microRNA-181 promotes prostate cancer cell proliferation by regulating DAX-1 expression

microRNAs (miRNAs) are a class of short noncoding RNA molecules that have a critical role in the initiation and progression of types of human cancer, including prostate cancer. In the present study, the expression of miR-181 in prostate cancer tissues was evaluated and was demonstrated to be significantly upregulated in prostate cancer tissues compared with that in adjacent normal tissues. The results of in vitro MTT and BrdU incorporation assays, as well as cell-cycle analysis, indicated that miR-181 overexpression markedly promoted the proliferation of LNCaP cells. Furthermore, miR-181 overexpression was found to promote the progression of LNCaP tumor growth in nude mice. Mechanistic studies demonstrated that dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1), a negative regulator of androgen receptor in prostate cancer, was inhibited by miR-181 overexpression. Therefore, the results from the present study suggest that miR-181 functions as a growth-suppressive miRNA during prostate cancer development.


Introduction
Prostate cancer, one of the most common types of cancer in males, has become a major public health concern (1). The molecular pathogenesis of prostate cancer is complicated and remains poorly understood (2). Therefore, the identification of novel molecular mechanisms may help to develop strategies for its diagnosis, treatment and prognosis.
Recent studies have shown that microRNAs (miRNAs) have a critical role in the development of numerous different types of human cancer (3,4). miRNAs regulate multiple genes by targeting mRNAs, resulting in mRNA degradation or translation repression (5). For example, miR-888 is a miRNA secreted by prostate cells that promotes prostate cell growth and migration through the repression of the levels of protein produced by the tumor suppressor genes RBL1 and SMAD4 (6).
Previous studies have demonstrated that the upregulation of hepatic miR-181 promotes the growth, clonogenic survival, migration and invasion of hepatocellular carcinoma cells (7,8). Furthermore, the expression level of miR-181 is significantly associated with overall survival in hematological malignancies and may be an important clinical prognostic factor for patients with hepatocellular carcinoma (9). However, the expression and function of mi-181 in prostate cancer has yet to be elucidated. Therefore, in the present study, the expression of miR-181 was determined in prostate cancer tissues. In addition, the proliferation of prostate cancer cells overexpressing mi-R181 was analyzed in vivo and in vitro. Furthermore, the targets of miR-181 were investigated in order to determine the underlying mechanism of miR-181 in prostate cancer.

Materials and methods
Tissue samples and cell culture. A total of 20 prostate cancer samples and adjacent normal tissues were obtained from patients who underwent surgery at Huashan Hospital Affiliated to Fudan University (Shanghai, China). The present study was approved by the hospital institutional review board and written informed consent was obtained from each patient. LNCaP cells were provided by the Institute of Biochemistry and Cell Biology of Chinese Academy of Science (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies), 100 IU/ml penicillin and 100 µg/ml streptomycin sulfate. Cells were incubated at 37˚C with 5% CO 2 .
Luciferase reporter assay. cDNA fragments corresponding to the entire 3'-untranslated region (UTR) were amplified by qPCR from the total RNA extracted from LNCaP cells with KpnI and EcoRI linkers. The PCR products were cloned downstream of the Renilla luciferase open reading frame of the pMir-Report (Qiagen), which also contained a constitutively expressed firefly luciferase gene that was used to normalize the transfections. For the luciferase reporter assays, the cells were seeded in 24-well plates and harvested 48 h after transfection. The wild-type and mutant 3'-untranslated region fragments from the human DAX-1 gene were cloned into pMir-Report (Qiagen). Mutations were introduced in potential miR-181 binding sites using a site-directed mutagenesis kit (Qiagen). Luciferase values were determined using the Dual-Luciferase Reporter assay system (Promega Corporation, Madison, WI, USA).
Tumor growth assay. Male BALB/c nude mice, aged 4 weeks, were purchased from the animal center of the Second Military Medical University (Shanghai, China). A total of 2x10 5 LNCaP cells stably expressing miR-181 or NC were injected subcutaneously into the dorsal flank of the mice. The mice were observed over 5 weeks for tumor formation. The mice were then sacrificed and the tumors were recovered and the wet weight of each tumor was determined. The tumor volume (mm 3 ) was calculated according to the following formula: Volume (mm 3 ) = 1/2 x length x width 2 . The experimental protocol was approved by the Experimental Animal Care Commission of Huashan Hospital Affiliated to Fudan University.
Statistical analysis. Differences between groups were analyzed using a Student's t-test and expressed as the mean ± standard deviation from ≥three independent experiments. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using GraphPad Prism version .0 software (GraphPad Software, Inc., La Jolla, CA, USA).

miR-181 is upregulated in prostate cancer tissues.
The expression of miR-181 was analyzed in prostate cancer tissues and adjacent normal tissues using qPCR. It was found that miR-181 is significantly upregulated in cancer tissues compared with that in normal adjacent tissues, as shown in Fig. 1.

miR-181 overexpression promotes prostate cancer cell proliferation in vitro.
Since miR-181 was found to be upregulated in prostate cancer tissues, the effect of miR-181 on prostate cancer cell growth was investigated. LNCaP cells were transfected with miR-181 mimics or NC (Fig. 2A). The results demonstrated that cell growth was significantly increased in miR-181-overexpressing cells compared with that of their corresponding controls, measured using the MTT and BrdU assays ( Fig. 2B and C). Furthermore, miR-181 overexpression decreased the percentage of cells in the G1 phase and increased the percentage of cells in the S phase (Fig. 2D).

miR-181 overexpression promotes tumor growth in vivo.
To further investigate the function of miR-181 on tumor growth in vivo, LNCaP cells with stable overexpression of miR-181 were generated and injected subcutaneously into the dorsal flank of nude mice. Tumor growth was closely monitored for 5 weeks. The tumor size and volume were markedly increased in mice injected with LNCaP cells overexpressing miR-181 compared with those in control mice (Fig. 3A and B). In addition, the average tumor weight was significantly increased by miR-181 overexpression (Fig. 3C), suggesting that miR-181 may promote tumor growth in vivo.

miR-181 targets the DAX-1 3'-untranslated region (3'-UTR)
and downregulates its expression. In order to understand the underlying mechanism, potential targets of miR-181 were determined using TargetScan software. DAX-1 was identified as a potential target of miR-181. Notably, the 3'-UTR of DAX-1 mRNA was observed to contain a complementary site for the seed region of miR-181 (Fig. 4A). To investigate  whether DAX-1 may be directly targeted by miR-181, a luciferase reporter vector was constructed, containing the putative miR-181 binding sites within the DAX-1 3'-UTR. The results showed that miR-181 overexpression significantly decreased the luciferase activity, and mutations in the miR-181 binding site from the DAX-1 3'-UTR abolished this effect, suggesting that miR-181 directly inhibited DAX-1 expression by targeting the 3'-UTR (Fig. 4B). Furthermore, miR-181 mimics decreased the endogenous protein levels of DAX-1, as indicated by western blot analysis (Fig. 4C), while the DAX-1 mRNA levels remained unchanged (Fig. 4D). Therefore, these results suggest that miR-181 may negatively regulate DAX-1 expression at the translational level in LNCaP cells. DAX1 has been previously demonstrated to repress the transcriptional activity of the androgen receptor (AR) in LNCaP cells (10). In the present study, elevated expression levels of AR target genes and proteins, including prostate-specific antigen, cyclin-dependent kinase (CDK) 1 and CDK2, was observed in LNCaP cells overexpressing miR-181 (Fig. 5). In combination, these results further confirm that DAX-1 is an important target gene of miR-181 in prostate cancer cells.

Discussion
It has been previously demonstrated that several miRNAs are dysregulated in prostate cancer tissues or cell lines, and they have been shown to be associated with prostate cancer progression and disease outcome (11)(12)(13). In the present study, it was demonstrated for the first time, to the best of our knowledge, that miR-181 overexpression may promote cell proliferation and cell-cycle progression in LNCaP cells. In addition, miR-181 overexpression was observed to promote the growth of LNCaP tumors in nude mice. Therefore, miR-181 may be an onco-miRNA in the development of prostate cancer.
Furthermore, in the present study DAX-1 was identified as a direct target of miR-181 in prostate cancer cells. DAX-1, a

A B
member of the orphan nuclear receptor family, is known to have an important role during development, particularly in gender determination and steroidogenesis (14,15). In humans, DAX-1 gene mutations usually lead to the X-linked congenital adrenal hypoplasia and primary adrenal insufficiency associated with hypogonadotropic hypogonadism (16,17). With regard to the types of cancer observed in humans, DAX-1 expression has been reported in endocrine and sex steroid-dependent neoplasms, including adrenocortical, pituitary, endometrial and ovarian tumors (18)(19)(20). For example, DAX-1 overexpression has been demonstrated to repress estrogen-dependent breast cancer cell proliferation via the inhibition of aromatase expression (19). In addition, DAX-1 expression has been observed to be significantly downregulated in prostate cancer (10). In a previous study, at the molecular level, DAX-1 was demonstrated to interact with the AR and inhibit its nuclear localization. As a result, DAX-1 was found to repress androgen-dependent gene transcription in prostate cancer cells (10). The results from the present study are in accordance with these findings. They demonstrate that miR-181 overexpression causes the upregulation of AR target genes, suggesting that the proliferative role of miR-181, at least in part, may be dependent on androgen signaling.
In conclusion, the present study provides a novel role for miR-181 in prostate cancer cell proliferation. The results suggest that miR-181 may be a potential therapeutic target for the treatment of prostate cancer in the future.