The expression of miR-93 in PC tissues and their adjacent healthy tissues (n = 16) was determined by real-time PCR (qPCR). After we set the expression level of miR-93 in each adjacent tissue sample to 1.0, the expression of miR-93 in each PC tissue sample was >1.5-fold greater than that in the adjacent tissues, except in cases 6 and 12. The average expression level of miR-93 in the PC group was 3.14-fold greater compared with that of the adjacent tissues, showing statistically significant variation (p < 0.01; Figure 1).Figure 1

Expression of miR-93 in Human Prostate Cancer Tissues and Their Adjacent Tissues

The expression of miR-93 was considered 1; the average expression level of miR-93 in the PC group was 3.14-fold compared with that of the adjacent tissues showing statistically significant variation.

The expression of miR-93 in four PC cell lines and normal prostate epithelial cells was detected by qPCR. The four PC cell lines expressed miR-93 significantly more strongly than did the normal prostate epithelial cell line RWPE-1; this was especially true for PC cell lines LNCaP (human prostate adenocarcinoma cells that are androgen-sensitive) and DU145 (approximately 6.5-fold higher expression; Figure 2). Therefore, LNCaP and DU145 cells were chosen for further experiments.Figure 2

Expression of miR-93 in Human Prostate Cancer Cell Lines and Normal Prostate Epithelial Cells

The four-mouse model with prostate cancer expressed significantly higher miR-93 levels than the normal prostate epithelial cell line RWPE-1.

After the binding of miR-93 to its target was blocked by transfection with the miR-93 antisense oligonucleotide (ASO), the cell growth and colony formation ability of LNCaP and DU145 cells remarkably decreased, especially on days 4 and 5 after transfection (Figure 3).Figure 3

Effect of Inhibited miR-93 on the Growth of Prostate Cancer Cells

(A) Cell activity of LNCaP after transfected with miR-93 ASO determined by real-time PCR. (B) Cell activity of DU145 after transfected with miR-93 ASO determined by real-time PCR. (C) Colony formation experiment revealed that reduced expression of miR-93 by ASO markedly inhibited the growth of prostate cancer cells. (D) The histogram of colony formation experiment results.

To understand the influence of miR-93 on the invasiveness of PCCs, we conducted a Transwell invasion experiment. The Transwell invasive cell number of the LNCaP cells transfected with the miR-93 ASO was 87 ± 12.21, which was significantly higher than that of the LNCaP cells transfected with a control ASO (39 ± 7.19; p < 0.05). Similarly, the Transwell invasive cell number of DU145 cells transfected with the miR-93 ASO was higher than that of the DU145 cells transfected with the control ASO (65 ± 10.37 versus 35 ± 6.85; p < 0.05; Figure 4). These results showed that downregulation of miR-93 inhibited the invasiveness of PCCs.Figure 4

Effect of Reduced miR-93 Expression on the Invasiveness of Prostate Cancer Cells

(A) The image of transwell invasive cells of LNCaP and DU145 in miR-93 ASO and control ASO. (B) The Transwell invasive cell number of the LNCaP and DU145 cells transfected with miR-93 ASO and control ASO.

It has been reported that the influence of miRNAs on the phenotypes of tumor cells can be elucidated by evaluating the regulation of their specific target genes. Therefore, we identified three target genes of miR-93, namely, TGFΒR2, ITGB8, and LATS2, by means of a bioinformatic analysis. We performed western blot analysis (WBA) to detect the expression levels of TGFBR2, ITGB8, and LATS2 in LNCaP and DU145 cells transfected with the miR-93 ASO and found that the expression levels of all three target genes were significantly lower than those in LNCaP and DU145 cells transfected with the control ASO (Figure 5). Therefore, it could be inferred that miR-93 might promote the proliferation and invasiveness of PCCs by regulating these three target genes.Figure 5

Effect of Reduced miR-93 Expression on Its Target Genes TGFBR2, ITGB8, and LATS2

(A) The expression levels of TGFBR2, ITGB8, and LATS2 in LNCaP and DU145 cells transfected with miR-93 ASO and control ASO by WBA. (B) The protein expression levels of TGFBR2, ITGB8, and LATS2 in LNCaP and DU145 cells transfected with miR-93 ASO and control ASO.

To assess the effect of miR-93 on the tumor growth of PC, we inoculated LNCaP cells stably transfected with the miR-93 ASO into nude mice by subcutaneous injection to induce the formation of human PC. The growth rate and volume of these tumors induced by LNCaP cells stably transfected with the miR-93 ASO were significantly lower than those of tumors induced by LNCaP cells transfected with the control ASO (Figure 6A), suggesting that miR-93 might promote the growth of PC. After that, we determined the expression levels of miR-93 target genes TGFBR2, ITGB8, and LATS2 by WBA in both above-mentioned groups. When miR-93 was blocked, the expression levels of TGFBR2, ITGB8, and LATS2 significantly diminished (Figure 6B). These results suggested that miR-93, along with its target genes, may participate in the promotion of tumor growth.Figure 6

In Vivo Effect of Reduced miR-93 on the Growth of Prostate Cancer

(A) The tumors induced by LNCaP cells stably transfected with miR-93 ASO were significantly smaller than those transfected with control ASO. (B) The expression levels of TGFBR2, ITGB8, and LATS2 in all groups by WBA.

Healthy female BALB/c nude mice with immunodeficiency (n = 12, aged 6 weeks) were obtained from the Laboratory Animal Center of the Chinese Military Academy of Medical Sciences. The human normal prostate cell line RWPE-1 and human PCCs, including cell lines PC-3, LNCaP, DU145, and 22RV1, were procured from the conserved cell bank of our laboratory. Fresh human PC tissues and their corresponding adjacent normal tissues were collected at the Yongchuan Hospital affiliated with Chongqing Medical University. All of the tissue samples were histopathologically confirmed and used after acquisition of informed consent from each patient. A liposomal transfection reagent (Lipofectamine 2000) and TRIzol were purchased from Invitrogen (Shanghai). An M-MLV kit and RNase inhibitors were bought from Promega (USA). A qPCR master mix kit was purchased from TaKaRa (Japan). Primer pairs for miR-93 and U6 were synthesized by Invitrogen (Shanghai). Antibodies employed in this study were purchased from Abcam (USA). miR-93 ASO and its specific negative control, as well as miR-93-inhibited plasmid (miR-93 ASO/pGCMV/EGFP/miR/Blasticidin), were acquired from Genepharma (Shanghai, China). This study was approved by the Ethics Committee of Yongchuan Hospital affiliated with Chongqing Medical University. Written informed consent was obtained from all participants.

RWPE-1, PC-3, LNCaP, DU145, and 22RV1 cells (Bioleaf, Shanghai, China) were cultured in the RPMI 1640 medium (GIBCO, USA) supplemented with 10% of fetal bovine serum (FBS; HyClone, USA), 100 U/mL penicillin, and 100 mg/L streptomycin in an incubator at 37°C and 5% CO₂.

Cells in the exponential growth phase were harvested, and RNA extraction was conducted by means of the TRIzol reagent. Similarly, total RNA was extracted from fresh human PC tissues and their adjacent normal tissues using the TRIzol reagent. The quality and purity of RNAs were determined by agarose gel electrophoresis and UV spectrophotometry. To obtain cDNA, we subjected total RNA (2 μg per tissue sample) to reverse transcription, and the resultant cDNA was amplified with specific primers by qPCR. The primer sequences used in this study were as follows: miR-93: 5′-TGCGGTTTGGCACTAGCACAT-3′ (Forward), 5′-CCAGTGCAGGGTCCGAGGT-3′ (reverse); and internal control U6: 5′-TGCGGGTGCTCGCTTCGGCAGC-3′ (forward), 5′-CCAGTGCAGGGTCCGAGGT-3′ (reverse). The reaction conditions of qPCR were the following: one cycle at 94°C for 4 min for initiation; 40 cycles at 94°C for 15 s, 55°C for 30 s, and 72°C for 30 s for extension; and at 72°C for 10 min for melting curve analysis. All of the reactions were carried out in triplicate on an ABI 7500 PCR machine. Data analysis was performed by calculating the relative expression levels via the formula 2^−ΔΔCt.

Cells in the exponential growth phase were seeded in 96-well plates and incubated at 37°C and 5% CO₂ overnight. The miR-93 ASO and control ASO were transfected into LNCaP and DU145 cells. At 48 hr after transfection, 10 μL of 5 mg/L methyl thiazolyl tetrazolium (MTT) was added into each well and incubated at 37°C and 5% CO₂ for 4 hr. After centrifugation at 2000 × g for 5 min, the supernatant was discarded, and 100 μL of DMSO was added into each well to stop the reaction. After shaking for 10 min, the absorbance value at 570 nm was detected on a μQuant microplate reader (Bio-Tek). All of the experiments were conducted in triplicate.

At 48 hr after transfection, cells were washed with PBS and digested using 0.1% trypsin. These cells were seeded in 12-well plates at a density of 120 cells per well and incubated at 37°C and 5% CO₂ for 10 days. The medium was replaced every 3 days. After 10 days, the cells were fixed with methanol for 10 min and stained with 2 g/L crystal violet for 10 min. Cell colonies were counted under an inverted microscope. Cell clusters with ≥50 cells were considered a colony. All of the experiments were conducted in triplicate.

Transwell chambers were pretreated with 35 μL of Matrigel and dried at 37°C and 5% CO₂ for 6 hr in 24-well plates. At 48 hr after transfection, the cells were harvested, and a suspension at a concentration of 5 × 10⁵ cells/mL in FBS-free RPMI 1640 was prepared. Then the cell suspension (200 μL) was seeded in the upper chamber, and the RPMI 1640 medium containing 10% FBS (600 μL) was added into the lower chambers. After regular incubation for 24 hr, the Transwell chambers were fixed with methanol for 10 min. The cells present above the membrane were carefully removed with a cotton swab, whereas cells present below the membrane were washed thrice with PBS and stained with 2 g/L crystal violet for 15 min. Finally, the cells present below the membrane were examined under a microscope.

PC tissue samples and their adjacent normal tissue samples, as well as the cultured cells, were lysed by precooled RIPA (radioimmunoprecipitation assay) buffer containing a proteinase inhibitor. After centrifugation at 12,000 rpm for 10 min, the supernatants were collected to perform SDS-PAGE and subsequent WBA. Proteins were resolved by SDS-PAGE and transferred onto a nitrocellulose membrane (NCM). After blockage with BSA for 2 hr, the NCM was incubated with primary antibodies at 4°C overnight, followed by incubation with a horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 hr. Prior to incubation at each step, the NCM was thoroughly washed with 1× TBST (a mixture of tris-buffered saline [TBS] and polysorbate 20). Specific protein bands were developed using an enhanced chemiluminescence (ECL) reagent for 2 min in the dark. GAPDH served as a loading control. The gray intensity value of each protein band was analyzed on a LabWorks gel scan imaging system.

Healthy female BALB/c nude mice (n = 12, aged 6 weeks) were housed in a specific pathogen-free environment. The mice were randomly subdivided into two groups. A dose of 0.1 mL of a suspension of PCCs stably transfected with the miR-93 ASO/pGCMV/EGFP/miR/Blasticidin plasmid or its control plasmid (10⁷ cells/mL) was inoculated into the bilateral axillary of nude mice. The long (b) and short (a) diameters of each tumor were measured once every day after the seventh day of inoculation. The tumor volume was calculated as V = 1/2 × b × a2. After measurement of these parameters for 7 days, the mice were euthanized, and each tumor was isolated, weighed, and photographed. Subsequently, the final tumor volume was measured.

Data are presented as the mean ± SD and were analyzed in SPSS v16.0 statistical software. Comparisons between groups were performed by ANOVA or Student’s t test. p < 0.05 indicated statistical significance.