Background
Colorectal cancer (CRC) is one of the leading causes and gives rise to large amounts of cancer-related deaths around the word every year [
1,
2]. Currently, surgery and chemotherapy are the most common methods for CRC treatment [
3]. Although some advances have been achieved on CRC treatment over recent decades, the overall survival rate of patients with advanced or metastatic CRC is still below 50% [
4,
5]. The main cause is the increasing resistance to many anti-cancer agents in most CRC patients [
6]. The therapeutic efficacy becomes disappointing. Accumulating studies have showed that some important genes regulate CRC development, such as APC and KRAS [
7,
8]. However, the molecular mechanism that controls CRC development and progression still remains largely unknown. Therefore, to develop novel and effective approaches for CRC therapy, it is very necessary to define the molecular mechanism of CRC tumorigenesis.
Recent evidence demonstrates that nearly 98% of the genome transcripts in human are noncoding RNAs (ncRNA) [
9,
10], among which long noncoding RNAs (lncRNAs) are transcripts of longer than 200 nucleotides and have no protein coding potential [
11,
12]. More and more reports showed that lncRNAs have many kinds of biological functions involved in embryo development, immunoregulation, and tumor development [
13‐
15]. Aberrant expression of lncRNAs is closely related with human diseases, especially in cancers [
16,
17]. For example, long noncoding RNA PVT1 is up-regulated in hepatocellular carcinoma, nonsmall cell lung cancer, osteosarcoma, esophageal squamous cell carcinoma, cervical cancer, breast cancer and so on [
18‐
23]. Furthermore, lncRNAs may control the resistance of tumor cells to drug. For instance, BCAR4 enhances cisplatin resistance in gastric cancer patients [
24]. In colorectal cancer, many lncRNAs, including a large number of uncharacterized lncRNAs, are also abnormally expressed [
25]. We showed that DLEU1 was up-regulated in CRC tissues compared to normal tissues. Previous research demonstrated that DLEU1 promotes ovarian carcinoma and gastric cancer development [
26,
27]. Nevertheless, the roles of DLEU1 in other tumors including colorectal cancer remain elusive.
In this study, we found that DLEU1 was up-regulated in CRC tissues. Furthermore, overexpression of DLEU1 promoted CRC cell proliferation, migration and invasion in vitro and in vivo. In terms of mechanism, we found that DLEU1 co-localized with SMARCA1 in colorectal cancer cells. And DLEU1 is indispensible for the deposition of SMARCA1 at the promoter of KPNA3 gene. Collectively, DLEU1 recruited SMARCA1 to epigenetically activate downstream gene KPNA3, thereby promoting proliferation and migration in colorectal cancer. Therefore, our results propose a model for DLEU1-mediated cell proliferation in CRC.
Methods
Patient samples
100 pairs of CRC tissues and adjacent non-tumor tissues were obtained from The First Affiliated Hospital of Harbin Medical University. Three pathologists evaluated all specimens according to the World Health Organization (WHO) guidelines and the pTNM Union for International Cancer Control (UICC) pathological staging criteria. The samples were frozen in liquid nitrogen and stored at − 80 °C until use. Informed consent was obtained from all patients. The protocol was approved by The First Affiliated Hospital of Harbin Medical University. All methods involving human patients were performed in accordance with the relevant guidelines and regulations of The First Affiliated Hospital of Harbin Medical University.
Cell lines and cell culture
The human colon cell lines (CCD18-Co, FHC and HCoEpiC) and human colorectal cancer cell lines (HCT116, HT29, SW480, SW620, DLD-1, LoVo, HCT8, RKO, and CaCo2) were purchased from the American Type Culture Collection (ATCC) and cultured according to their instructions. Cells were incubated at 37 °C in a humidified atmosphere with 5% CO2.
Cell transfection
DLEU1, SMARCA1 and KPNA3 were cloned into pCDNA3 plasmid. shRNAs were synthetized by invitrogen and cloned into pGPH1/Neo (GenePharma, Shanghai, China) as described before [
28]. Transfection was conducted with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) and stably DLEU1-silenced cell lines were screened out as previously described [
28]. The shRNA sequences are as follows: shDLEU1: 5′-CACTTAAGCCTCGGAACAA-3′; shSMARCA1: 5′-TTGCCAGTTCCAGTGTATT-3′; shKPNA3: 5′-GTCTCAGTCACTTTGCAGT-3′.
Antibodies
Anti-PCNA (13110), anti-MMP2 (87809), anti-TWIST (46702), anti-SMARCA1 (12483), anti-GAPDH (5014) and anti-CYCLIN D1 (2922) were purchased from Cell Signaling Technology. Anti-KPNA3 (HPA046852) was from Sigma.
Apoptosis analysis
Cell apoptosis were analyzed by flow cytometry (FACScan; BD Biosciences) using CellQuest software (BD Biosciences).
The 6-week-old male athymic BALB/c nude mice were maintained under specific pathogen-free conditions and manipulated according to protocols approved by the Medical Experimental Animal Care Commission at The First Affiliated Hospital of Harbin Medical University. A volume of 0.1 ml of 4×106 suspended cells was respectively subcutaneously injected into the posterior flank of each mouse. Tumor volumes and weight was measured at indicative time points.
MTT assay and clone formation were used for evaluated cell viability and proliferation. Cell proliferation was documented following the manufacturer’s protocol every 24 h. For the colony formation assay, cells were seeded in a fresh six-well plate and maintained in media containing 10% FBS, replacing the medium every 4 days. After 14 days, methanol and stained with 0.1% crystal violet (Sigma-Aldrich) fixed cells and count clones.
In vitro migration and invasion assay
In the transwell migration assay, 5×104 cells were placed in the top chamber of each insert (Millipore, Billerica, MA) with an uncoated membrane. For the invasion assay, 8×104 cells were placed in the upper chamber of each insert coated with 100 μl Matrigel (BD Biosciences, MA) to form a matrix barrier. For both assays, cells were trypsinized and resuspended in 200 μl DMEM, and 500 μl DMEM supplemented with 10% FBS was added to the lower chamber. After incubation at 37 °C, any cells remaining in the top chamber or on the upper membrane of the inserts were carefully removed. After fixation and staining in a dye solution containing 0.1% crystal violet, the cells adhering to the lower membrane of the inserts were counted and imaged with an IX71 inverted microscope (Olympus Corp., Tokyo, Japan).
Real-time quantitative PCR
Total RNAs were extracted with TRIzol according to the manufacturer’s protocol. Then cDNA was synthesized with the M-MLV reverse transcriptase (Promega). Then mRNA transcripts were analyzed with ABI 7300 qPCR system using specific primer pairs. Relative expression levels were calculated and normalized to endogenous GAPDH for mRNA and U6 for DLEU1. The primer sequence information is available if requested.
Northern blot
Total RNA was extracted from sample cells with TRIzol. 10 μg RNA from each sample was subjected to formaldehyde-denaturing agarose electrophoresis followed by transferring to positively charged NC film with 20 × SSC buffer. Membrane was UV cross-linked and incubated with hybrid buffer for a 2 h prehybridization, followed by incubation with biotin-labeled RNA probes. Biotin signals were detected with HRP-conjugated streptavidin according to the manufacturer’s instruction.
In situ hybridization
Samples were fixed and embedded with paraffin. Then sample sections were incubated in graded alcohols and incubated in 3% hydrogen peroxide (H2O2) for 30 min. Biotin-conjugated probes and streptavidin-HRP conjugate were used for ISH. The samples were finally stained with haematoxylin. The probe sequences for DLEU1 were as follows: 5′-ACGATGATTCTGCGCATGTG-3′ and 5′-CTGGTAGCTATAAGACGACC-3′.
DNA FISH
Cells were fixed with 4% PFA containing 10% acetic acid for 15 min at room temperature, followed by replacement with 70% ethanol at − 20 °C. Cells were then incubated in buffer containing 100 mM Tris-HCl (pH 7.5), 150 mM NaCl, followed by cytoplasm digestion in 0.01% pepsin/0.01 N HCl for 3 min at 37 °C. Cells were further fixed in 3.7% PFA and replaced with ethanol to a final concentration of 100%. Cells were air dried and washed with 2×SSC, followed by blocking with buffer containing 100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20, 3% BSA for 20 min. Cells were then denatured in 70% formamide/2×SSC, and incubated with fluorescence-labeled DNA probes overnight. Cells were counterstained with DAPI for nucleus post washing with PBS.
RNA pulldown
Biotin-labeled RNAs were transcribed in vitro with the Biotin RNA Labeling Mix (Roche Diagnostics) and T7 RNA polymerase (Roche Diagnostics), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA). Next, whole-cell lysates were incubated with 3 μg of purified biotinylated transcripts for 1 h at 25 °C. Complexes were isolated with streptavidin agarose beads (Invitrogen). The beads were washed briefly three times and boiled in sodium dodecyl sulfate (SDS) buffer, and the retrieved protein was detected by western blot or mass spectrum.
RNA immunoprecipitation (RIP)
We performed RNA immunoprecipitation (RIP) experiments using the Magna RIP™RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer’s instructions. The co-precipitated RNAs were detected by reverse-transcription PCR. The total RNAs were the input controls.
Chromatin immunoprecipitation (ChIP)
We conducted ChIP using the EZ ChIP™Chromatin Immunoprecipitation Kit for cell line samples (Millipore, Bedford, MA). Briefly, we sonicated the crosslinked chromatin DNA into 200- to 500-bp fragments. The chromatin was then immunoprecipitated using primary antibodies. Normal IgG was used as the negative control. Quantification of the immunoprecipitated DNA was performed using qPCR with SYBR Green Mix (Takara).
Statistical analysis
All statistical analyses were performed using the Statistical Package for the Social Sciences version 20.0 software (SPSS Inc., Chicago, IL, USA). Survival curves were calculated using the Kaplan-Meier method and were analyzed using the log-rank test. For comparisons, one-way analyses of variance and two-tailed Student’s t-tests were performed, as appropriate. P < 0.05 was considered statistically significant.
Discussion
Accumulating evidences have demonstrated the importance of lncRNAs in various human tumors, including CRC [
33‐
35]. The expression of lncRNAs is often abnormal in human cancers [
36]. Therefore, many lncRNAs are reported to serve as a biomarker for tumor diagnosis [
37]. Seeking the key lncRNAs and understanding their functional mechanism are a matter of great significance for diagnosis, therapy and prognosis of different cancers. However, lncRNAs in CRC are still an emerging field, only a few lncRNAs have been defined in CRC and should be further explored as predictive biomarkers. In our study, we found that the expression of DLEU1 was remarkably up-regulated in CRC tissues and correlated with clinical severity.
Our data proved that DLEU1 knockdown significantly inhibited cell proliferation both in vitro and in vivo, whereas overexpressing DLEU1 promoted tumor growth. Depletion of DLEU1 led to decreased cell division. Fewer cells entered into S phase after DLEU1 knockdown. Accumulating evidences showed that lncRNAs regulate tumorigenesis and cancer progression by various mechanisms including epigenetic regulation and transcriptional regulation [
38‐
40]. To reveal the underlying mechanism, we performed RNA pulldown and mass spectrum assays. We identified SMARCA1 as an interactive protein of DLEU1. SMARCA1 is an essential subunit of the chromatin remodeling NURF complex [
41,
42]. NURF complex promotes target gene expression by remodeling chromatin accessibility. However, the role of SMARCA1 in CRC has not been explored. In our study, we found that DLEU1 interacted with SMARCA1 directly in CRC cells and regulated cancer cell proliferation.
Many evidences proved that lncRNAs may regulate the expression of their neighbor genes [
31]. To define the downstream target gene of DLEU1 in CRC, we analyzed the expression of the neighbor genes of DLEU1 by RT-qPCR. We found that DLEU1 knockdown significantly down-regulated the expression of KPNA3. The function of KPNA3 remains elusive in CRC. In our study, we found that the expression of KPNA3 was significantly up-regulated in CRC tissues compared to non-tumor tissues. Our data demonstrated that DLEU1 and SMARCA1 deposited at the same promoter region of KPNA3. Moreover DLEU1 (nt 1~ 400) is indispensible to recruit SMARCA1 at KPNA3 promoter. DLEU1 and SMARCA1 cooperated to promote KPNA3 activation in CRC. Furthermore, KPNA3 knockdown remarkably inhibited cell proliferation, migration and invasion, and vice versa. Nevertheless, how KPNA3 exerts roles in CRC progression still requires to be further investigated. KPNA3 is a subunit of the nuclear pore complex (NPC) and involved in nuclear protein import [
43]. KPNA3 might regulate protein transfer to promote CRC growth, metastasis and relapse.