br MicroRNAs miRNAs are small non coding RNAs
MicroRNAs (miRNAs) are small non-coding RNAs (21 to 23 nu-cleotides) that play an important role in cancer processes like cell proliferation, apoptosis and metastasis (Cho, 2007). miRNAs regulate the expression of target genes by binding to 3′ untranslated regions (UTRs) of its target mRNAs and inhibit mRNA translation or promote mRNA degradation (Bushati and Cohen, 2007). Recent studies have shown that miR-34 family may be vital miRNAs in the progress of cancer, which contain three members: miR-34a, miR-34b, and miR-34c. miR-34a gene is located on chromosome 1p36.22, while miR-34b and miR-34c are located on chromosome 11q23.1. miR-34a is the key
Available online 19 September 2018
member of miR-34 family; the expression of miR-34a is higher than that of miR-34b and miR-34c in most human tissue (Maroof et al., 2014). miR-34a has been identified as an master tumor suppressor in cancer. CpG methylation of miR-34a displays in kinds of cancer including prostate cancer. Seventy nine percent of primary prostate cancers de-tected with miR-34a CpG methylation (Lodygin et al., 2008). It has been shown that miR-34a regulates multiple gene expression and in-volves in cell cycle, apoptosis, metastasis and chemoresistance in cancer (Misso et al., 2014). The proto-oncogene c-myc is frequently found to be activated in human malignancies, which activates the transcription of Z VAD FMK promoting genes, negatively regulates cell cycle arrest genes and results in cell cycle progression (Chen et al., 2014; Cole, 2014). Recently, c-myc was reported to be a direct target of miR-34a. miR-34a targeted c-myc and inhibited cell proliferation (Christoffersen et al., 2010; Yamamura et al., 2012).
In the present study we first revealed that BBP, one of the typical phthalate esters, altered the expression of cell cycle related gene and promoted cell proliferation of LNCaP and PC-3 prostate cancer cells. Furthermore, we showed that BBP induced the proliferation of prostate cancer cells through miR-34a/c-myc axis.
2. Materials and methods
2.1. Chemicals and reagents
Cell culture reagents were purchased from Gibco (Carlsbad, CA, USA) unless otherwise stated. Fetal bovine serum (FBS) was obtained from PAA Laboratories (Pasching, Austria). BBP was purchased from Sigma-Aldrich (St. Louis, MO, USA). miR-34a mimic, miR-34a inhibitor, control mimic and control inhibitor were purchased from Applied Biological Materials Inc. (Richmond, BC, Canada). The primary anti-bodies for anti-cyclin D1, anti-PCNA, anti-p21, anti-c-myc and anti-GAPDH were obtained from Cell Signaling Technology (Beverly, MA, USA).
Human prostate cancer LNCaP and PC-3 cells were obtained from Chinese Academy of Typical Culture Collection Cell Bank. PC-3 cells were cultured and maintained in DMEM-F12, supplemented with 10% FBS and 100 U /mL penicillin and 100 μg/mL streptomycin. LNCaP cells were cultured and maintained in RPMI 1640, supplemented with 10% FBS, 100 U /mL penicillin and 100 μg/mL streptomycin. All cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The fresh medium was changed every other day.
Cells were seeded in 96-well plates at a density of 1 × 103 cells/well in 100 μL of medium without any treatment for 24 h. Twenty four hours later, cells were treated with various doses of BBP (0,10−4,10−5,10−6,10−7 and 10−8 mol/L) every other day for 6 con-secutive days. Cell viability was assessed using MTT assay. BBP is not colored nor MTT reducer. Twenty microliter of methylthiazolete-trazolium (Sigma-Aldrich, St. Louis, MO, USA) assay solution (5 mg/ mL) was added to each well and the plates were further incubated for 4 h at 37 °C. medium containing MTT was removed and precipitants were solubilized in DMSO. Absorbance was measured at 490 nm using a microplate reader (Titertek, Huntsville, AL, USA). All measurements were performed in triplicate.
2.4. Western blot analysis
0.4% Triton X-100, 10% glycerol, 5 μg/mL leupeptin, 50 μg/mL of phenylmethanesulphonylfluoride, 1 mM benzamidine, 5 mg/mL apro-tinin and 1 mM sodium orthovanadate], then centrifuged at 4 °C for 30 min. Protein concentrations were measured with the BCA protein assay (Beyotime, China). Forty to eighty micrograms of proteins were fractionated by electrophoresis through 7.5–10% SDS-PAGE and were transferred to PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% defatted milk and subsequently probed with primary antibody overnight at 4 °C, and then incubated with horseradish peroxidase-conjugated secondary antibody. GAPDH was served as the loading control.
2.5. Transfection of miR-34a mimic and inhibitor