Expression of Micro-RNA-492 (MiR-492) in Human Cervical Cancer Cell Lines is Upregulated by Transfection with Wild-Type P53, Irradiation, and 5-Fluorouracil Treatment In Vitro

Abstract

BACKGROUND The status of p53 is critical to the chemoradiosensitivity of cervical cancer cells. Wild-type p53 is essential to orchestrate the cellular response to cytotoxic stimuli. Our previous data illustrated that cervical cancer patients whose specimens overexpressed microR-492 (miR-492) were highly sensitive to concurrent chemoradiation. Although p53 activation has been reported to upregulate miR-492 by a miRNA profiling assay in lung cancer cells, the transcriptional regulation of miR-492 in cervical cancer cells remains poorly understood. Therefore, we aimed to decipher the relationship between p53 and miR-492 in cervical cancer cells. MATERIAL AND METHODS The expression of p53 and miR-492 in cervical cancer cell lines was measured by western blot and real-time PCR. After cells were transfected with wild-type p53 plasmid or were treated by irradiation and 5-fluorouracil (5-FU), the expression changes of p53 as well as miR-492 were examined by western blot and real-time PCR. The putative p53 binding site of miR-492 was first analyzed by bioinformatics tools, then validated by chromatin immunoprecipitation and dual-luciferase reporter assays. RESULTS We found that miR-492 was upregulated in cells with wild-type p53 compared to cells with mutant p53. Transfection of wild-type p53 plasmid or treatments with cytotoxic reagents including irradiation and 5-FU all induced miR-492 overexpression. Bioinformatics analysis and experimental validations further proved p53 interacted with miR-492 promoter directly. CONCLUSIONS In cervical cancer cells, p53 activated miR-492 expression transcriptionally.

Figure 1

Expression of p53 and miR-492 in cervical cancer cell lines. (A) The protein expression of p53 in C-33A, SiHa, and HeLa cells was detected by western blot; β-actin was used as an internal control. Quantification of p53 expression was performed by using Gel-Pro Analyzer 3.1 software. (B) miR-492 expression was examined by qRT-PCR with U6 as an internal control. All values were represented as means ±SD from at least 3 individual experiments.

Figure 2

MiR-492 expression was induced by p53. (A, B) HeLa and SiHa cells were transiently transfected with wild-type p53 expression plasmid pCMV-Neo-Bam p53 wt or its control vector, and the levels of p53 and miR-492 were examined by western blot and qRT-PCR. Expression changes of p53 and miR-492 were also detected when SiHa cells were treated with 8 Gy with 6 MV x-ray irradiation for 24, 48, or 72 hours (C, D), or exposed to different concentrations of 5-FU for 20 hours (E, F) by using western blot and qRT-PCR. The densitometric measurement was conducted with each western blot analysis. All data were shown as means ±SD from at least 3 individual experiments. * P<0.05.

Figure 3

Features of the miR-492 promoter region. (A) Schematic representation of p53 BS position in the miR-492 promoter region. The sequence of putative p53 response element of miR-492 was compared with the consensus p53 BS in detail. Besides, p53 BS of miR-492 was conserved among 7 primate species. The sequences were derived from the UCSC Genome Browser, and the tandem repeats of consensus p53 binding motifs in miR-492 promoter were highlighted with blue. (B) Promoter sequence 2 kb upstream and 1 kb downstream of the pre-miR-492 was visualized on the UCSC Genome Browser. Upper panel: The histone post-transcriptional modifications including H3K4me1, H3K4me3, and H3K27Ac of miR-492 promoter were presented from GM12878, H1-hESC, and HSMM cell lines, as determined by the ChIP-seq assays from ENCODE data. Lower panel: DNase cluster tracks showed DNase I hypersensitivity areas. The black or gray box indicated a higher degree of DNase I hypersensitivity. The number of cell lines hypersensitive to DNase I in the putative p53 BS was shown, as well. The locus of p53 BS was highlighted with purple. BS – binding site; ChIP – chromatin immunoprecipitation.

Figure 4

MiR-492 expression was directly regulated by p53. (A) ChIP analysis of p53 enrichment at the promoter region of miR-492 was performed with SiHa and HeLa cells. The chromatin segments bound to p53 or control IgG were purified and further analyzed by semi-quantitative PCR with primers surrounding the p53 BS. (B) p53 BS transcriptional activity with or without 5-FU induction was validated by dual-luciferase reporter assays. pGL3-basic construct harboring wild-type or mutant p53 BS or the control vector was transfected into SiHa cells together with pRL-SV40 as an internal control with or without 300 μM 5-FU for 20 hours. Data were represented as means ±SD from 3 independent experiments, * P<0.05. ChIP – chromatin immunoprecipitation; 5-FU – 5-fluorouracil.