br Quantitative real time PCR qRT PCR analysis br
2.3. Quantitative real-time PCR (qRT-PCR) analysis
Total RNA was extracted from tissues and cell lines using TRIZOLTM Reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized using QIAGEN qPCR Kit (QIAGEN, Dusseldorf, Germany) and the sequence of specific hsa-miR-93 reverse transcrip-tion primer was shown as follow: miR-93 forward 5′-CAAAGUGCUGU UCGUGCAGGUAG-3′ and reverse unknown (secret information and re-served by QIAGEN); U6 forward, 5′-ATT GGA ACG ATA CAG AGA AGA TT-3′ and reverse 5′-GGA ACG CTT CAC GAA TTT G-3′; DAB2 forward
F: 5′-TGGGAGTGAGGCCCTAATGA-3′ and reverse 5′-AATCCATCTGG TCAACACCCG-3′. Reverse transcription was performed using Super M-MLV Reverse Transcriptase (BioTeke, Beijing, China) or the miScript re-verse transcription kit (QIAGEN, Dusseldorf, Germany) for mRNA or miRNA expression analysis, respectively. Finally, Real-time PCR was done on a Bio-rad CFX 96 Real-Time PCR System. The PCR conditions were initial denaturation at 95 °C for 3 min, followed by 40 cycles of
2.4. In situ hybridization
The expression of miR-93 in PC and human benign prostatic hyper-plasia tissues was analyzed using in situ hybridization with the probe of human miR-93 (Bioster, Wuhan, China) according to the manufactur-er's instruction. Finally, the slides were stained with DAB (ZSGB-BIO, Beijing, China) for 5 min and analyzed with a light microscope (×200).
miR-93 mimics (Maji, Shanghai, China) and miR-NC (Maji, Shanghai, China), alone or in combination were transiently transfected into PC-3 cells by using Lipofectamine 2000 Kit (QIAGEN) according to the proto-col. miR-93 inhibitor (Maji, Shanghai, China) and miR-NC (Maji, Shang-hai, China) were transiently transfected into DU145 cells in the same way. All transfections were performed 24 h before MTT, colony forma-tion, invasion and migration assays.
4 h in a humidified LNicotine containing 5% CO2 at 37 °C. Subse-quently, the cell-free supernatant was discarded and replaced with di-
methyl sulfoxide (200 μl/well) to dissolve the formazan product. Finally, the optical density (OD) at 490 nm was measured using a micro-plate reader (Bio-Tek Instruments, Winooski, VT, USA), and each exper-iment was performed with three times.
For colony formation assay, transfected cells were seeded in six-well plates (2 × 103 cells/ml) containing 2 ml growth medium and were cul-tured for 14 days. Colonies were fixed using 1% formaldehyde for 30 min, followed by the addition with 1 ml of 1.0% crystal violet for 2 min, and then the number of colonies formed was counted using a mi-croscope (Olympus, Tokyo, Japan).
The invasion capability of PC cells was determined using a transwell chamber (Corning Incorporated, Toledo, NY, USA) pre-coated with Matrigel (Invitrogen, Shanghai, China). Briefly, 48 h after transfection,
200 μl of serum-free medium containing (1 × 106 cells) was placed into the upper chamber. Meanwhile, 500 μl growth medium containing 15% Fetal Bovine Serum (FBS) was placed into the lower chamber. After
24 h of incubation, non-invasive cells remaining on the upper chamber were gently removed by a cotton swab. The invasive cells on the lower filter side were then fixed with 10% formalin solution, stained with 0.1% crystal violet (Sigma) and accounted under a microscope (Olympus). The mean number of invaded cells was counted at 200× magnification from five different fields per membrane.
For cell migration assay, transfected cancer cells were allowed to grow for 24 h in 24-well plates at a concentration of 5 × 105 cells per well. After being confluent, a uniform scratch was made across the cen-ter of the cell monolayer using a 1-ml sterile plastic pipette tip, followed by washing twice with phosphate-buffered saline (PBS). Then, serum-free medium was added to the respective wells for further incubation. After 24 h, cell migration was observed and photos were performed using the Nikon inverted microscope. The migration distances between the leading edge of the migrating cells and the edge of the wound were compared. The migration rate was quantified as the following equation:
migration rate = [(migration distance of transfected cells / migration distance of control cells) × 100%]. Experiments were independently performed at least four times. The migration rate of the control was set as 100%.
2.6.5. Tumorigenic studies in nude mice
Animal experiments were approved by the Animal Care and Use Committee of Hunan Provincial People's Hospital (Changsha, China). 200 μl of PC-3 cells (5 × 107 cells/ml) suspension were injected subcu-taneously in the flank of male nude mice. The nude mice were observed every other day after inoculation, and the growth of the tumor was re-corded. In the meantime, the mental state of the vaccinated mice was also observed. When tumor reached a size of approximately 150 mm3, the treatment was initiated. The mice were assigned to two groups: miR-93 mimics group and miR-NC control group, and ten mice in each group. miR-93-expressing plasmid or empty vector was injected into the tumor mass one time every other day. The mice were sacrificed four weeks later by cervical dislocation and the xenografts from the mice were removed. Tumor growth was measured every other day and tumor volume (V) was calculated using the following formula: V = L × W2 × 0.52, where L represents largest superficial diameter and W represents smallest superficial diameter as previously described by Xin et al. (2012) .