MicroRNA-25 regulates small cell lung cancer cell development and cell cycle through cyclin E2

Abstract

Purpose: We intended to examine the underlying mechanism of microRNA-25 (miR-25) in regulating small cell lung cancer (SCLC).

Methods: The miR-25 expression was measured by quantitative RT-PCR (qRT-PCR) in 5 SCLC cell lines and 9 human SCLC tissues. In SCLC cell line H510A cells, endogenous miR-25 was downregulated by stable transfection of antisense oligonucleotide of miR-25 (miR-25-as). Then the effects of miR-25 downregulation on SCLC growth, invasion and chemoresistance were assessed by MTT, migration and cisplatin assays, respectively. Furthermore, the effects of miR-25 downregulation on cancer cell cycle arrest, production of cell cycle proteins cyclin E2 and CDK2 were examined by cell cycle assay, western blot and luciferase assays, respectively. Finally, cyclin E2 was over-expressed in H510A cells to investigate its effect on miR-25 mediated SCLC regulation.

Results: In both SCLC cells and human SCLC tumor tissues, miR-25 was overexpressed. Down-regulation of miR-25 in H510A cells significantly reduced cancer cell growth, invasive capability and resistance to cisplatin. Also, it induced G1 cell cycle arrest and downregulated cell cycle related proteins cyclin E2 and CDK2. Luciferase assay demonstrated cyclin E2 was directly targeted by miR-25. Overexpression of cyclin E2 in H510A cells reversed the cell cycle arrest and restored invasive capability impaired by miR-25 downregulation.

Conclusions: Our study shows miR-25 is overexpressed in SCLC and acting as oncogenic regulator by regulating cyclin E2.

Keywords: CDK2; SCLC; cyclin E2; miR-25.

Figure 1

MiR-25 is upregulated in SCLC cell lines and human SCLC tissues. A. qRT-PCR was used to assess miR-25 expression levels in MRC5, H146, H209, H446, H510A and H889 cells. All data were normalized to MRC5 (*, P < 0.05). B. qRT-PCR was used to compare the miR-25 expression levels between 9 pairs of tumor tissues (T) and adjacent non-tumor tissues (ANT). All data were normalized to ANT of each case (*, P < 0.05).

Figure 2

Downregulating MiR-25 reduced proliferation, migration and chemoresistance in H510A cells. H510A cells were transfected with either miR-25 inhibitor (miR-25-as, 100 nM) or control miRNA (miR-Ctl, 100 nM) for 24 hours. (A) Transfection efficiency was measured by qRT-PCR (*: P < 0.05). (B) 24, 48, 72 and 96 hours after transfection, A MTT assay was used to assess proliferation in H510A cells. (*: P < 0.05). 12 hours after transfection, an invasion assay was conducted (C), and migrated cells were quantified (*: P < 0.05) (D). (E) 24 hours after transfection, H510A cells were treated with 1, 2, 5, 10, 20, 50, 100 and 200 μg/ml cisplatin for additional 12 hours, followed by a MTT assay to measure cell proliferation (*, P < 0.05).

Figure 3

Downregulating MiR-25 induced G0/G1 arrest through cyclin E2 in H510A cells. A. H510A cells were transfected with either miR-25-as (100 nM) or miR-Ctl (100 nM). Three days after transfection, a cell cycle assay was conducted. MiR-25 downregulation significantly induced cell cycle G0/G1 arrest in H510A cells (*: P < 0.05). B. Western blotting was conducted to examine the cell cycle related proteins CDK2 and cyclin E2, using β-actin as loading control. C. Wild type (WT) cyclin E2 3’-UTR, with putative corresponding miR-25 binding site was shown. A mutated (MT) cyclin E2 3’-UTR with modified fragments was also shown. Both were inserted into the pGL3 luciferase vector and validated by DNA sequencing. D. Relative luciferase activity was measured after co-transfection of miR-25-as or miR-Ctl with the luciferase vectors, along with an endogenous control Renilla luciferase vector in H510A cells (*: P < 0.05).

Figure 4

Overexpressing cyclin E2 restored cell cycle and invasion impaired by miR-25 down-regulation in H510A cells. A. H510A cells were transfected with recombinant plasmid eukaryotic expression vector pcDNA3.1-CyclinE2 to over-express CDC42, or empty control vector (pcDNA3.1-Ctl). Two days later, cyclin E2 expressions were examined by qRT-PCR (*: P < 0.05). B. H510A cells were transfected miR-25-as for 24 hours, followed by another transfection of either pcDNA3.1-CyclinE2 or pcDNA3.1-Ctl. A cell cycle assay was conducted 72 hours after last transfection. (*: P < 0.05). C. Twenty-four hours after last transfection, a Matrigel migration assay was conducted to assess the invasive capability of cells. D. Quantification showed significantly more cells invaded into lower chambers while cyclin E2 was overexpressed in H510A cells (*: P < 0.05).