Figure 4. Increase of cell apoptosis and decreases of cell viability, migration, and invasion induced by knockdown of antisense non-coding RNA in the INK4 locus (ANRIL), were reversed by miR-99a inhibition. Non-treated cells acted as control. A, Expression level of miR-99a measured by qPCR. U6 acted as an internal control. B, Rate of apoptotic cells by flow cytometry. C and D, Cell viability was determined by CCK-8 assay. The migration (E) and invasion (F) were measured by Transwell assay. shANRIL: pENTR^TM/U6 vector carrying small hairpin RNA targeting ANRIL; shNC: pENTR^TM/U6 vector carrying a non-targeting sequence; qPCR: quantitative real-time PCR; CCK-8: Cell Counting Kit-8; miR-99a: microRNA-99a; Data are reported as means±SD. *P<0.05; **P<0.01; ***P<0.001; ^###P<0.001 (two-tailed Student's t-test). In panels C and D, *indicates significant difference between the shNC + inhibitor control group and the shANRIL + inhibitor control group, and ^#indicates significant difference between the shANRIL + inhibitor control group and the shANRIL + miR-99a inhibitor group.