MiR‐605 represses PSMD10/Gankyrin and inhibits intrahepatic cholangiocarcinoma cell progression

The aberrant expression of PSMD10 has important functions in various malignancies. This study showed that PSMD10 was highly expressed and inversely correlated with the expression of miR‐605 in intrahepatic cholangiocarcinoma (ICC) specimens. MiR‐605 directly targeted and repressed PSMD10 expression. In addition, over‐expression of miR‐605 inhibited ICC cell progression both in vitro and in vivo. This effect of miR‐605 on ICC cells was similar to that of PSMD10 knock‐down by RNAi. Moreover, restoration of PSMD10 could reverse the phenotypic alteration caused by miR‐605 in ICC cells. These results suggest a new therapeutic strategy in ICC by restoring miR‐605, which is regulated by p53.


Introduction
Intrahepatic cholangiocarcinoma (ICC) is the second most common primary epithelial tumor arising from the epithelium of the intrahepatic bile duct. ICC is a treatment refractory malignancy with a high mortality and increasing incidence worldwide [1][2][3]. However, the molecular etiology of ICCs remains unclear. The proto-oncogene, proteasome 26S subunit non-ATPase 10 (PSMD10), which encodes a subunit of the PA700/19S complex, has important functions in the pathogenesis of cancer. Dysregulation of PSMD10 expression contributes to cancer progression, and has essential functions in cell survival, migration, and proliferation [4][5][6]. Early studies have shown that PSMD10 enhances the ubiquitylation and degradation of p53, which is often mutated in ICC [7][8][9]. PSMD10 has become an important therapeutic target for ICC. Therefore, a promising therapeutic strategy against ICC should be developed, and a better understanding of the molecular regulatory mechanism of PSMD10 may be helpful for the treatment of ICC.
MicroRNAs (miRNAs) are a class of small non-coding RNAs that negatively regulate gene expression at the post-transcription level by mRNA degradation and translation inhibition through binding the 3 0 untranslated regions (UTRs) of human mRNAs [10]. In the past decade, miRNA has been reported to be dysregulated in tumors. MiRNAs act as potent oncogenes or tumor suppressor genes [11][12][13]. The dysregulation of miRNAs has also been reported to be responsible for ICC tumor progression. For example, miR-204 and miR-320 are lowly expressed in ICC, and over-expression of miR-204 and miR-320 can induce chemotherapeutic drugtriggered apoptosis and down-regulate the expression of BCL-2 and Mcl-I, respectively [14]. Down-regulation of miR-214 contributes to ICC metastasis by targeting Twist [15]. However, little is known about PSMD10-related miRNAs in ICC.
In this study, we aimed to investigate whether the expression of PSMD10 is regulated by miR-605, which is a target of p53 for transcriptional activation [16]. Furthermore, the biological function of dysregulated miR-605 and PSMD10 in ICC was investigated. The results of this study could elucidate the mechanism of miRNA in ICC. Medical University with documented informed consent in each case. All samples confirmed by pathology were collected and frozen in liquid nitrogen. RBE cells were purchased from the Chinese Academy of Sciences Type Culture Collection (Shanghai, China), and QBC939 was obtained from the XiangYa Central Experiment Laboratory (Hunan, China). Cells were maintained in RPMI 1640 medium (GIBCO, NY, USA) containing 10% fetal bovine serum (FBS; Sigma-Aldrich, St Louis, USA). Cells were grown in an incubator at 37°C and 5% carbon dioxide. All transfections were performed using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer's instructions.

RNA extraction and quantitative reverse transcription PCR (qRT-PCR)
Total RNA from cells or tissue samples was isolated using Trizol (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. The RNAs were mixed with oligo (dT) or miRNA-specific stem-loop RT primers, and reverse transcribed to cDNA using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA). These cDNAs were used to analyze the expression of miR-605, miR-875-3p, and PSMD10 by qPCR using an SYBR premix Ex Taq kit (TaKaRa, Dalian, China). Paired primers (PSMD10-semi-F and PSMD10-semi-R) were used to determine PSMD10 mRNA expression, and the level of GAPDH mRNA was used as an endogenous control. After RT by miRNA specific RT primers, miRNA specific forward primers (miR-605-Fwd and miR-875-3p-Fwd) and the universal reverse primer (Rev) were used to analyze the expression of miR-605 and miR-875-3p. U6 snRNA was used for normalization. All reactions were performed in an ABI 7500 system (Applied Biosystems, Foster, CA, USA) in triplicate, and the levels of gene expression were calculated using the 2 ÀDDCt method. All primers, purchased from Genewiz Biotechnologies (Suzhou, China), are shown in Table 1.

Western blot analysis
Total proteins from clinical tissues and ICC cells were prepared using RIPA buffer (Beyotime, Jiangsu, China). The proteins were quantified and separated in 10% SDS-PAGE gel, and transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were probed with the following antibodies: mouse anti-PSMD10 monoclonal antibody (Abcam, Cambridge, UK), mouse anti-p53 monoclonal antibody (Abcam, Cambridge, UK), and mouse anti-GAPDH monoclonal antibody (Abcam, Cambridge, UK). GAPDH was used as a loading control. After incubation with HRP-conjugated goat anti-mouse secondary antibody (Santa Cruz, CA, USA), the proteins were visualized by an enhanced chemiluminescence kit (Amersham Corp, Buckinghamshire, United Kingdom) and exposed to chemiluminescent film.

Cell proliferation assay
RBE and QBC939 cells (5 Â 10 3 cells/well) were plated in 96-well plates (Costar, CA, USA). After 24 h, cells were transfected and incubated for another 24, 48, and 72 h. Cell viability was measured by MTT assays. For colony formation analysis, transfected Table 1 Primers used in the experiments.

Primer name
Primer sequence cells were plated in six-well plates at a concentration of 2 Â 10 3 cells/well. After 10 d, cells were washed and stained with crystal violet. Each group was repeated in triplicate.

Cell apoptosis
Cells were plated in six-well plates at a final concentration of 6 Â 10 5 cells/well. After 24 h, cell were transfected and grown continuously for 48 h. After transfection, cells were washed in cold PBS and trypsinized for apoptosis analysis using an Annexin V-PE/ 7-amino actinomycin D detection kit (BD Pharmingen, San Diego, CA, USA) following the manufacturer's protocol.

Cell migration assays
Cell migration analysis was performed using transwell cell culture inserts (Invitrogen, USA) coated with Matrigel. A total of 5 Â 10 4 cells (in 0.2 ml of RPMI 1640 with 5% FBS) were seeded into the upper chamber with 600 ll of RPMI1640 containing 5% FBS.
The bottom chamber was filled with DMEM containing 20% FBS. After incubation for 16 h, chambers were washed with PBS and stained with crystal violet solution. The cells that migrated across the membrane were counted in five random visual fields. All assays were repeated in triplicate.

In vivo tumorigenesis assay
BALB/c nude mice (five-week old) were purchased from the Animal Center of the Chinese Academy of Science (Shanghai, China), and randomly assigned to one of four groups (10 mice per group). RBE or QBC939 cells (5 Â 10 6 cells) transiently transfected with pcDNA3.1/pri-miR-605 or pcDNA3.1 were suspended in 100 ll of RPMI 1640, and injected subcutaneously in the right posterior of mice. After 10 d, tumor growth was monitored and recorded every 3 d. After 31 d, mice were sacrificed for evaluation.

Target prediction and luciferase activity assay
The DNA fragments of wild-type and mutant PSMD10 3 0 UTR containing miR-605 binding region were cloned into the PmeI and XbaI sites of pmirGLO vector (Promega, Madison, WI, USA). For reporter assays, cells were transfected with 3 0 UTR or mutant 3 0 UTR luciferase reporters only, or co-transfected with miR-605 expression vector and luciferase reporter. At 48 h after transfection, cells were harvested and analyzed using luciferase activity in a Dual-Glo Luciferase Assay System (Promega, WI, USA). Normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) for each construct was compared with that of the group of pmirGLO vector with the wild-type PSMD10 3 0 UTR. Each assay was performed in triplicate.

Statistical analysis
Each experiment was performed in triplicate. All data are presented as mean ± S.D. The difference between means was analyzed with Student's t-test, Non-parametric Mann-Whitney U test or ANOVA. All tests performed were two-sided. P < 0.05 was considered to be statistically significant.

MiR-605 negatively regulates PSMD10 and is inversely correlated with its expression in ICC
To investigate the candidate miRNAs that regulate the expression of PSMD10, computer algorithms were used. Four miRNAs, namely, miR-559, miR-605, miR-875-3p, and miR-1254, were found to contain the complementary nucleotides to the PSMD10 3 0 UTR (Fig. 1A). We employed a gene over-expression approach to examine the effect of these four miRNAs on PSMD10 protein expression. RBE cells were transfected with miRNA over-expression vector, and PSMD10 protein expression was measured by Western blot analysis. As shown in Fig. 1B, over-expression of miR-605 and miR-875-3p significantly suppressed PSMD10 expression. Further studies on the expression of these two miRNAs and PSMD10 by qRT-PCR indicated that only miR-605 was lowly expressed in PSMD10-up-regulated ICC specimens (Fig. 1C). To validate whether miR-605 directly binds to PSMD10 and leads to its dysregulation, luciferase reporter assay was used. We constructed the pMIR reporter luciferase vectors containing the wild-type or mutant putative target site of the PSMD10 3 0 UTR, and transfected them into RBE with miR-605 expression plasmid or pcDNA3.1 control vector. The results show that miR-605 could significantly down-regulate the luciferase activities of the PSMD10 3 0 UTR reporter but not the mutant PSMD10 3 0 UTR reporter relative to the negative control group (Fig. 1D). Furthermore, the correlation of miR-605 and mRNA level of PSMD10 also indicate that miR-605 was inversely correlated with PSMD10 mRNA (Fig. 1E, r = À0.6738) in ICC tissues. These observations demonstrate that miR-605 could directly bind to the 3 0 UTR of PSMD10 and repressed its expression.

MiR-605 inhibits ICC cell proliferation and invasion in vitro
To investigate the function of miR-605 dysregulation in ICC, we investigated the influence of miR-605 on the cell proliferation, apoptosis, and migration of ICC cells by increasing the expression level of miR-605 in RBE and QBC939 cell lines. qRT-PCR was performed to analyze the expression of miR-605 in RBE and QBC939 cell lines (Fig. 2A). MTT (Fig. 2B) and colony formation (Fig. 2C) results indicate that over-expression of miR-605 suppressed ICC cell proliferation. We also detected the effect of miR-605 on ICC cell migration by transwell assay. The results show that the number of migrating cells decreased after transfection with miR-605 expression vector (Fig. 2D). Furthermore, results of FACS analysis indicate that the potency of apoptosis cells was higher in miR-605 over-expressing ICC cells than that in the control group (Fig. 2E). These data indicate that miR-605 inhibited the cell proliferation, induced cell apoptosis, and suppressed the migration of ICC cells in vitro.

Knock-down of PSMD10 represses cell proliferation and induces apoptosis of ICC cells
We discovered that miR-605 directly repressed PSMD10 expression and inhibited ICC cell progression. If this effect of miR-605 is mediated by PSMD10, down-regulation of PSMD10 in ICC cells will have similar results to miR-605. In this study, we transfected PSMD10-specific shRNA plasmid to repress its expression in ICC cells (Fig. 3A). The results of MTT (Fig. 3B), colony formation assay (Fig. 3C), and transwell analysis (Fig. 3D) indicate that down-regulation of PSMD10 inhibited ICC cell proliferation. In addition, FACS analysis results show that the quantity of apoptotic cells was higher in down-regulated PSMD10 groups than that in the control groups (Fig. 3E).

Restoration of PSMD10 reverses the phenotypic alteration caused by miR-605 in ICC cells
To further confirm whether the effect of miR-605 on ICC cells is dependent on the repression of PSMD10 expression, we transfected PSMD10 expression vector into miR-605 over-expressing ICC cells. Western blot analysis was performed to detect the expression of PSMD10 (Fig. 4A). Moreover, the cell proliferation and apoptosis of cells were analyzed. The ectopic expression of PSMD10 could rescue the increase in cell apoptosis (Fig. 4B), and inhibition of migration (Fig. 4C) and colony formation ability (Fig. 4D) caused by over-expression of miR-605. These data suggest that the effects of miR-605 on ICC cells were achieved by directly inhibiting PSMD10 expression.

MiR-605 suppresses ICC tumor growth in vivo
To test whether miR-605 has the same effect in vivo, RBE and QBC939 cells transfected with miR-605 expression vectors or pcDNA3.1 vectors were injected subcutaneously into BALB/c nude mice, respectively. As shown in Fig. 5A and B, the tumor volume in mice with high miR-605 levels was smaller compared with those in pcDNA3.1-transfected cells. The expression level of miR-605 in tumor tissues of miR-605 expression vector transfection groups was higher than that in the control groups (Fig. 5C), which was consistent with the expression of PSMD10 (Fig. 5D). These data suggest the suppression of miR-605 on ICC cell growth in vivo, and indicate the tumor suppressor function of miR-605.

P53 indirectly inhibits PSMD10 expression in ICC cells
miR-605 has been confirmed to be regulated by P53. To test whether PSMD10 joins P53-miR-605 network. P53 expression plasmid was transfected independently or co-transfected with miR-605 ASO or control ASO into RBE cells. Cells transfected with pcDNA3.1 vector only was served as control. AS it shown in Fig. 6A and B, The ectopic expression of P53 could up-regulate P53 level but repress the expression of PSMD10 in RBE cells. While the transfection of miR-605 ASO could restorate PSMD10 expression in P53 transfected cells. These data indicated that PSMD10 is a member of P53_miR-605_PSMD10 feedback loop and regulate ICC cell progression (Fig. 6D).

Discussion
Dysregulation of the PDMD10 pathway and miRNA has been reported to be crucial for the development of tumor [4,5,17,18]. MiR-605 is located in chromosome 10q21.1. It has been reported to be positively regulated by p53, and forms a p53-miR-605-Mdm2 feedback loop in response to stress [16]. Early studies have been shown that miR-605 was lowly expressed in lung cancer and the mutant of miR-605 might be increase the risk of lung cancer among males [19]. miRNAs are a class of non-coding RNAs that regulate cancer cell progression through their downstream target genes. One miRNA can regulate various gene expression. Here we have predicted some candidate target genes of miR-605. For example, PSMD10, PDCD2, TGFBR1, RABL5, P21, CADM1, MDM2. These genes are well known and play important role in Tumor biological processes. Here we chose PSMD10, one crucial regulator in ICC, as our research target in this work. In this study, we found that miR-605 was lowly expressed in ICC tissues and inversely correlated with the expression of PSMD10. Luciferase reporter assays Western blot analysis showed that the expression of miR-605 could inhibit the expression of PSMD10 in ICC cells by directly binding the 3 0 UTR of PSMD10. In addition, Ectopic expression of miR-605 resulted in the induction of cell apoptosis and inhibition of cell proliferation and migration ability in vitro. Furthermore, the results of xenograft indicate that miR-605 significantly suppressed ICC tumor growth in vivo, similar to the decreased level of PSMD10 protein. Thus, miR-605 may serve as a tumor suppressor in ICC cells by inhibiting the proto-oncogene PAMD10.
PSMD10 is a component of the 26S proteasome, which is required for ubiquitin-dependent protein degradation. The aberrant expression of PSMD10 has been found in colorectal cancer, breast cancer, and liver cancer [4,[20][21][22]. Aberrant PSMD10 expression has a function in tumor metastasis, proliferation, and drug resistance [4,6,[22][23][24]. The results of the present study suggest that PSMD10 was directly targeted by miR-605. To verify that the effect of miR-605 in ICC cells was mediated by PSMD10, RNAi technique was used to repress PSMD10 expression. Knock-down of PSMD10 induced ICC cell apoptosis and inhibited cell proliferation and migration. In addition, restoration of PSMD10 protein expression counteracted the effects caused by over-expression of miR-605. Thus, the anti-tumor function of miR-605 in ICC was mediated by PSMD10.
P53 is a novel tumor suppressor gene and plays important role in cancer genesis, proliferation and differentiation. The mutation of P53 were reported in various type of cancer and may play a role in the tumor growth pattern [25][26][27][28]. In contrast, some studies also indicated the over-expression of P53 in ICC patients [29,30]. miR-605 is positively regulated by P53, then miR-605 represses Mdm2 expression, which results in the increase in p53 transcriptional activity. In addition, PSMD10 can bind to Mdm2 and promote p53 degradation [7]. In the present study, we measured the status of miR-605 and PSMD10 in ICC cells after transfection of the p53 expression plasmid. The results show that increased p53 levels up-regulated the miR-605 levels and inhibited PSMD10 expression. Therefore, PSMD10 joined the p53 network to form a p53:miR-605:PSMD10/Mdm2 positive feedback loop, and regulated tumor progression.
In this study, we found that miR-605 was down-regulated and directly inhibited the expression of PSMD10 in ICC. We also showed that miR-605 inhibited cell proliferation, induced cell apoptosis, and suppressed cell migration, suggesting a tumor suppressor function in ICC mediated by PSMD10. These findings will be helpful to improve understanding of p53-dependent tumor regulation, and may represent a new therapeutic target for the treatment of ICC.