[Effects of miR-135a on HOXA10 expression, proliferation and apoptosis of ovarian cancer cells].

OBJECTIVE

To investigate the effects of miR-135a on HOXA10 expression, proliferation and apoptosis of SKOV3 cells.

METHODS

(1) Through computer-aided algorithms,the predicted target gene of miR-135a (HOXA10)were determined. (2) miR-135a mimics, miR-135a inhibitor and negative control were transfected into SKOV3 cells, respectively.Reverse transcription (RT)-PCR, western blot analysis were used to examine the expression levels of HOXA10 at different times (24, 48 and 72 hours). (3) A luciferase reporter assay was used to confirm the direct regulation between miR-135a and HOXA10. (4) SKOV3 cells proliferation at different times (24,48 and 72 hours) was detected by methyl thiazolyl tetrazolium (MTT) assay [quantified by absorbance(A)]. Western blot was used to examine the expression of apoptosis-associated protein bcl-2, bax and caspase-3 in SKOV3 cells after 48 hours transfection.

RESULTS

(1) HOXA10 was predicted to be the target gene of miR-135a by computer-aided algorithms. (2) RT-PCR shown that HOXA10 mRNA levels were decreased over time (24, 48 and 72 hours) after miR-135a mimics transfection in SKOV3 cells (0.94 ± 0.04 vs 0.78 ± 0.03 vs 0.70 ± 0.03, P < 0.05). While, the expression of HOXA10 mRNA was increased over time after miR-135a inhibitor transfection (1.14 ± 0.05 vs 1.16 ± 0.03 vs 2.60 ± 0.08,P < 0.05). After transfected with miR-135a mimics or miR-135a inhibitor over 48 and 72 hours, the HOXA10 expression levels in SKOV3 cells were significantly lower or higher than each control group, respectively (all P < 0.01). Western blot analysis of HOXA10 expression in SKOV3 cells confirmed the results of RT-PCR detected. (3) After cotransfection of miR-135a plasmid and pMIR-REPORT luciferase plasmid containing HOXA10, luciferase reporter assays showed that the luciferase activity reduced by 67.8% (P < 0.01). (4) MTT showed that SKOV3 cells growth after miR-135a mimics transfection for 48 and 72 hours were significantly lower than those in control group (0.38 ± 0.03 vs 0.52 ± 0.05, 0.67 ± 0.05 vs 0.75 ± 0.06;respectively,all P < 0.05).While, SKOV3 cells transfected with miR-135a inhibitor for 72 hours grew significantly faster than that in control group (0.95 ± 0.05 vs 0.75 ± 0.06, P < 0.01). After miR-135a mimics transfection, the level of bcl-2 protein was significantly lower than that in control group (0.28 ± 0.06 vs 0.76 ± 0.09,P < 0.01). The activity of caspase-3 was significantly higher than that in control group (115.0 ± 2.4 vs 95.4 ± 2.1, P < 0.01). While, there was no statistical difference of bax expression (P = 0.142). However, after miR-135a inhibitor transfection, the expression level of bcl-2 protein was significantly higher than that in control group (0.92 ± 0.03 vs 0.76 ± 0.09, P = 0.037) and the activity of caspase-3 was significantly lower than that in control group (59.5 ± 4.1 vs 95.4 ± 2.1, P < 0.01). There was also no statistical difference of bax expression (P = 0.066).

CONCLUSION

miR-135a may play an important role in cell proliferation and apoptosis of ovarian cancer cells by regulating HOXA10 and its downstream pathways.