Transfection and apoptosis assay

Cells were placed in six-well plates at 70–90% confluency 24 h before transfection in serum free media. Cells were transfected with various plasmids (2 μg/well) by using Lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacture’s protocol. After 72 h, cells were collected by trypsinization, washed with phosphate-buffered saline and dual stained with the viability dye 7-amino-actinomycin D and Annexin V- fluorescein isothiocyanate using Annexin V- fluorescein isothiocyanate /7-amino-actinomyicin D kit (Beckman Coulter, Brea, CA) according to the manufacturer’s protocol. Stained cells were immediately analyzed using a flow cytometer (Cell Lab Quanta SC; Beckman Coulter).

RNA and DNA extraction

Total RNA was extracted from microdissected FFPE tissue and cultured cells using a miRNeasy FFPE Kit (Qiagen, Valencia, CA) for FFPE tissues and RNeasy mini kit (Qiagen) for cultured cells. Total DNA was extracted from 21 microdissected FFPE prostate cancer tissues and adjacent normal tissues.

5-Aza-2′-deoxycytidine treatment

Cells were plated in six-well plates on Day 0, and treated with a final concentration of 5 μM 5-aza-2′-deoxycytidine (Sigma, St Louis, MO) daily beginning from Day 1, until Day 4. The treated cells were harvested on Day 5.

Quantitative real-time PCR

Complementary DNAs were synthesized by using specific-miRNA primers (Applied Biosystems, Foster City, CA). Gene expression levels were measured by real-time quantitative PCR using an Applied Biosystems 7500 Fast Sequence Detection and gene-specific Taqman assay kits. RNU48 was used as endogenous control to normalize expression data. The thermal cycling conditions were according to the TaqMan Fast Universal PCR protocol. Each sample was analyzed in quadruplicate.

Bisulfite modified DNA sequencing

Methylation status of CpG sites in the miR-145 promoter was determined by NaHSO₃-sequencing method. DNA was treated with NaHSO₃ and amplified by PCR with the following primer set: forward, 5′GTGTAGATAGTAGAGGGTAGTTT; reverse, 5′TCCCACATCCAACCTCACAAA. This PCR product, which covers the miR-145 promoter region from −224 to +194, was sequenced on an ABI sequencer with dye terminators (Applied Biosystems). The quotient of C over C + T at each CpG site indicates the percentage of methylation.

Methylation-specific PCR

The bisulfite-treated genomic DNA was amplified by PCR with a set of primers for the unmethylated reaction and with another set of primers for the methylated reaction: (a) unmethylated forward primer (5′GGGTTTTTGGTATTTTTTAGGGTAATTGAAGTTTT) and reverse primer (5′AACCAAAATAAAATACCACACATCACCA); (b) methylated forward primer (5′GGGTTTTCGGTATTTTTTAGGGTAATTGAAGTTTC) and reverse primer (5′TAAAATACCACACGTCGCCG). PCR was performed at 95°C for 5 min, 40 cycles at 94°C for 30 s, 56°C (unmethylated primer) or 64°C (methylated primer) for 30 s and 72°C for 30 s followed by a final extension at 72°C for 10 min in a PTC-100 thermal cycler (MJ Research, Waltham, MA). The PCR products were loaded on 2.0% agarose gel for analysis.

Sequence analysis of the p53 gene

DNA was amplified by PCR using five pairs of primers corresponding to sequences of exons 4–8 of p53 genes (4forward, 5′AAGCTCCCAGAATGCCA and 4reverse, 5′TGAAGTCTCATGGAAGCCAG; 5forward, 5′GTGCCCTGACTTTCAACTCT and 5reverse, 5′CAACCAGCCCTGTCGTCTCT; 6forward, 5′GCCTCTGATTCCTCACTGAT and 6reverse, 5′CCAGAGACCCCAGTTGCAAA; 7forward, 5′CAAGGCGCACTGGCCTCAT and 7reverse, 5′CAGTGTGCAGGGTGGCAAGT and 8forward, 5′GACCTGATTTCCTTACTGCCT and 8reverse, 5′GAATCTGAGGCATAACTGCAC). The PCR product was sequenced on an ABI sequencer with dye terminators (Applied Biosystems).

Electrophoretic mobility shift assay

A DNA probe containing the p53 response element at 5′ upstream site (−829 to −810) of miR-145 was obtained by annealing two oligos. A DNA probe containing consensus p53 response element (5′ GCCGAGACATGCCTAGACATGCCTCAA) was obtained by annealing two oligos. EMSA was performed using an EMSA kit (Molecular Probes; Invitrogen) according to the manufacturer’s instructions.

Laser captured microdissection (LCM) and RNA extraction from FFPE human prostate cancer samples

Microdissection was performed using the AutoPix System from Arcturus. Sections (8 μm) were placed on glass slides, deparaffinized, stained with hematoxylin, dehydrated and placed in the AutoPix instrument for microdissection. Areas of interest were captured with infrared laser pulses onto CapSure Macro LCM Caps (Figure 1).

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Fig. 1.

Laser capture microdissection (LCM) in prostate tissues. Upper and lower panels are two prostate tissues. The left panels show targeted glands and stroma before LCM. The center panels show gland cells captured by LCM. The right panels show residual tissues left behind after LCM.

In situ hybridization

Non-radioactive in situ hybridization was performed on 5 μm sections from FFPE prostate cancer tissue blocks using the digoxigenin-labeled locked nucleic acid modified detection probe (20 μM; 5′-AGGGATTCCTGGGAAAACTGGAC-3′, Exiqon, Tustin, CA) complementary to mature miR-145 and IsHyb in situ hybridization Kit (BioChain, Hayward, CA) following the protocol from Exiqon. Briefly, 5 μm sections were deparaffinized, hydrated, treated with proteinase K (10 μg/ml; Qiagen) at 37°C for 5 min then with 0.2% glycine for 1 min and fixed with 4% paraformaldehyde for 10 min. The sections were incubated in pre-hybridization solution at 50°C for 3 h and then in hybridization solution with digoxigenin -labeled, locked nucleic acid-modified miR145 probe at 57°C for 16 h. The sections were incubated in the phosphate-buffered saline diluted anti- digoxigenin -AP antibody (1:200) at 4°C for 16 h, washed with 2× standard saline citrate, 1.5× standard saline citrate, 0.2× standard saline citrate, incubated in blocking solution for 1 h at room temperature, washed with phosphate-buffered saline three times, then washed with 1× alkaline phosphatase buffer twice. BM purple (Roche South San Francisco, CA) was used for visualization of hybridization signal.

miR-145 expression in prostate cancers is correlated with p53 status and methylation in the upstream sequence of miR-145 in prostate cancer

miR-145 contains several CpG sites and p53 response element (Figure 3A). p53 status and methylation in the upstream sequence of miR-145 were examined in 21 prostate cancer and adjacent normal samples. Prostate cancer samples (17 of 21, 81%) showed lower miR-145 expression compared with the adjacent normal regions. In these 17 samples, 6 had both p53 mutations and hypermethylation of miR-145, 5 had only p53 mutations, 3 had only hypermethylation of miR-145, whereas only 3 (18%) had neither p53 mutation nor hypermethylation (Table I, supplementary Figure 1 is available at Carcinogenesis Online). In four prostate cancer samples with no downregulation of miR-145, neither p53 mutation nor hypermethylation of miR-145 was detected (P = 0.022) (Table I, supplementary Figure 1 is available at Carcinogenesis Online). BPH samples (9 of 10) with high expression of miR-145 showed no methylation in the upstream region of miR-145 by methylation-specific PCR. Only one BPH sample showed partial methylation (Figure 3B).

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Fig. 3.

Methylation of miR-145 in prostate cancer, adjacent normal and BPH. (A) A schematic description of the putative miR-145 promotor. (B) Methylation of miR-145 in BPH samples determined by methylation-specific PCR (U, product from unmethylated primers; M, product from methylated primers).

In order to further confirm the correlation of miR-145 level with p53 status and methylation, we measured miR-145 expression levels with p53 status and miR-145 methylation in 47 cancer cell lines, including 4 prostate cancer cell lines, 4 kidney cancer cell lines, 11 breast cancer cell lines, 20 colorectal cancer cell lines and 8 pancreatic cancer cell lines. We observed a significant correlation of miR-145 expression with p53 gene status and miR-145 methylation (P < 0.001). In 34 cell lines with low miR-145 expression, 16, 12 and 5 cell lines had both p53 mutations and miR-145 methylation, only p53 mutation, or methylation of miR-145, respectively. p53 mutation and miR-145 methylation were not detected in only one cell line. In 13 cell lines with high miR-145 expression, p53 mutation and miR-145 methylation were not present in 10 cell lines (supplementary Table 1 is available at Carcinogenesis Online ).

5-Aza-2′-deoxycytidine treatment induces the expression of miR-145

To confirm the silencing effect of miR-145 methylation, seven cancer cell lines with methylated miR-145 promoter (PC3, LNCaP, Du145, HT29, C, Caki2 and A498) and two cell lines with no methylation of miR-145 (SW48, RKO) were treated with DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine. MiR-145 expression was induced in all seven cell lines with methylated miR-145, whereas miR-145 was not as much induced in cell lines with no miR-145 methylation (Figure 4).

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Fig. 4.

MiR-145 levels were upregulated by 5-aza-2′-deoxycytidine treatment. P53 status and methylation of miR-145 promoter region is shown below each cell line name. In each cell line, left bar is the untreated control.

Methylation of miR-145 interferes with the binding of p53 to the p53 response element upstream of miR-145

To investigate the relationship of methylation and p53 in miR-145 expression, we performed EMSA with a DNA probe containing the p53 response element at the 5′ upstream site (−829 to −810) of miR-145. A band was observed by the binding of probe with LNCaP cell nuclear extract [containing wild-type p53 (WT p53)]. The band was not observed when anti-p53 antibody was added, indicating the nuclear protein was recognized by the anti-p53 antibody. As many investigators showed, in EMSA, the addition of antibodies can lead to either slower mobility of band (supershift) or loss of the shifted band due to the interfering with the formation of protein/DNA complex by the interaction between epitopes and antibodies (http://www.piercenet.com/proteomics, http://www.promega.com/guides). The band was not formed when the methylated probe was mixed with LNCaP cell nuclear extract (Figure 5A), indicating that methylation of the miR-145 promoter region interferes with p53 binding. To confirm the shifted band is the complex of p53 and p53 element in miR-145 promoter, we performed EMSA with a probe of consensus p53 response element (Figure 5B). The band was shifted as in Figure 5A, and the shifted band was not observed when anti-p53 antibody was added. The methylation also interfered with the formation of protein/DNA complex as in Figure 5A.

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Fig. 5.

WT p53 binds to miR-145 promoter, regulates its expression and increases apoptosis of cells. (A) The binding of p53 to the p53 response element upstream of miR-145 gene shown by EMSA. The binding of p53 to the p53 response element is indicated by the arrow. (B) The binding of p53 to the consensus p53 response element shown by EMSA. The binding of p53 to the p53 response element is indicated by the arrow. (C) miR-145 expression levels are up regulated after transfection of PC-3 cells with WT p53 but not mutant p53 by real-time PCR. Empty vector pCMV was used as a control. (D) Apoptotic cells are significantly increased after transfection of PC-3 cells with WT p53 (12.72%), but not with MUT p53 (5.59%). Empty vector pCMV is used as a control (2.75%).

We thank Dr Roger Erickson for his support and assistance with the preparation of the manuscript.

Conflict of Interest Statement: None declared.