MicroRNA Expression Profiling in Human Ovarian Cancer: miR-214 Induces Cell Survival and Cisplatin Resistance by Targeting PTEN

miR-214 negatively regulates PTEN through binding to 3′-UTR of the PTEN. A, sequence alignment of human miR-214 with 3′-UTR of PTEN. The seed sequence of miR-214 (top) matches 3′-UTR of PTEN (middle). Bottom, mutations of the 3′-UTR of PTEN for creating the mutant luciferase reporter construct. B, rows 1 to 4, miR-214 reduces PTEN protein but not mRNA levels. HIOSE-80 cells (left) were transfected with pcDNA3.1/V5-His-Topo-miR-214, pcDNA3.1/V5-His-Topo-miR199a*, and vector alone and immunoblotted with indicated antibodies; rows 5 and 6, the expression of miR-214 and miR-199a* was determined by qRT-PCR; row 8, PTEN mRNA level was measured by RT-PCR. U6 (row 7) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; row 9) were used for controls. Middle and right, knockdown of miR-214 inducing PTEN expression. A2780CP cells were transfected with antisense 2′-O-me oligonucleotide targeting miR-214 at concentration of 150 pmol/L/well (6-well plate) with Lipofectamine 2000. Anti-miR199a* and scramble 2′-O-me oligonucleotide were used as controls. Middle, after incubation of 72 h, cells were lysed and immunoblotted with indicated antibodies; right, inhibition of miR-214 and miR-199a* expression by 2′-O-me oligonucleotide in A2780CP cells was shown by qRT-PCR. GSK3β, glycogen synthase kinase 3β. C, miR-214 inhibits wild-type but not mutated PTEN-3′-UTR reporter activity. miR-214–positive A2780CP cells (left) and miR-214–negative HIOSE-80 cells (right) were transiently transfected with indicated plasmids. Following 36 h of incubation, cells were subjected to luciferase assay. Columns, mean of three independent experiments; bars, SD. D, rows 1 and 2, representative tumor and normal tissue lysates were analyzed by Western blot with indicated antibodies; row 3, expression of miR-214 was analyzed by qRT-PCR; row 4, U6 was used as a control.

Ectopic expression of miR-214 induces ovarian cancer cells resistant to cisplatin-induced apoptosis. A, top, RNase protection analysis of miR-214 expression in ovarian cancer cell lines and immortalized human ovarian surface epithelial cells; bottom, 5S was used as control. B, ectopic expression of miR-214. A2780S and OV119 cells, which express low levels of endogenous miR-214, were transfected with pcDNA3.1/V5-His-Topo-miR-214 or vector alone. Following G418 selection, cells were subjected to qRT-PCR analysis for expression of miR-214 (top) and U6 (bottom). C and D, expression of miR-214 renders A2780S and OV119 cells resistant to cisplatin. The vector (Topo)-transfected and miR-214–transfected cells were treated with cisplatin or DMSO for different time points. C, cell viability was detected by MTT assay. D, after 48 h of the treatment, cells were labeled with Annexin V and analyzed by flow cytometry.

Knockdown of miR-214 sensitizes A2780CP cells to cisplatin. A, A2780CP, a cisplatin-resistant cell line and expressing elevated levels of endogenous miR-214, was transfected with 2′-O-me-anti-miR-214 or scramble 2′-O-me oligonucleotides and assayed with qRT-PCR with primers of miR-214 (top) and U6 (bottom). B, MTT assay. The 2′-O-me-anti-miR-214–transfected or scramble 2′-O-me–transfected A2780CP cells were treated with 20 μmol/L of cisplatin or DMSO vehicle for the indicated times and examined for cell viability. C, flow cytometry. Indicated cells were treated with cisplatin or DMSO for 12 h and the sub-G₁ population was identified by flow cytometry.