Background
On a global scale, colorectal cancer (CRC) is one of the most common malignant [
1]. Annually, approximately 1.2,000,000 new cases were diagnosed and 60,000 death cases happened [
2]. The overall occurrence and mortality are declining due to the advance in new techniques and therapeutic methods for CRC predication and treatment [
3]. However, the overall 5-year survival rate is still lower than anticipation as metastatic CRC frequently occurred in patients at the time of diagnosis [
4,
5]. Hence, it is paramount to reveal the underlying mechanism behind the progression of CRC, so as to identify potential novel diagnostic and prognostic biomarkers.
The newly-identified non-coding RNAs (ncRNAs) are a cluster of RNAs with the ability of transcriptional control and post-transcriptional mediation, but they lack the potential to encode proteins [
5‐
8]. Long non-coding RNAs (lncRNAs) are a kind of ncRNAs containing over 200 nucleotides in length. They have been extensively studied in the carcinogenesis of many diseases, including cancer. Moreover, recent findings manifested that lncRNAs are involved in a series of biological processes, such as cell proliferation, apoptosis, invasion and migration [
9,
10]. Furthermore, lncRNAs have been identified as new biomarkers of many malignancies, since they are associated with the complicated pathogenesis of malignancies [
11,
12]. In addition, the prevalent competing endogenous RNA (ceRNA) mechanism has revealed the crucial regulatory role of lncRNAs on downstream RNAs, consequently impacting various physiological and pathophysiological activities within cells [
13]. To date, the pathologic roles of most lncRNAs remain undiscovered, which indicates the extensive application potential of lncRNAs in the prediction and treatment of different cancers.
In CRC, the expression status and functional role of some lncRNAs were investigated. For instance, lncRNA XIRP2-AS1 was discovered significantly lowly expressed in CRC tissues and could serve as a favorable biomarker for CRC patients [
14]. Silencing lncRNA AWPPH significantly curbed CRC cell proliferation via down-regulating the expression of GLUT-1 [
15]. FEZF1-AS1 has been reported to accelerate the progression of CRC via up-regulating the expression of NT5E through sponging miR-30a-5p [
16].
RHPN1-AS1, as a newly identified lncRNA, has been revealed to promote the carcinogenesis of head and neck squamous cell carcinoma [
17]. Furthermore, it has been reported as a potential clinical biomarker and participated in important biological processes and pathways in non-small cell lung cancer (NSCLC) [
18]. Nevertheless, the molecular role of RHPN1-AS1 behind the carcinogenesis and development of CRC has not been explored yet. Therefore, the aim of present study is to explore the cellular function of RHPN1-AS1 in CRC, which might contribute to providing some novel thoughts about finding effective treatment targets for CRC patients.
Materials and methods
Cell culture
Normal human colorectal mucosal cell (FHC) and CRC cells (SW620, SW480, HCT-116, HT29) were bought from Chinese Academy of Sciences (Beijing, China). Human kidney cell (293T) was gained from Genechem (Shanghai China). Cells were grown in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) adding 10% fetal bovine serum (FBS; Invitrogen), 1% penicillin/streptomycin (Sigma-Aldrich, Milan, Italy) and cultured in a 5% CO2 incubator at 37 °C.
Cell transfection
HCT-116 and HT29 cells were transfected with specific shRNAs against RHPN1-AS1 (sh-RHPN1-AS1#1/#2), STAT3 (sh-STAT3), OGT (sh-OGT#1/#2) and their corresponding control group (sh-Ctrl), and pcDNA3.1/STAT3, pcDNA3.1/OGT and the empty pcDNA3.1 vector, respectively. The miR-7-5p inhibitor, miR-7-5p mimics, NC mimics and NC inhibitor were synthesized by GenePharma (Shanghai, China). Lipofectamine 2000 (Invitrogen) was used for executing transfection experiments for 48 h.
Quantitative real time polymerase chain reaction (qRT-PCR)
TRIzol reagent (Invitrogen) was employed to obtain total RNA from cells. Reverse Transcription Kit (Applied Biosystems, CA, USA) or Taqman Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientic) was used to reverse transcribe total RNA into cDNA. qRT-PCR was performed on Bio-Rad CFX96 System utilizing the ABI StepOnePlus system (Applied Biosystems). Fold expression changes were calculated with 2−∆∆Ct method, and GAPDH/U6 was endogenous control.
Cell counting kit-8 (CCK-8) assay
1 × 103 cells were added to 96-well plates with culture media. The medium of every well was replaced by a fresh culture media containing 10 µL CCK8 at specific time points. The cells were cultivated for additional 4 h. The absorbance at 450 nm was measured via a microplate reader (Olympus, Tokyo, Japan).
EdU incorporation assay
EdU (5-ethynyl-2′-deoxyuridine) assay was performed to detect cell proliferation ability using the Click-iT EdU Imaging Kit (Invitrogen) in line with manufacturer’s directions. Transfected HT29 and HCT-116 were maintained into 96-well plate. After incubation of 48 h, 100 μl medium with 50 μM EdU was added into all wells. Cells underwent 2 h incubation at 37 °C and were fixed with 4% paraformaldehyde. Nuclei were dyed in DAPI for 30 min. EdU-positive cells were visualized using a microscope (Olympus). Proliferation rate was determined by calculating the ratio of the fluorescent positive cells to total cells.
Flow cytometry analysis
HCT-116 and HT29 cells were reaped and cultured in 6-well plates after transfection. Followed by rinsing with PBS, Annexin V-FITC and Propidium iodide (PI) were used for double staining. Apoptosis rate was measured via a flow cytometry (BD Biosciences, Beijing, China).
Transwell assay
HCT-116 and HT29 cells were seeded into the upper chamber (Corning, NY, USA) with or without pre-coated Matrigel (Becton–Dickinson, San Jose, CA, USA) for invasion or migration analysis. 5 × 104 cells with serum-free medium were added into the top compartment, while medium containing 10% FBS was placed into the bottom compartment later. After incubation for 24 h (migration) or 48 h (invasion), cells on the underside of membrane were immobilized and dyed in methanol or crystal violet. Finally, cells were counted in 5 random chosen fields under the microscope (Olympus).
Western blot
Total proteins were extracted with RIPA lysis buffer (Invitrogen). Protein concentrations were examined by BCA kit. There was 10% SDS-PAGE for the isolation of proteins. And then proteins were moved on PVDF membranes. After being blocked with PVDF, membranes were cultured with primary antibodies for E-cadherin (ab194982), N-cadherin (ab202030), Vimentin (ab193555), Slug (ab51772), Twist (ab187008), OGT (ab96718) and GAPDH (ab8245) from Abcam (Cambridge, CA, USA). Then, secondary antibodies were added for 1 h cultivation. The amount of protein was determined using chemiluminescence detection system.
Subcellular fractionation
Both nuclear and cytoplasmic RNA from CRC cells were separated with PARIS Kit (Ambion, Austin, TX, USA). Subsequently, qRT-PCR was employed for detecting the relative expression level of RHPN1-AS1. GAPDH/U6 was used as cytoplasmic/nuclear control.
Fluorescence in situ hybridization (FISH) assay
RHPN1-AS1 FISH probe was constructed by Ribo Bio Technology (Guangzhou, China). Cells were fixed in 4% paraformaldehyde, and then permeabilized in PBS which was mixed with 0.5% Triton X-100. After that, cells were hybridized with Cy3 labeled RNA FISH probe at 37 °C. Subsequently, cells were stained by the use of Hoechst. The presence of RHPN1-AS1 was visualized using confocal microscopy (Olympus).
Luciferase reporter assay
The wild-type (WT) and mutant (MUT) binding sites of miR-7-5p in RHPN1-AS1 sequence or OGT 3′-UTR was sub-cloned into pmirGLO dual-luciferase vector to construct RHPN1-AS1-WT/MUT, OGT-WT/MUT, and then co-transfected with miR-7-5p mimics or NC mimics into HCT-116, HT29 and HEK-293T cells, respectively. The pGL3-RHPN1-AS1 promoter was co-transfected with pcDNA3.1/STAT3, pcDNA3.1, sh-STAT3 or sh-Ctrl into HEK-293T cell. Dual-Luciferase Reporter Assay System (Promega, USA) detected the luciferase activity.
RNA pull down assay
The RHPN1-AS1 WT, RHPN1-AS1 MUT or NC was biotin labeled into Biotin-RHPN1-AS1 WT, Biotin-RHPN1-AS1 MUT or Bio-NC, severally. Afterwards, cell lysates were incubated with biotinylated RNA overnight and M-280 streptavidin magnetic beads (Sigma-Aldrich) were added for cultivation for 48 h. Relative enrichment of miR-7-5p was assayed by RT-qPCR.
Chromatin immunoprecipitation (ChIP) assay
Cells were subjected to chromatin immunoprecipitation based upon an EZ-ChIP chromatin immunoprecipitation kit (Millipore, Bedford, MA, USA). Briefly, cells were cross-linked by formaldehyde. Cell lysates were gathered and sonicated to shear DNA. After that, chromatin was immunoprecipitated using anti-STAT3 (Millipore), while IgG was the negative control. The relative enrichment of RHPN1-AS1 was evaluated by qRT-PCR.
RNA immunoprecipitation (RIP) assay
EZ-Magna RIP kit (Millipore) was applied for conducting RIP assays. HCT-116 and HT29 cells were lysed in RIP lysis buffer, and incubated with anti-Ago2 (Millipore) and IgG (Millpore). The relative expression levels of miR-7-5p, RHPN1-AS1 and OGT were examined using qRT-PCR analysis.
Tumor xenograft experiment
Female nude mice were gained from Shi Laike Company (Shanghai, China). Cells infected with sh-RHPN1-AS1#1 or sh-Ctrl was injected subcutaneously into the hip back of mice. 4 days later, tumor growth was recorded by measuring their volume. After 4 weeks, nude mice were euthanized to weigh the tumors. This study was progressed with the approval of the Ethics Committee of Henan Provincial People’s Hospital.
Statistical analysis
Experimental data were manifested as mean ± SD. All statistical analyses were achieved using the GraphPad software 6.0 (GraphPad Inc., San Diego, CA, USA). Each experiment was done at least three times. The variance among groups was analyzed via Student’s t-test or one-way ANOVA. Differences were seen as statistically significant when P < 0.05.
Discussion
Mounting evidence has demonstrated that lncRNAs are closely associated with normal physiological progresses and pathological change [
26]. The aberrantly expressed lncRNAs could be applied as prediction biomarkers for many diseases, including cancers [
27‐
29]. In present study, we found that RHPN1-AS1, as a newly discovered lncRNA, was aberrantly up-regulated in CRC cells than non-tumor one. On the one hand, knockdown of RHPN1-AS1 curbed CRC cell proliferation and promoted apoptosis. On the other hand, RHPN1-AS1 knockdown greatly inhibited cell migration and invasion capacities. These results indicated that RHPN1-AS1 played an oncogenic role in the development of CRC in vitro. STAT3 signal transduction pathway has been reported to be an important pathway in CRC [
30]. In present study, transcription factor STAT3 was found to be closely related to the abnormal up-regulation of RHPN1-AS1 in CRC. We identified that STAT3 could activated the transcription of RHPN1-AS1 via ChIP and luciferase reporter assays.
The competing endogenous RNAs (ceRNAs) network has revealed that lncRNAs could restore the expression and biological function of mRNAs via competitively binding to shared miRNAs. This lncRNA-miRNA-mRNA regulatory mechanism exerted significant impacts on malignant tumor progression [
31]. In this study, we found that miR-7-5p could interact with RHPN1-AS1. Contrary to the expression status of RHPN1-AS1, miR-7-5p was aberrantly down-regulated in CRC cell lines compared with normal colorectal epithelial cell line. Furthermore, miR-7-5p inhibition could abrogate the suppressing effects of RHPN1-AS1 knockdown on CRC cell proliferation, apoptosis, migration and invasion. This indicated that RHPN1-AS1 was a sponge of miR-7-5p in CRC and its oncogenic function was fulfilled by sponging miR-7-5p expression.
MiRNAs can bind to 3′UTR of target gene with micro response elements (MREs) and further inhibit the cellular function of target gene [
32]. OGT was identified a target gene of miR-7-5p by multiple means, including qRT-PCR, luciferase reporter and RIP assays. We found that OGT expression down-regulated most apparently by miR-7-5p overexpression compared with other potential combinable mRNAs. It has been found that OGT is significantly upregulate in colon tissues and may play crucial role in colon cancer carcinogenesis and progression [
33]. Furthermore, OGT has been found to promote CRC metastasis by negatively regulating miR-101 and increasing EZH2 stability [
34]. In this study, OGT was also significantly up-regulated in CRC cell lines compared with normal colon cell line. Silencing OGT inhibited CRC malignant progresses, including cell proliferation, migration and invasion. Besides, silencing OGT facilitated CRC apoptosis. Rescue experiments revealed that OGT overexpression could abrogate the repressing effects of RHPN1-AS1 down-regulation on CRC cell proliferation, migration and invasion. Overall, these findings initially revealed that lncRNA RHPN1-AS1 promoted CRC progression via acting as ceRNA to up-regulating OGT by sponging miR-7-5p.
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