Fig 4. Functional impacts of HOTAIR knockdown on survival activity of ovarian clear cancer cells.

(A) Specific miRNAs (set A to D) against HOTAIR were prepared and introduced into ovarian clear cancer cells, ES-2 and TOV-21G. Gene knockdown efficiency of each miRNA construct was verified by QPCR using scramble miRNA as the control. Data were averages of triplicates and presented as means ± S.D. (B) BrdU uptake assay was performed in cells transfected with scramble control or the mixture of anti-HOTAIR miRNA-B and miRNA-D (miRNA-B/D) (left). Positive cells were counted and averaged as means ± S.D. from 200 nuclei (right). (C) MTT assay and (D) wound healing assay were performed to investigate the impacts of HOTAIR knockdown by miRNA-B/D on cell growth and migration, respectively. (E) Apoptotic cells were detected by annexin V staining in cells 72 hrs after transfection. Data in (C) and (D) were averages of five replicates and presented as means ± S.D. (F) The expression levels of HOXD10 and HOXA5 were analyzed by QPCR in transfected cells 24 hrs after HOTAIR knockdown. Cells treated with scramble were utilized as the controls. Data were averages of triplicates and presented as means ± S.D. For (A), (B), (E) and (F), data from miRNA-B/D treated cells were compared to the ones from scramble control by using t-test. For (C) and (D) time course studies, two-way ANOVA was utilized to analyze statistical significance. The p values were presented as *: p value < 0.05, **: p value < 0.01, and ***: p value < 0.001.