Effect of CASC19 on proliferation, apoptosis and radiation sensitivity of cervical cancer cells by regulating miR-449b-5p expression

Abstract

Objective: To investigate the effects of cancer susceptibility candidate gene 19 (CASC19) regulating the expression of microRNA-449b-5p (miR-449b-5p) on the proliferation, apoptosis and radiation sensitivity of cervical cancer cells. Methods: (1) HeLa cells of cervical cancer cell line were cultured. HeLa cells were irradiated with X-ray at different doses (0, 2, 4, 6, 8 Gy, respectively), then the expression level of CASC19 mRNA and miR-449b-5p were detected by real-time quantitative PCR. (2) HeLa cell proliferation, apoptosis, radiation sensitivity (expressed as a survival fraction) and its related protein expression included cyclin D1, cleaved-caspase-3, and histone variant H2AX (γ-H2AX) were examined after different treatment including silencing CASC19 expression, over-expressing miR-449b-5p, down-regulating miR-449b-5p and silencing CASC19 expression. (3) The dual luciferase reporter gene experiment and real-time quantitative PCR technology were used to verify the targeting relationship between CASC19 and miR-449b-5p. Results: (1) With the increase of X-ray irradiation different dose (0, 2, 4, 6, 8 Gy), the expression level of CASC19 mRNA in HeLa cells gradually increased (F=502.681, P=0.000), and the expression level of miR-449b-5p gradually decreased (F=202.936, P=0.000).(2) After silencing CASC19 expression or over-expressing miR-449b-5p, the survival rate of HeLa cells was significantly reduced (P<0.05), the apoptosis rate was significantly increased (P<0.05), the survival fraction was significantly reduced (P<0.05), the expression level of cyclin D1 protein was significantly reduced (P<0.05), and the expression levels of cleaved-caspase-3 and γ-H2AX protein were significantly increased (P<0.05). After down-regulating miR-449b-5p and silencing CASC19 expression, the survival rate of HeLa cells was significantly reduced (P<0.05), the apoptosis rate was significantly increased (P<0.05), the survival fraction was significantly reduced (P<0.05), the expression levels of cyclin D1 and γ-H2AX protein were significantly increased (P<0.05), and the expression level of cleaved-caspase-3 was significantly decreased (P<0.05). (3) Over expression of miR-449b-5p could significantly reduce the luciferase activity of CASC19 wild type (1.00±0.09 versus 0.37±0.05, P<0.01), but there were no significant effect on the luciferase activity of CASC19 mutant type (0.92±0.07 versus 0.94±0.05, P>0.05). After the expression of CASC19 was silenced, the expression level of miR-449b-5p in HeLa cells increased significantly (1.00±0.12 versus 4.84±0.49, P<0.05). After overexpression of CASC19, the expression level of miR-449b-5p in HeLa cells was significantly reduced (1.00±0.09 versus 0.38±0.04, P<0.05). Conclusion: CASC19 in HeLa cells negatively regulates the expression of miR-449b-5p, and down-regulating the expression of miR-449b-5p could partially reverse the effects of silencing CASC19 on HeLa cell proliferation, apoptosis and radiation sensitivity.