microRNA-150 promotes cervical cancer cell growth and survival by targeting FOXO4

Abstract

Background: Dysregulation of microRNA-150 (miR-150) is commonly observed in solid tumor and has been reported to be involved in multiple important biological processes, such as cell proliferation, apoptosis, and metastasis. Elevated miR-150 level was also detected in cervical carcinoma, whereas its function in cancer progression has not been studied yet.

Methods: The expression of miRNA-150 in cervical carcinoma was compared with normal cervical tissue and using qRT-PCR. The effects of miR-150 on cell cycle and apoptosis, as well as the expression of cycle- and apoptosis-related genes, were determined using flow cytometry, TUNEL assay, qRT-PCR, and Western blot, respectively. The direct target of miR-150 was confirmed using 3' untranslated region (UTR) luciferase reporter assay.

Results: miR-150 promotes cervical cancer cell survival and growth, while the inhibition of miR-150 suppresses these actions. miR-150 also induced the cell cycle progression from G1/G0 to S phase, resulting in an enhancement of growth. Several cell cycle- and apoptosis-related genes, CyclinD1, p27, BIM, and FASL were modulated by miR-150. Moreover, miR-150 directly reduced the expression of FOXO4, which regulates the expression of CyclinD1, p27, BIM, and FASL, by targeting its 3' UTR.

Conclusion: Taken together, our data demonstrated that elevated miR-150 targets FOXO4 expression and therefore regulates multiple genes expression, resulting in cervical cancer cell growth and survival.

Fig. 1

Cervical cancer cells express higher level of miR-150. a miR-150 expression was measured in cervical carcinoma and para-carcinoma tissues from the patients (n = 10) by RT-PCR. The miR-150 expression in cervical carcinoma tissue normalized to that in paired para-carcinoma tissues is presented by a histogram. b miR-150 expression in cervical tissue from healthy donors (normal, n = 13) or in cervical carcinoma tissue from patients (n = 108) was determined by RT-PCR and the relative miR-150 expression is presented after normalization to healthy donors. c The miR-150 expression in cervical tissue from healthy donors (n = 13) or in cervical carcinoma tissue from 62 patients at different stages (23 at grade II, 19 at grade III, 20 at grade IV), as well as in a human cervical carcinoma cell line C-33A, was evaluated by RT-PCR

Fig. 2

miR-150 promotes the survival of cervical carcinoma cells. a C-33A cells were transfected with siRNA control, or miR-150 mimics (miR-150) or inhibitors for 48 h and the apoptosis was determined by TUNEL assay and PI staining. Representative pictures of the TUNEL assays are presented. b The percentage of TUNEL-positive cells was counted and presented by a histogram. Mean values ± standard deviation (SD) for 3 independent experiments is shown

Fig. 3

miR-150 promotes the growth of cervical carcinoma cells. a The expression of miR-150 in non-transduction C-33A cells (mock) or stable cells after transduction with control virus (control) or virus containing miR-150 mimics (miR-150) was analyzed by RT-PCR. The bar represents 3 independent experiments. b Same number of C-33A cells expressing miR-150 mimics or inhibitors, or empty vector-transducted C-33A cells (control) were seeded into 24-well plate and cultured for 4 days. The cell number was counted every day. Mean values ± SD for 3 independent experiments is shown. c The cell cycle analysis in the three stable C-33A sub-cell lines was performed after PI staining and representative DNA content profiles of cell cycle are presented. d The percentages of cells in different cell cycle stages from 3 independent experiments were analyzed and presented by a histogram

Fig. 4

miR-150 affects the expression of proteins involved in cell proliferation and apoptosis. a The mRNA expression of cell cycle-related genes CyclinD1 and p27 in the three C-33A sub-cell lines (control, miR-150, miR-150 inhibitor) was measured by RT-PCR. Mean values ± SD for 3 independent experiments is shown. b The mRNA expression of apoptosis-related genes FASL and BIM in these three sub-cell lines from 3 independent experiments was evaluated by RT-PCR. Mean values ± SD for 3 independent experiments are shown. c The expression of Cyclin D1, p27, GAPDH, and phophorylated pRb (p-pRb) in the sub-cell lines was determined by western blot, and the pixel densities of these proteins were analyzed and presented by histograms (bottom). Mean values ± SD for 3 independent experiments is shown. d The expression of FASL, BIM, and GAPDH in the sub-cell lines was determined by western blot, and the pixel densities of these proteins were analyzed from 3 independent experiments and presented by histograms (bottom)

Fig. 5

miR-150 directly targets the 3′UTR of FOXO4. a The expression of FOXO4 in three C-33A sub-cell lines was determined by western blot. b The sequence of the wild type and mutant 3′ UTR of FOXO4 (mut-3′UTR) for luciferase reporter assay are shown. c Three C-33A sub-cell lines were transfected with reporter plasmids containing wild type or mutant 3′UTR of FOXO4 for 48 h and the luciferase activity was measured. Mean values ± SD for 3 independent experiments is shown. d Hypothetic illustration of how miR-150 promotes cervical cancer cell cycle progression and survival by targeting FOXO4