetic and pathogenetic studies, the molecular mechanisms of choriocarcinoma oncogenesis remain elusive.Homeodomain protein DLX4 is a transcriptional factor which is found lately. Recently, it has been reported that DLX4 plays an important role in oncogenesis, but little is known about the relationship between DLX4 and choriocarcinoma. Our experiments began with an investigation of DLX4 expression in choriocarcinoma cells and normal placental tissues. RNA interfence knocked down DLX4 expression in choriocarcinoma JEG-3 cells, and subsequently, western blot, RT-PCR, immunohistochemistry, flow cytometric assessment, gelatin zymography, matrigel invasion assay and wound healing assay were applied to explore the effect of DLX4 on JEG-3 cells apoptosis and invasion. The results of our experiments suggest that abnormal expression of DLX4 is one of the factors that affect apoptosis and invasion of choriocarcinoma.These experiments are divided into 3 parts, as following: (1) Expression of DLX4 in choriocarcinoma cells; (2) DLX4 RNAi promotes apoptosis in choriocarcinoma cells; (3) The role of DLX4 in motiolity and invasion of choriocarcinoma cells.Section I Expression of DLX4 in choriocarcinoma cellsObjective To examine the expression of DLX4 in human choriocarcinoma cell lines JAr and JEG-3 cells and explore the relationship between theaberrant expression of this gene and the tumorigenesis of the chor i ocarc i noma.Methods RT-PCR was used to investigate the expression of DLX4 mRNA (isoforml and isoform2) and western blot was used to detect the protein of it in JAr and JEG-3 cells, comparing with 30 first trimester placental tissues and 8 third trimester placental tissues.Results (1) Both isoforms of DLX4 were expressed in all the cells and tissues, their levels were higher in JEG-3 and JAr than in placental tissues (p<0. 001) ; the level of isoform2 was higher than isoforml in JAr, JEG-3 and term placental tissues, but the expression of isoforml was higher than isoform2 in the first trimester placental tissues. (2) The expression of DLX4 protein was higher in JAr and JEG-3 than in placental tissues, and the highest expression was in JEG-3 cells. The results were consistent with RT-PCR.Conclusions Overexpression of DLX4 may be relatd to the oncogenesis of choriocarcinoma and DLX4 isoform2 may play a more important role in this process.Section II DLX4 RNAi promotes apoptosis in choriocarcinoma cellsObjective To investigate the role of DLX4 in apoptosis of choriocarcinoma cells and the related mechanicsms.Methods We chose JEG-3 cells, which has higher DLX4 expression, to be targeted cells. Two DLX4 specific siRNAs (SI and S2) and one DLX4 scrambled siRNA were designed, and cloned into pRNA plasmid vectors. The recombinants were transfected into JEG-3 cells and RNA interference was used to knockdown DLX4 expression. Inhibition of DLX4 expression by RNAi was assessed by semi-qutitative RT-PCR and western blot. The effect of DLX4 inhibition on apoptosis was determined by flow cytometry and M30immunohistochemical staining. Moreover, we investigated the expression of caspase-3, caspase-8 and Bax, which were closely related with apoptosis.Results (1) Both of DLX4 siRNAs knocked down expression of DLX4 specifically and effectively. 24 h-72 h post-transfection, compared with negative control groups, DLX4 mRNA levels in cells transfected with SI were decreased 25-88% (DLX4 isoforml, p<0. 05 or p<0. 01) and 41-100% (DLX4 isoform2, p<0.01); DLX4 mRNA levels in cells transfected with S2 were knocked down 28-92% (DLX4 isoforml, p<0. 05 or p<0. 01) and 35-85% (DLX4 isoform2, p<0.05 or p<0.01); the expression of DLX4 protein was down-regulated 35-82% and 13-90% in cells transfected with SI and S2 respectively. (2) The inhibition was time-dependent and the maximal inhibition was 72 h after transfection. (3) Comparing with negative controL groups, knock-down of DLX4 expression by RNAi increased apoptosis in JEG-3 cells (p<0.01). (4) The expression of caspase-3 and caspase-8 was increased after DLX4 knock-down, but DLX4 RNAi did not influence the level of Bax.Conclusions These results demonstrate that inhibition of DLX4 could induce apoptosis of JEG-3 cells and the mechanicsm may be due to transcriptional regulation on caspase-3 and caspase-8.Section HI The role of DLX4 in motility and invasion of choriocarcinomacellsObjective To investigate the role of DLX4 in invasion of choriocarcinoma cells.Methods We chose JEG-3 cells, which has higher DLX4 expression, to be targeted cells. pRNA-DLX4 siRNA (SI) and pRNA-DLX4 scrambled siRNA which have been designed and constructed in section IIwere used to transfect JEG-3 cells. The cells were cultured in medium with G418 for 10-14 days...