Background
Long non-coding RNAs (lncRNAs) are untranslated transcripts longer than 200 nucleotides baring many of the structural characteristics of mRNAs, including a polyA tail, 5′-capping, and a promoter structure, but no conserved open reading frame [
1]-[
6]. Many lncRNAs are expressed at specific times and in specific tissues during development, and exhibit a variety of slicing patterns. It has been proposed that lncRNAs are involved in the epigenetic regulation of coding genes, and thus exert a powerful effect on a number of physiological and pathological processes, including the pathogenesis of many human cancers [
7]-[
11].
MicroRNAs (miRs) are small noncoding RNAs usually 20–22 nucleotides long. To date, close to 1000 human miRs have been identified. Collectively, miRs are thought to regulate more than 50% of all human genes by binding to mRNA sequences and repressing expression, either by inhibiting translation or promoting RNA degradation [
12]-[
17].
Given the structural similarly with mRNAs, lncRNAs may be another important member of the non-coding RNA family [
18]. The interaction between lncRNAs and miRs has been linked to the invasion and metastasis of tumors [
19]. For example, the
miR-29a epigenetically modulated expression of the lncRNA
MEG3 in hepatocellular carcinoma (HCC) through promoter hypermethylation [
20]. Loss of miR-31 expression in triple-negative breast cancer (TNBC) lines is attributed to hypermethylation of its promoter-associated CpG islan.
MicroRNA-31 anchors the novel lncRNA
LOC554202 and adjusts its transcriptional activity [
21]. Moreover, the lncRNA
HULC can inhibit the expression of the tumorigenic
miR-372[
22].
Prostate cancer gene expression marker 1 (
PCGEM1) is part of a novel class of androgen-regulated lncRNAs [
23]. Overexpression in prostate cancer (PCa)-derived LNCaP cells promotes proliferation and a dramatic increase in colony formation [
24],[
25]. Many miRs function as oncogenes or tumor suppressors in human cancers [
26]-[
32]. Downregulation of miR-145 has been reported in PCa, suggesting that
miR-145 functions as a tumor suppressor [
33]. Using the biology information software RegRNA (
http://regrna.mbc.nctu.edu.tw/), we predicted that 48 distinct miRs bind to
PCGEM1. Further online comprehensive analysis (
http://cbio.mskcc.org/cancergenomics/prostate/data/) indicates that 96 miRs are associated with PCa. Clustering intersection analysis also linked
miR-145 with PCa. Significantly,
miR-145 has a binding site for lncRNA; thus, reciprocal regulation of
PCGEM1 and
miR-145 may promote or suppress PCa cell proliferation [
34]. In this study, we explored possible mutual regulation of
PCGEM1 and
miR-145 expression in prostate cancer and the impact on PCa cell proliferation and invasive capacity.
Materials and methods
Materials
Non-cancerous RWPE-1 cells, HEK293T cells and LNCaP cells were purchased from the Shanghai Institute of Cell Biology (Shanghai, China). RPMI 1640 medium, fetal bovine serum (FBS), and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, CA, USA). The restriction enzymes NotI and XhoI, T4 DNA ligase, and reagents for RT-PCR were purchased from TaKaRa (Takara BioInc, Shiga, Japan). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), annexin-V-FITC, and propidium iodide (PI) were purchased from Sigma Chemical (USA), and negative control sequences and negative control inhibitor sequences were purchased from Ruibo Company (Shanghai, China).
Design and construction of eukaryotic expression vector for hsa-miR-145
The mature hsa-miR-145 sequence (5′-GUCCAGUUUCCCAGGAAUCCCU-3′) is available from the miRNA Registry (MIMATOOOO437). To prevent formation of a termination signal, TTGGCCACTGACT was selected as the region in a miR expression vector template. The sequence TGCT was added to the 5′ positive-sense strand template of the miR expression vector and GTCC to the 5′ antisense strand template. Further, a nonspecific sequence was designed and sent to Shanghai GenePharma Co, Ltd. for synthesis. The assay was according to previously described [
35]. The eukaryotic expression vector plasmid targeting hsa-miR-145 was named
pmiR-145.
Design and synthesis of siRNA
siRNAs are methylated 21 bp double-stranded RNA oligonucleotides. It uses gene-specific targets for RNAi analysis and reports up to 10 top scoring siRNA targets. The freeze-dried siRNAs were dissolved in RNase-free water and stored as aliquots at –20°C. The siRNA sequence of PCGEM1 (sense: 5′-GCCCUACCUAUGAUUUCAUAU-3′, antisense: 5′-AUAUGAAAUCAUAGGUAGGGC-3′) and negative control sequence (sense: 5′-UUCUCCGAACGUGUCACGUUUC-3′ antisense: 5′-GAAACGUGACACGUUCGGAGAA-3′) were synthesized by Shanghai GenePharma (Shanghai, China).
Grouping and cell transfection
The experimental culture groups included 1) untransfected LNCaP and RWPE-1 cells (control groups), 2) cells transfected with pmiR-145 or miR-145 mimics (1.6 μg/ml and 50 nM, respectively), 3) cells transfected with the scrambled nucleotide sequence and empty vector (negative control or NC groups, 50 nM), 4) cells transfected with a miRNA inhibitor (NI group, 100 nM), 5) a negative control for NI (NCI group, 50 nM), 6) cells transfected with siRNA PCGEM1 sequence (siRNA PCGEM1 group, 50 nM). Cells in log phase growth were seeded on 6-well culture plates (2 × 105 cells/well) and transfected when the cell fusion rate reached 70%. The DNA Lipofectamine 2000 or RNA Lipofectamine 2000 compound was added according to the manufacturer’s instructions (Invitrogen). After 6 h, the transfection medium was discarded. Cells were washed with serum-free RPMI 1640 and then cultured in RPMI 1640 supplemented with 10% FBS.
Luciferase reporter assay
The whole mRNA sequences of the PCGEM1 gene were obtained by PCR amplification and cloned separately into multiple cloning sites of the psi-CHECKTM-2 luciferase miRNA expression reporter vector. HEK293T cells were transfected with miR-145 mimic, miR-145 inhibitor, a control miRNA, a miRNA inhibitor control, or empty plasmid using Lipofectamine 2000 according to the manufacturer’s instructions. Nucleotide-substitution mutation analysis was carried out using direct oligomer synthesis of PCGEM1 sequences. All constructs were verified by sequencing. Luciferase activity was measured using the dual luciferase reporter assay system kit (Promega Co, Madison, WI, USA) according to the manufacturer’s instructions on a Tecan M200 luminescence reader.
Quantitative real-time RT-PCR
Total RNA samples were extracted using Trizol (Invitrogen, CA) according to the manufacturer’s instructions. Real-time quantitative PCR analysis was performed using an Applied Biosystems 7500 Real-Time PCR Systems (Applied Biosystems, Foster City, CA). The expression level of 18S was used as an internal control for mRNAs, and U6 level as an internal control for miRNAs. Primers used in quantitative real-time PCR analysis were: U6 (forward: 5′-CTCGCTTCGGCAGCACA -3′, reverse: 5′- AACGCTTCACGAATTTGCGT-3′); 18S (forward: 5′-CCTGGATACCGCAGCTAGGA-3′, reverse: 5′-GCGGCGCAATACGAATGCCCC-3′); miR-145 (RT primer: 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTTCCCAT-3′, forward: 5′-ACACTCCAGCTGGGGTCCAGTTTTCCCAGGAA-3′, reverse: 5′-CTCAACTGGTGTCGTGGA-3′); PCGEM1 (forward: 5′-CACGTGGAGGACTAAGGGTA-3′, reverse: 5′-TTGCAACAAGGGCATTTCAG-3′); The expression level was calculated using CT and 2-ΔΔCt..
MTT assay
The viability of LNCaP and RWPE-1 cells was determined by MTT assay. Briefly, cells at 5 × 104/ml were transfected with siRNA PCGEM1 (siRNA PCGEM1 groups, 50 nM), empty plasmid and scramble sequence (negative control group, 1.6 μg/ml), or pmiR-145 (pmiR-145 group, 1.6 μg/ml) in the presence of Lipofectamine 2000 and serum-free RPMI 1640 media for 6 h. Cells were plated in 96-well plates in medium containing 10% FBS for another 24, 48, or 72 h. MTT stock solution (20 μl, 5 mg/ml) was added to each well for a final MTT concentration of 0.45 mg/ml and the plate was incubated for 4 h at 37°C. Media was then removed and dimethylsulfoxide (DMSO) (150 μl added to dissolve the blue formazan crystals (the product of MTT conversion by viable cells) at room temperature for 30 min. The relative change in viable cell number was estimated by absorbance at 570 nm on a Bio-Rad microtiter plate reader (Hercules, CA, USA).
Flow cytometry assay
LNCaP and RWPE-1 cells were seeded at 1.0 × 106/ml in 24-well plates (Costar) and transfected in 500 μl media/well siRNA PCGEM1 (siRNA PCGEM1 groups, 50 nM), empty plasmid and scramble sequence (negative control group, 1.6 μg/ml), or pmiR-145 (pmiR-145 group, 1.6 μg/ml) by Lipofectamine 2000 reagent (Invitrogen) in serum-free RPMI 1640 for 6 h. After transfection, 500 μl of the appropriate growth medium containing 20% FBS were added to each well. Cells were incubated for another 48 h then harvested, washed twice with PBS, fixed with 70% ethanol, and treated with RNase A (1 mg/ml). Finally, the cells were double-stained with FITC-conjugated annexin-V and propidium iodide (PI) solution (50 μg/ml). For each sample, data from approximately 10000 cells were recorded in the list mode on logarithmic scales. Apoptosis and necrosis were analyzed by quadrant statistics on double negative, annexin-V-positive/PI-negative, annexin-V-negative/PI-positive, and double-positive cells.
Migration and invasion assay
Cells were transfected with siRNA PCGEM1 (siRNA PCGEM1 groups, 50 nM), empty plasmid and scramble sequence (negative control group, 1.6 μg/ml), or pmiR-145 (pmiR-145 group, 1.6 μg/ml) by Lipofectamine 2000 reagent in serum-free RPMI 1640 for 6 h. One day after transfection, 1 × 105 cells were collected, resuspend in 100 μl basal medium, and transferred to the transwell chamber. A 600 μl volume of complete medium was added to the well and the chamber inserted. Plates were incubated at 37°C for 48 h. Remaining cells were swabbed from the top transwell membrane filter and the chamber submerged in 4% paraformaldehyde for 20 min. Cells in the well were stained with crystal violet for 10 min, washed in PBS buffer, then counted by light microscope to determine transwell migration.
The transwell assay was performed as above except with Matrigel in the wells of 24-well plates. The Matrigel was first incubated in pre-chilled basal medium (40 μl) at 37°C for 2 h. The excess medium was discarded, 100 μl basal medium added to the well with the Matrigel and 600 μl to the chamber, following by incubation at 37°C overnight. Transfected cells (1 × 105) were resuspend in 100 μl basal medium and transfer to the transwell chamber. Then, 600 μl complete medium was added to the well and the chamber inserted. The plates were incubated at 37°C for 24 or 48 h. Cells were stained with crystal violet for 10 min, washed with the PBS and counted using an inverted microscope.
In vivo treatment
BalB/c (nu/nu) mice from the Animal Center of Guangzhou Province (Guangdong, China) received subcutaneous injections of 2 × 106 LNCaP cells into each axilla area. When xenograft tumors became palpable (about 0.1 mm3), mice were randomly divided into the control group receiving PBS injection (100 μl), siRNA PCGEM1 (500 nM), negative control (plasmid, scramble sequence 16 μg), PmiR-145 (16 μg), with 6 mice each group. There was no difference in baseline tumor size between the groups. Tumor volume was calculated every 3 days according to the formula v = ab2π/6, where “a” is the maximum tumor diameter and “b” the minimum diameter. After treatment for 20 d, mice were euthanized and tumors were dissected and weighed.
Data analysis
All results are the averages of at least three independent experiments from separately treated and transfected cultures. Data are expressed as the mean × SD. Statistical comparisons were made by one-way analysis of variance (ANOVA). P < 0.05 was considered to indicate a statistically significant difference.
Discussion
Long non-coding RNAs (lncRNAs) are a new class of regulatory RNA [
36]. These mRNA-like molecules, which lack significant protein-coding capacity, were once thought to be a part of the genomic “dark matter”, but recent studies have implicated lncRNAs in a wide range of biological functions through poorly understood molecular mechanisms [
37]. Despite recent insights into how lncRNAs function in such diverse cellular processes as regulation of gene expression and assembly of cellular structures, by and large, the key questions regarding lncRNA mechanisms remain to be answered [
38]. The lncRNA Prostate cancer gene expression marker 1 (
PCGEM1) is overexpressed in PCa, suggesting roles in proliferation, metastasis, and invasion [
39]. In order to reveal the mechanisms regulating expression in PCa, we have predicted PCGEM1 interaction with miR-145 using billogical information (Figure
1C), and futher investigated a possible interaction with the tumor suppressor
miR-145. Co-transfection of LNCaP cells with
miR-145 mimics or miR-145 inhibitor with psiCHECK-2-
PCGEM1 significantly inhibited reporter gene activity but only miR-145 suppressed reported gene expression when transfected with empty psiCHECK-2 (Figure
1A, B). Thus,
miR-145 may regulate
PCGEM1 expression by directly binding to target sites within the
PCGEM1 sequence.
We then demonstrated a mutual inhibitory control relationship between
PCGEM1 and
miR-145 by selective siRNA-mediated
PCGEM1 knockdown and miR-145 overexpression. Expression of
PCGEM1 (locus 2q32) was detected in the androgen receptor-positive cell line LNCaP but not in noncancerous prostate lines or androgen-receptor negative Pca lines [
23].
PCGEM1 overexpression in LNCaP cells promotes cell proliferation and a dramatic increase in colony formation, suggesting a role in cell growth regulation [
24]. In contrast, miR-145 expression was low in all the prostate cell lines tested (PC3, LNCaP, and DU145) compared to the normal cell line RWPE-1, and in cancerous regions of human prostate tissue compared to adjacent normal prostate tissue [
39]. To test the possibility of mutual negative regulation of
PCGEM1 and
miR-145, we design a small interfering RNA targeting
PCGEM1 and a vector for
miR-145 overexpression (
pmiR-145) and transfected these into LNCaP cells and normal RWEP–1 cells. RT-PCR results showed that knockdown of
PCGEM1 in LNCaP cells increased
miR-145 expression (Figure
2A) and that miR-145 overexpression reduced
PCGEM1 expression (Figure
2B). Inhibition of
PCGEM1 reduced LNCaP proliferation (Figure
3A), transwell migration and invasive capacity into Matrigel (Figures
5A,
6A), and the growth of solid tumors, possibly by promoted early apoptosis (Figure
4A). However, altering
PCGEM1 expression had no significant effect on RWPE-1 cell growth, migration, or invasion (Figures
3,
4,
5,
6B). The proliferation, colony formation, and soft agar growth of liver cancer cells was reduced by inhibiting expression of the lncRNA TUC339 using an siRNA [
40], while silencing HULC expression in hepatoma effectively inhibited the growth of liver cancer cells [
41]. In contrast, siRNA gene silencing of
MEG3 expression promote cell proliferation, whereas overexpression inhibited proliferation and promoted apoptosis [
42]. Thus, individual lncRNAs can either promote or inhibit carcinogenesis. Selective knockdown and overexpression of lncRNAs may be feasible strategies to reduce tumor growth in a variety of tissue. Specifically,
miR-145 is a well documented tumor suppressor [
43]-[
46], and we successfully constructed an overexpressing vector that suppressed PCa cell growth with no observable effects on noncancerous prostate cells. Similarly, gain-of-function assays revealed that
miR-145 transfection inhibited cell proliferation, migration and invasion of PC3 and DU145 PCa cell lines [
47].
While
PCGEM1 is known to be overexpressed in PCa, it is unknown if overexpression directly causes hyperproliferation and (or) metastasis [
24]. Fu [
48] found that overexpression of
PCGEM1 attenuated doxorubicin-induced expression of
p53 and
p21Waf1/Cip1, and inhibited apoptosis of LNCaP cells. Petrovics [
24] revealed that elevated
PCGEM1 expression increased cell proliferation and Rb phosphorylation. However, to the best of our knowledge, no study has investigated
PCGEM1 regulation by
miR-145. An siRNA
PCGEM1 inhibited LNCaP cells growth and reduced migration and invasion, likely by raising the expression levels of
miR-145.
Indeed, we confirmed direct binding of miR-145 to the PCGEM1 and demonstrated reciprocal regulation of these two transcripts. Moreover, miR-145-mediated suppression of PCGEM1 suppressed tumor growth in vivo and PCa cell proliferation and invasive capacity in vitro. In turn, Reciprocal regulation of PCGEM1 and miR-145 promote proliferation of LNCaP prostate cancer cells.
In conclusion, our study demonstrates reciprocal negative control of PCGEM1, a tumor-promoting long noncoding RNA, and the tumor suppressor miR-145. This study highlights the interrelationship between two classes of non-coding RNAs. Both downregulation of PCGEM1 or overexpression of the miR-145 reduced the proliferation and invasive capacity of prostate cancer cells in vitro and in vivo.
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Competing interests
The authors declare that they have no competing interests.