Background
MicroRNAs (miRNAs) belong to a class of conserved and small noncoding RNAs that repress protein expression through base pairing with the 3′-untranslated region (3’-UTR) of target mRNA [
1,
2]. It is well documented that miRNA aberrations may be an important factor in cancer development. The potential connection between miRNA regulation and cancer has been made at several levels, suggesting that miRNAs play critical roles in cellular growth and differentiation, which are two cellular processes that are commonly defective in tumor cells [
3‐
6].
Prostate cancer (PCa) is one of the most frequently diagnosed cancers among men and is the third leading cause of male cancer-related death in the United States [
7]. Despite the initial success of surgery and radiation therapy for localized prostate cancer, > 30% patients experience biochemical recurrence and emergence of advance-stage disease particularly, metastatic progression [
8‐
10]. Therefore, a more thorough understanding of the mechanisms underlying PCa pathogenesis will help to develop more effective therapeutic strategies, for which there is an urgent need. Alteration of miRNA expression is observed in PCa that have been collected from different study cohorts [
11‐
13]. Furthermore, several miRNAs (eg, miR-1, miR-135a, miR-21, miR-96, miR34a, miR-203 and miR-205) have been shown to regulate PCa cell growth, apoptosis, migration and/or invasion [
9,
14‐
17], suggesting a dysfunction of miRNA may be associated with prostate carcinogenesis. Clearly, more comprehensive research is required to elucidate the role of miRNAs during PCa progression and to identify those miRNAs that could serve as novel prognostic predictors and therapeutic targets for PCa.
Previous profile studies of miRNA expression have noted the downregulation of a series of miRNAs,including miR-1-3p in PCa tissues. It has been shown that ectopic expression of miR-1 inhibits prostate cancer cell growth, epithelial-mesenchymal transition and bone metastasis [
18,
19]. In addition, miR-1-3p has been reported to suppress tumor growth in colon carcinomas [
20], decrease cellular proliferation and migration of oral squamous cell carcinoma [
21], inhibit cell proliferation and invasion and induce apoptosis in bladder cancers [
22]. These data indicate a potential tumor suppressive function of miR-1-3p. However, the role of miR-1-3p in prostate carcinogenesis and the molecular mechanisms by which it functions and modulates the malignant phenotypes of PCa cells remain to be delineated.
In this study, we report that deregulation of miR-1-3p in PCa is important in the development of an aggressive phenotype and is correlated with a poor prognosis. Ectopic overexpression of miR-1-3p in PCa cells is sufficient to inhibit cell invasion, both in vitro and in vivo. More importantly, for the first time, we provide evidence that miR-1-3p directly targets two central cell cycle genes, the E2F transcription factor 5 (E2F5) and PFTAIRE Protein Kinase 1 (PFTK1) mRNA, to suppress cell proliferation. Collectively, the results of this study provide an explanation for the aggressiveness of PCa and link it mechanistically to interactions between miR-1-3p, E2F5 and PFTK1. Our results also suggest that miR-1-3p could be employed as a new prognostic marker and/or as an effective therapeutic target for PCa.
Methods
Patients and tissue samples
PCa samples and adjacent normal tissue samples were collected during radical prostatectomy from PCa patients between 2008 and 2014 at the Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology in Wuhan, China. The PCa cases selected were based on a clear pathological diagnosis, follow-up data, and absence of androgen deprivation therapy, chemotherapy, radiotherapy or other anticancer treatment before surgery. All specimens had confirmed pathological diagnosis and were classified according to the WHO criteria. The clinicopathological patient information was collected and summarized in Table
1. All protocols were approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and informed consent was obtained from all patients before surgery. All in vivo protocols were approved by the Institutional Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.
Table 1
Relationship between miR-1-3p and clinicopathologic variables in patients with prostate cancer
Age |
≥ 65y | 70 | 30 (42.9%) | 40 (57.1%) | 0.860 |
< 65y | 54 | 24 (44.4%) | 30 (55.6%) |
Serum PSA |
≥ 10 ng/ml | 74 | 35 (47.3%) | 39 (52.7%) | 0.306 |
< 10 ng/ml | 50 | 19 (38.0%) | 31 (62.0%) |
Gleason score |
≥ 7 | 76 | 42 (55.3%) | 34 (44.7%) | 0.001 |
< 7 | 48 | 12 (25.0%) | 36 (76.0%) |
pT stage |
< T3 | 35 | 23 (65.7%) | 12 (34.3%) | 0.002 |
≥ T3 | 89 | 31 (34.8%) | 58 (65.2%) |
Lymph node metastasis |
Presence | 31 | 20 (64.5%) | 11 (35.5%) | 0.012 |
Absence | 93 | 34 (36.6%) | 59 (63.4%) |
Seminal vesicle invasion |
Presence | 33 | 19 (57.6%) | 14 (42.4%) | 0.164 |
Absence | 81 | 35 (43.2%) | 46 (56.8%) |
Biochemical recurrence |
Presence | 14 | 9 (64.3%) | 5 (35.7%) | 0.097 |
Absence | 110 | 45 (40.9%) | 65 (59.1%) |
Cell culture
22RV1 and LNcaP cells (ATCC) were maintained in RPMI-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) and the normal prostate epithelial cells RWPE-1 (ATCC) were maintained in Keratinocyte-SFM (Gibco, GrandIsland, NY, USA). All cells were cultured in a humidified atmosphere of 5% CO2 maintained at 37 °C.
Oligonucleotide, lentivirus production and cell transfection
All small RNA molecules were ordered from RiboBio Co., Ltd.(Guangzhou, China), including miR-1-3p mimics, mimics negative controls (mimics-NC), miR-1-3p inhibitor, inhibitor negative controls (inhibitor-NC),siE2F5 (sense:5′-CAGAUGACUACAACUUUAATT-3′; antisense:5′-UUAAAGUUGUAGUCAUCUGTT-3′) and siPFTK1 (sense: 5’-GTTCATTCTTTACCACATT-3′;antisense: 5’-AGGTTGCATCTTTGTTGAA-3′). MiR-1-3p mimics are double-stranded RNA molecules containing the miR-1-3p sequence, while miR-1-3p inhibitors are single stranded RNA molecules containing the miR-1-3p reverse complement sequence, which can competitively bind to endogenous miR-1-3p. For lentiviral-mediated overexpression, viral particles were harvested 48 h after transfection of 293FT cells with pCDH-CMV-miR-1-3p, –E2F5 or –PFTK1 and the packaging plasmids pRSV/pREV, pCMV/pVSVG and pMDLG/pRRE using Lipofectamine 2000 (Invitrogen). MiR-NC (TTCTCCGAACGTGTCACGT) was cloned into the same backbone and the resulting construct Lenti-miR-NC served as a negative control. The transfection or infection efficiencies were detected by RT-qPCR. Recombinant lentivirus-transducing units were used to infect LNcaP cells in the presence of 8 mg/ml Polybrene (Sigma, St Louis, MO, USA). Cells were plated in growth medium at a density of 45% to 70%. The transfection was carried out using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA) 24 h later according to the manufacturer’s protocol. The final concentration of RNAs was 75 nM for each well. All cell lines were tested and found to be free of mycoplasma contamination.
RNA isolation and quantitative real-time PCR
Total RNA of cells was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Reverse transcription of microRNA and mRNA were done using RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania) and miProfile™ miRNA qPCR Primer (GeneCopoeia, Guangzhou,China). RT-qPCR analysis of miRNA was performed with the Platinum SYBR Green qPCR Supermix UDG kit (Invitrogen, Carlsbad, CA) using synthesized primers from GeneCopoeia (Guangzhou, China). The U6 primers were obtained from GeneCopoeia. All experiments were done in triplicate. The expression level values were normalized to those of the small nuclear RNA U6 as a control. Several primer sequences used are available as follows:
The colony formation assay was conducted as previously described. Briefly, exponentially growing cells were plated at approximately 2000 cells per well in 6-well plates after transfection. Culture medium was changed every 3 days. Colony formation was analyzed 12 days following infection by staining cells with 0.05% crystal violet solution for 30 min. The number of colonies was counted using an inverted microscope (Olympus, Japan). Cell proliferation was assessed by using the CellTiter 96 Aqueous One Solution Cell Proliferation. Assay kit (Promega, Madison, WI, USA) as previously described. Briefly, RNA transfected cells were grown in 96-well plates at a density of 2000 cells/well. Cell growth was measured daily for 4 days. At each time point, 20 μl of CellTiter 96 Aqueous One Solution was added and incubated. Absorbance was detected by a microplate reader (Bio-Rad, Berkeley, CA, USA) at 490 nm.
Cell cycle
At 72 h after transfection, cells were fixed in 70% cold ethanol, incubated with RNase A (Sigma,St. Louis, MO, USA) and stained by propidium iodide (PI) (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) staining solution. After staining, the cells were analyzed on a FACSort flow cytometer (BD Biosciences, San Diego, CA, USA). The data were processed by CELL quest software (BD Biosciences).
Western blot analysis
Cells were harvested at 72 h following transfection. Proteins were separated by 10% SDS/PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking, the membranes were incubated overnight at 4 °C with appropriate dilutions of specific primary antibodies as follows: E2F5 (1:1000) (Abcam, ab44996), PFTK1 (1:1000) (Abcam, ab104150), CDK4 (1:1000) (Cell Signaling Technology, 12,790), CDK2 (1:1000) (Cell Signaling Technology, 2546), GAPDH (1:500) (Boster, Wuhan, China). Next, membranes were incubated with corresponding second antibody and detected by enhanced chemiluminescence (ECL) assay kit (Millipore).
Luciferase reporter assay
E2F5 and PFTK1 3’UTR reporter and control constructs were purchased from GENECHEM. Tumor cells overexpressing miR-1-3p and miR-NC cultured in 48-well plates were co-transfected with 1.5 mg of firefly luciferase reporter and 0.35 ng Renilla luciferase reporter with Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA). 24 h post transfection, firefly luciferase activities were measured using the Dual Luciferase Assay (Promega) and the results were normalized with Renilla luciferase according to the manufacturer’s protocol.
Xenograft model of PCa in nude mice
Two groups of five male BALB/c nude mice at 6 weeks of age each were injected subcutaneously with prepared cells (1 × 106 LNcaP cells stably expressing miR-1-3p or miR-NC) at the same site. Tumor onset was measured with calipers at the site of injection every 3–4 days by two trained laboratory staff members at different times on the same day, starting 12 days after injection when appreciable tumor formed subcutaneously. Tumor volume was calculated using the formula, V = 0.5ab2, where a represents the larger and b represents the smaller of the two perpendicular indexes. Animals were euthanized and xenografts were harvested 40 days after injection and tumors were weighed. Formalin-fixed, paraffin-embedded PCa xenografts were assessed by hematoxylin and eosin (HE) and Ki-67 staining and evaluated for target gene expression. Nude mice were manipulated and cared for according to NIH Animal Care and Use Committee guidelines in the Experiment Animal Center of the Tongji Medical College of Huazhong University of Science and Technology, China.
Immunohistochemistry (IHC)
Formalin-fixed, paraffin-embedded tissue sections (5 μm) were deparaffinized in xylene and rehydrated with gradient concentrations of ethanol. The tissue sections were stained with specific antibodies against E2F5 (1:400) (Abcam, ab203124) and PFTK1 (1:400) (Abcam, ab224098). Sections incubated with secondary antibodies in the absence of primary antibodies were used as negative control. Hematoxylin was used for counterstaining. Slides were viewed and photographed under a light microscope.
Statistical analysis
Statistical analysis was performed using SPSS software (SPSS Standard version 19.0, SPSS Inc. Chicago, IL). Differences between variables were assessed by the χ2 test or Fisher’s exact test. Continuous data were compared using the Student’s two-tailed t test. For survival analysis, Kaplan-Meier method, and log-rank tests were used to identify the potential differences between progression-free-survival in the two patient cohorts. Hazard ratios (HRs) were estimated with a multivariate Cox proportional hazards model. X-tile software version 3.6.1 (Yale University School of Medicine, New Haven, CT, USA) with a built-in validated feature was used to define the cutoff point [
23]. Data are represented as the mean ± SEM. In all cases,
p-values < 0.05 were considered to be statistically significant.
Discussion
Accumulating studies have described that miRNA can function as tumor suppressors or oncogenes by regulating target gene expression levels in various human cancers [
5,
26]. Multiple miRNAs have been observed to be involved in several crucial processes in PCa, such as cell proliferation, apoptosis, invasion and metastasis [
9,
12,
14,
15,
17]. The tumor suppression activity of miR-1-3p during tumorigenesis has been well-characterized in several cancers, including colorectal cancer [
20], bladder cancer [
22] and prostate cancer [
18,
19]. However, the underlying molecular mechanisms by which miR-1-3p modulates PCa carcinogenesis and the clinical significance of miR-1-3p in PCa patients remain poorly understood. In this study, we observed that downregulation of miR-1-3p is a frequent occurrence in PCa tissues and cell lines. Low-level expression of miR-1-3p was significantly associated with a more aggressive tumor phenotype and a short progression-free survival time for patients with PCa. In functional studies, proliferation and colony formation of PCa cells in vitro, and tumor growth in vivo
, were dramatically suppressed upon reintroduction of miR-1-3p. These findings suggest that miR-1-3p plays a crucial role in the proliferation and/or cell cycle progression of PCa.
It is known that proliferation is one of the most important hallmarks of malignant tumors, and is the foremost fatal factor directly correlated with mortality in human cancers. Therefore, the identification of proliferative and/or cell cycle progressive factors as well as exploration of the underlying molecular mechanisms involved in miR-1-3p regulation of PCa progression in tumor growth are critical. We predicted its target genes using publicly available online algorithms, and identified that E2F5 and PFTK1, which have been demonstrated to have an important role in the cell proliferation, are potential functional targets of miR-1-3p. In our study, it was suggested that miR-1-3p binds to a complementary site, which is conserved among most vertebrates on the 3′-UTR of E2F5 and PFTK1, resulting in down-regulation of its target genes E2F5 and PFTK1 expression in PCa cells, as determined by luciferase assays and Western blot analyses. In addition, we also demonstrated that both E2F5 and PFTK1 were functionally involved in miR-1-3p-mediated suppression of proliferation and cell cycle progression in PCa cells. In addition, an inverse correlation between the levels of miR-1-3p and mRNA expression of E2F5 and PFTK1 was evaluated in our PCa cell lines and tissues. These observations provide the first line of evidence, to the authors’ knowledge, that miR-1-3p mechanistically acts through the regulation of both E2F5 and PFTK1 in PCa. It has been observed that E2F transcription factor 5 (E2F5) and/or PFTAIRE Protein Kinase 1 (PFTK1, also known as CDK14) are upregulated in various types of human cancers, including prostate cancer [
27‐
29]. Furthermore, patients with high E2F5 and/or PFTK1 expression are associated with a more aggressive tumor phenotype [
30,
31]. These results are consistent with our findings that miR-1-3p downregulation is associated with a more aggressive and/or poor prognostic PCa phenotype. These results are consistent with our findings that miR-1-3p downregulation is associated with a more aggressive and/or poor prognostic PCa phenotype.
Moreover, as mentioned above, E2F5 belongs to E2F family and is well-known for its role in cell proliferation and cell cycle progression by binding pocket proteins in the G1 phase [
24]. Furthermore, previous studies have shown that E2F5 is negatively regulated by multiple miRNAs, such as miR-34a [
32], miR-613 [
33] andmiR-128-2 [
34]. PFTK1 is a novel member of the Cdc2 family and can regulate the expression of cyclins and the cell cycle [
25]. In addition, related studies have demonstrated that PFTK1 protein either activated or was involved in Wnt signaling and promoted migration and invasion [
29]. It does appear, therefore, that in our PCa cells, miR-1-3p modulates cell proliferation via regulation of E2F5 and PFTK1. In our study, we observed further that silencing E2F5 and PFTK1 largely mimicked the proliferation and cell cycle progression-inhibiting effect of miR-1-3p overexpression. Concomitant knockdown of miR-1-3p and E2F5 and PFTK1 substantially reversed the inhibitory effects of silencing either E2F5 or PFTK1 alone. These results support our theory that E2F5 and PFTK1 are predominant mediators of miR-1-3p suppression of PCa cell proliferation and cell cycle progression, suggesting that loss of function of miR-1-3p may result in an enhanced expression of E2F5 and PFTK1 and, in turn, the susceptibility of cells to proliferation. Similar to our study, Zhang et al. reported that tci-miR-1-3p is involved in cyflumetofen resistance by targeting TCGSTM4 in Tetranychus cinnabarinus [
35]. Frederico et al. also found that miR-1-3p participate in several alterations of TRIM63/FBXO32 gene/protein expression related to the pathophysiology of DM, including soleus muscle atrophy [
36]. Moreover, Shang and colleagues demonstrated that miR-1-3p suppresses the invasion and migration of bladder cancer by up-regulating SFRP1 expression [
22]. Clearly, our results, together with the findings of other groups, indicate that miR-1-3p may target multiple proteins that function spatiotemporally or in cooperation with different cellular processes.
Although we have shed new light on the molecular mechanism responsible for miR-1-3p in PCa progression, the detailed mechanism by which miR-1-3p is downregulated, such as through DNA promoter methylation [
37], interaction with long noncoding RNA [
38] or metabolic disorders (such as Hyperglycaemia, Hyperlipemia) induction [
39], still need to be elucidated in future studies. More importantly, E2F activity is regulated by the retinoblastoma (Rb) “pocket” protein family members Rb, p107, and p130, which bind and inhibit E2F5 and recruit repressive factors to E2F-driven promoters [
40]. However, mutation or genetic ablation of the Rb gene occurs commonly in prostate cancers and leads to dysfunction of RB-E2F pathway and increased proliferation [
41]. On the other hand, the protein p107 contains another growth suppression domains that interactions with CDK/ cyclin complexes [
42]. And interesting that CDK phosphorylation of p107 weakens the p107 C-terminal –E2F5–association [
40]. Therefore, future studies are warranted to further elucidate the relationship among miR-1-3p, PFTK1(CDK14), E2F5 and Rb pocket protein, and discover crucial miR-1-3p -target negative regulation pairs with network topological importance and evaluate their clinical significance in human PCa [
43].