We collected 19 cervical cancer and 19 normal tissue samples from the Department of Obstetrics and Gynecology, Peking University Third Hospital. The tumor tissues were derived from patients with a diagnosis of cervical cancer according to the FIGO criteria. The normal tissue samples were from patients with benign gynecological diseases such as uterine fibroids and chronic cervicitis. All patients voluntarily gave their signed informed consent before the operation. This study protocol was approved by the ethics committee of Peking University Third Hospital. Fresh tissues were stored in liquid nitrogen before RNA extraction. Total RNA was isolated by using Trizol reagent. The isolated RNA was dissolved in RNase-free water and stored at −80 °C.
Cell culture, treatment and reagents
The human cervical cancer cell lines C33A, HeLa, SiHa and Caski, which are kept in our lab, were cultured in DMEM with 10 % fetal bovine serum and 100 mg/ml penicillin/streptomycin at 37 °C in 5 % CO2 atmosphere. Cells were treated with TGF-β1(PeproTech, Rocky Hill, USA) at 5 ng/ml for the indicated times. Cell transfection involved use of Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) with miRNA mimics and inhibitors, pmir-GLO plasmid(Life Technologies) or DAB2 siRNA. The primer sequences were for miR-106b, 5′-AAGTGCTGACAGTG CAGATAA-3′, mature miR-106b mimic, 5′-CAAAGUGC UCAUAGUGCAGGUAG -3′ and miR-106b inhibitor, 5′-GUUUCACGAGUAUCACGUCCAUC-3′(Ribobio, Guangzhou, China). The primer sequence for DAB2 siRNA was sense, 5′-AACAAA GGAUCUGGGUCAATT-3′and antisense, 5′-UUGACCCAGAUCCUUUGU UTT-3′ (GenePharma, Shanghai). DAB2 and GAPDH antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Total RNA was extracted by the conventional Trizol method. Reverse transcription of RNA was performed using miRcute miRNA cDNA (KR201)(TIANGEN, Beijing). Mature miR-106b was detected by using a miRNA miRcute fluorescent quantitative detection kit (FP401) (TIANGEN, Beijing).
Western blot assay
Total protein was extracted and quantified by using the BCA detection kit. Cells were lysed in 5 × loading buffer, boiled for 5 min and denatured. Cell lysates (20 μg) were separated by 10 % SDS-PAGE and transferred to nitrocellulose membranes, which were blocked with 5 % nonfat milk and incubated with primary antibodies against DAB2(1:1000, rabbit anti-human polyclonalantibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. The goat anti-rabbit secondary antibody (ZSGB-BIO, Beijing) was incubated for 1 h at room temperature. The membranes were exposed by use of luminescent liquid (Millipore, Massachusetts, USA) in the darkroom, dried naturally. The densitometry of protein bands was quantified by use of Image J (NIH, Bethesda, MD, USA).
The paraffin sections of tissues were dewaxed as routine. Endogenous peroxidase of tissues was removed by 3 % H2O2 for 10 min. The tissues were blocked for 30 min by goat serum working fluid (ZSGB-BIO, Beijing) and stained with anti-DAB2 antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight, then goat anti-rabbitsecondary antibody (ZSGB-BIO, Beijing) for 1 h at room temperature. The tissues were stained with DAB color solution for an appropriate time (ZSGB-BIO, Beijing), dehydrated, fixed and photographed. Next, we conducted a semi-quantitative analysis by using Image-Pro Plus 6.0. First, five randomly selected fields (400×) of each stained section were photographed. The integrated optical density (IOD) and mean density (IOD SUM/area) value for positive areas of each photo were calculated. The mean density of each sample was the average of the mean density from five images. We further compared the mean density of 19 cervical cancer biopsy sections and 19 normal cervical tissues samples by t-test analysis.
In situ hybridization
In situ hybridization was performed by use of the miR-106b in situ hybridization labeling Kit. Paraffin sections of cervical cancer tissue were deparaffinized. Endogenous enzymes are inactivated by 3%H2O2. The exposure of mRNA nucleic acid fragment involved use of 3 % citric acid diluted fresh pepsin incubated for 15 min. Then, 1 % formaldehyde fixed for 10 min. After prehybridization for 4 h in a thermostat box at 38 °C, tissue was incubated with 5′digoxigenin-labeled oligonucleotide probe detecting miR-106b and hybridized overnight. Then, tissues were washed several times and blocked for 30 min, then, incubated with anti-digoxigenin antibody for 60 min at 37 °C. After rinsing, sections were incubated with SABC and Biotin peroxidase, then, stained with DAB color solution for the appropriate time and counterstained with nuclear. The relative level of miR-106b in cervical tissues was measured by Image-Pro Plus 6.0 as for the detailed content in immunohistochemistry.
Cell scratch and migration assays
In cell scratch assays, a 20-μl pipette tip was used to make three parallel wounds and one vertical wound in each well of 6-well plates with cells incubated at 1 × 106. Cells were cultured in serum-free mediumand photographed by inverted microscopy at 0 and 24 h. In the transwell assay, cells were seeded in the upper chamber of Costar transwell culture plates (24-well plates, 8 μm) and cultured in serum-free medium. DMEM with 20 % fetal bovine serum was added to the lower chamber. After 24 h, the chamber was washed 3 times with 1 × PBS. The membrane in the lower chamber was fixed in 4 % paraformaldehyde and stained with crystal violet. The cells on the upper membrane that did not migrate were wiped with a cotton swab. The number of migrated cells was counted under a microscope in at least five fields.
Dual-luciferase reporter assay
The DAB2 gene 3′UTR with 166 bp sequences including predicted miR-106b binding sites was amplified by PCR from human genomic DNA. The amplified sequences were inserted into cloning sites of the pmir-GLO vector at SacI and SalI sites and verified by sequencing. The mutant DAB2 3′UTR vector was constructed in the dual luciferase reporter gene pmir-GLO vector with miR-106b matching nucleotides “GCACTTT” replaced with “TATAGGG” (Life Technologies, Shanghai). HEK-293A cells in 24-well plates were transfected with the wild-type and mutant DAB2 luciferase reporter vector and miR-106b mimics. Luciferase assays involved use of the Luciferase Reporter Gene Assay Kit (Promega) and activities were normalized to Renilla luciferase activity.
Data are expressed as mean ± SEM and were analyzed by use of GraphPad Prism 5. Two groups were compared by Student’s t-test, and multiple groups by One Way ANOVA, with further two group comparison by Multiple Comparison Test Tukey’s test. When the variance of the two groups was not homogeneous, non parametric tests were used. Correlations were examined by Spearman correlation analysis. P < 0.05 was considered statistically significant.