Background
Pancreatic cancer (PC) is an aggressive tumor with devastating malignancy capability. The lack of effective early diagnostic and prognostic markers is the largest stumbling block in providing adequate treatment and consequently leads to a poor 5-year survival rate of less than 8% [
1]. PC patients are generally diagnosed at a more advanced-stage, with reports suggesting that approximately 50% of patients diagnosed are confirmed to have metastasis [
2]. Although existing therapeutic methods such as surgery and radio/chemotherapy are known to aid in lengthening survival and providing symptom relief, relatively few approaches provide a curative effect [
3]. Hence, it is of great importance that deeper knowledge pertaining to the underlying molecular mechanisms of PC carcinogenesis and progression are elucidated, in order to identify novel therapeutic and diagnostic targets for cancer treatment.
Long non-coding RNAs (LncRNAs) are involved in a large variety of biological processes, with reports linking the dysregulation of lncRNAs with cancer cell invasion, proliferation and metastasis [
4]. LncRNA actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) was reported to be up-regulated in nasopharyngeal carcinoma [
5], colorectal cancer [
6] and cholangiocarcinoma [
7]. The up-regulation of AFAP1-AS1 acts as an oncogene and has been demonstrated to result in poor prognoses accompanied by an elevated risk of metastasis [
8]. Importantly, pancreatic ductal adenocarcinoma (PDAC) also exhibits high expression of AFAP1-AS1 along with promoted PDAC cell proliferation, invasion and migration [
9]. Moreover, accumulating evidence has suggested that cancer stem cells (CSCs) are highly tumorigenic cancer cells with the abilities of self-renewal, differentiation and tumorigenesis [
10]. LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) has been linked with the maintenance of pancreatic CSC self-renewal [
11]. Moreover, lncRNA have been reported to regulate cancer stem cells by targeting microRNAs (miRNAs) [
12]. MicroRNA-384 (miR-384) has been reported to be a target of AFAP1-AS1 based on bioinformatics analysis, while the current study demonstrates that AFAP1-AS1 could decrease miR-384 expression by competitively binding to miR-384 in this study. Additionally, miR-384 has been demonstrated to be down-regulated in renal cell cancer (RCC), with the overexpression of miR-384 indicated to inhibit RCC cell growth and invasion through regulation of its target gene astrocyte elevated gene 1 [
13]. Bioinformatics analysis evidence has been provided confirming that Activin A receptor type 1 (ACVR1) is regulated by miR-384, which has been previously shown to play a role in the regulation of stem cell markers [
14]. The functional relationship between miR-384 and ACVR1 has been highlighted in breast cancer with studies providing evidence suggesting that miR-384 suppresses breast cancer progression by down-regulating ACVR1 [
15]. Based on our exploration of literature, we deem it highly plausible that AFAP1-AS1 could influence the biological characteristics of PC cells via the regulation of ACVR1 by competitively binding to miR-384. Hence, the potential role of AFAP1-AS1 in the initiation and development of PC was investigated in the current study, in order to ascertain as to whether AFAP1-AS1 functions in connection with the AFAP1-AS1/miR-384/ACVR1 pathway to affect PC cell self-renewal ability, tumorigenicity, invasion, migration and stemness.
Materials and methods
Ethics statement
All patients enrolled in the study signed informed written consent documents. All experiment protocols were approved by the Clinical Trials and Biomedical Ethics Committee of Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College (2017KT042). The experimental procedure and animal use program of the current study were approved by the Animal Ethics Committee.
Study subjects
Seventy-five PC tissues and adjacent normal tissues were collected from patients at the Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College between August 2014 and November 2016. There were 39 males and 36 females that were individually diagnostically confirmed post operatively by means of histopathology, among which there were 46 cases ≥50 years old and 29 cases < 50 years old. There were 28 cases with high levels of differentiation, 22 cases of moderate differentiation and 25 cases of poor differentiation. Lymph node metastasis (LNM) was detected in 37 cases, while a total of 38 cases did not exhibit any signs of LNM. Patients with PC were classified in accordance with the tumor-node-metastasis (TNM)-staging-system of the International Union Against Cancer (UICC) [
16]. The clinical stages were confirmed to be stage II and stage III. There were 42 stage II cases and 33 cases at stage III. All participating patients did not receive any drug therapy, chemo/radiotherapy or immunobiological treatment prior to inclusion to our study. The human PC cell line SW1990, Capan-1, AsPC-1, MIAPaCa-2, PANC-1 and human normal pancreas cell line HPC-Y5 purchased from Shanghai Yanhui Biological Technology Co., Ltd. (Shanghai, China) were resuscitated. The cell lines were further cultured in Dulbecco’s modified Eagles Medium (DMEM, Gibco Company, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco Company, Grand Island, NY, USA) and cultured in an incubator (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) with saturated humidity at 5% CO
2, 37 °C. When the cells were confirmed to have reached a density of 90%, they were treated with 0.25% pulmonary protease (T1300, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) followed by subculture at a ratio of 1: 3. The human PC cell lines were conducted in connection with reverse transcription quantitative polymerase chain reaction (RT-qPCR) methods for the detection of AFAP1-AS1 expression, with the cell lines screened prior to the subsequent experiments.
In situ hybridization
PC tissues as well as adjacent normal tissues were sectioned, dewaxed and water saturated. The sections were put on the hybridization oven with 200 μL prehybridization solution added in pre-hybridization at 65 °C for 1 h. During the In situ hybridization, 200 μL AFAP1-AS1 probe hybridization solution (DakoCytomation, Carpinteria, CA, USA) with a final concentration of 500 ng/mL was added. The sections were then cultured in a wet box at 55 °C for 1.5 h, followed by oscillation, washing with 55 °C water for 25 min, and then developed with 2–3 drops of nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate under conditions void of light for 1 h. When staining signal intensity was confirmed to be moderate, pure water was added to terminate the reaction. The sections were then counterstained for nuclei with 200 μL Nuclear Fast Red for about 1–5 min, and rinsed with running water for 10 min. Finally, the sections were dehydrated with 50, 75 and 100% ethanol (5 min/each), sealed with neutral balsam and analyzed for data collection purposes. The chromogenic agent BCIP/NBT was reflected by blue while the counterstaining agent Nuclear Fast Red was red. The staining results were scored in an independent fashion by two pathologists, and the cytoplasm stained with blue was considered to represent the positive cells [
17]. Five fields were randomly selected for each section, observed under a microscope (× 200) after which the percentage of positive cells was calculated. The percentage of positive cells < 5% was regarded as negative (−), while ≥5% as positive (+).
RT-qPCR
Total RNA was extracted using Trizol RNA extraction solution (Invitrogen Inc., Carlsbad, CA, USA). Reverse transcription was performed using Primescript TMRTreagent Kit (RRO37A, Takara Biotechnology Ltd., Dalian, Liaoning, China), according to the following steps: RNA precipitation was dissolved with 40 μL RNAase-free water, followed by adding 12 μL RNAase-free water, 2 μL OdT and 3 μL RNA sample in 200 μL RNAase-free pitot tube, well-mixed. After boiling at 70 °C for 5 min, the tubes were cooled on ice in a prompt manner for 2 min. Then, 1 μL deoxy-ribonucleoside triphosphate (dNTP), 1 μL guanidine isothiocyanate, 5 μL 5 × reverse transcription buffer and 1 μL Moloney Murine Leukemia Virus (MMLV) reverse transcriptase were applied and gently mixed using a pipette, and then further treated with a water bath at 37 °C for 90 min. The reaction was terminated by heating means at 70 °C for 5 min and conserved on an ice box. The target genes as well as internal reference genes were amplified by a fluorescence quantitative PCR instrument (ABI 7500, Applied Biosystems, Carlsbad, CA, USA). A total of 25 μL PCR reaction system was as follows:10 × PCR Buffer, 2.5 μL 25 mmol/L MgCl2, 1.5 μL 10 mmol/L dNTP, 0.5 μL 10 mmol/L Primer, 1 μL 1 nmol/L Probe, 0.25 μL Taq, 2.5 μL cDNA and 15 μL sterile distilled water. The reaction conditions included the following: pre-denaturation at 94 °C for 5 min, 40 cycles of denaturation at 94 °C for 30 s, denaturation at 58 °C for 45 s, denaturation at 72 °C for 30 s, and then extension at 72 °C for 10 min. All the reactions were preset with three replicates. U6 was regarded as the internal reference for AFAP1-AS1 and miR-384, while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the internal reference for the other genes. The AFAP1-AS1 and miR-384 expression levels as well as mRNA expression levels of ACVR1, Oct4, Nestin, CK19, CD133, and ABCG2 was calculated using the 2
-ΔΔCt method [
18], which reflected the ratio of the target gene expression between the experimental group and the control group. The formula applied was as follows: ΔCt = Ct
target genes - Ct
GADPH, ΔΔCT = ΔCt
experimental group - ΔCt
control group. Ct represented the amplification cycles when the real-time fluorescence intensity reached the set threshold value and when the amplification had entered a period of logarithmic growth. The experiment was repeated 3 times. The aforementioned methods were also applicable for the cell experiment. The primer sequences are displayed in Table
1.
Table 1
Primer sequences for RT-qPCR
AFAP1-AS1 | F: 5′-ACTGAAGAGGAACCAGGGACAG-3′ |
| R: 5′-GGGGAAACTGAAATGAATGAAG-3′ |
miR-384 | F: 5′-TGTTAAATCAGGAATTTTAA-3′ |
| R: 5′-TGTTACAGGCATTATGA-3′ |
ACVR1 | F: 5′-GTGAAGGTCTCTCCTGCGGTA − 3′ |
| R: 5′-GCCATCGTTGATGCTCAGTGA − 3′ |
Oct4 | F: 5’-CAAAGCAGAAACCCTCGTGC-3’ |
| R: 5’-AACCACACTCGGACCACTCG-3’ |
Nestin | F: 5’-ATCCCGTCAGCTGGAAAAGG-3’ |
| R: 5’-GGTGAGCTTGGGCACAAAAG-3’ |
CK19 | F: 5’-ACCATTGAGAACGCCAGGATT-3’ |
| R: 5’-TCCAGCACCCAAACACTCAA-3’ |
CD133 | F: 5′-TATAAAGCTTACCATGGCCCTCGTACTCGGCTC -3′ |
| R:5′-TATAGGATCCTCAATGTTGTGATGGGCTTGTC-3′ |
ABCG2 | F: 5′-CAGGTGGAGGCAAATCTTCGT-3′ |
| R: 5′-CTTGTACTCCGTCAGCGTGA-3′ |
GAPDH | F: 5’-ACAGTCCATGCCATCACTG-3’ |
| R: 5’-AGTAGAGGCAGGGATGATG-3’ |
U6 | F: 5’-TGCGGGTGCTCGCTTCGGCAGC-3′ |
| R: 5′-CCAGTGCAGGGTCCGAGGT-3′ |
Western blot analysis
The total protein of the cells was extracted using a radioimmunoprecipitation assay (RIPA) lysate (R0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) containing phenylmethylsulfonyl fluoride (PMFS). The cells were then incubated on ice for 30 min, centrifuged at 28985×g (4 °C) for 10 min followed by collection of the supernatant. The concentration of each protein sample was subsequently determined using bicinchoninic acid (BCA) kit (23,225, Pierce, Rockford, IL, USA), and adjusted with deionized water. Preparation of 10% SDS-PAGE gel was prepared (P0012A, Beyotime Biotechnology Co., Ltd., Shanghai, China). In the next step, each well was added with 50 μg protein sample, and the electrophoresis was at a stable pressure of 80 (Volt) V for 2 h. The protein was transferred onto polyvinylidene fluoride film (PVDF, Millipore, Billerica, MA, USA) via the wet transfer method at 110 V for 2 h. Membrane blockade was then performed with Tris-buffered saline with Tween 20 (TBST) containing 5% skimmed milk powder for 2 h. After the blocking solution was discarded, the film was washed once with TBST and incubated overnight at 4 °C with the primary rabbit antibodies against ACVR1 (1: 1000, ab155981), Oct4 (1: 1000, ab19857), Nestin (1: 2000, ab7659), CK19 (1: 1000, ab15463), CD133 (1: 500, ab16518), ABCG2 (1: 500, ab24115), and GAPDH (1: 2500, ab9485). All the above antibodies were purchased from Abcam Inc. (Cambridge, MA, USA). The film was then washed 3 times with TBST (10 min/time), and washed an additional 3 times with poly (butylene succinate-co-terephthalate) (PBST, 10 min/time). Finally, the film was developed with electrogenerated chemiluminescence (ECL) solution (WBKLS0100, Millipore, Billerica, MA, USA), with images then developed after exposure in a dark box. The relative protein expression was the ratio of target band to the internal reference GAPDH band. ImageJ (Bio-Rad Inc., Hercules, CA, USA) software was employed in the analysis of the grey value of protein bands. The experiment was repeated three times.
Flow cytometry
The culture medium was discarded when cell growth had reached the logarithmic phase. The cells were washed once with phosphate buffer saline (PBS), and further detached with ethylene diamine tetraacetic acid (EDTA)-free trypsin in an incubator at 37 °C for 2 min. When the cells were observed to have become round in shape, the digestion process was terminated by adding approximately 5 mL culture medium containing serum. Next, the cells were well mixed and placed into a sterilized centrifuge tube and centrifuged at 453×g for 5 min with the supernatant removed. The cells were then washed with PBS, suspended with 500 μL PBS, with CSC sorting performed on a flow cytometer. Finally, the sorted CSCs were maintained in DMEM/F-12 complete medium (containing 1% penicillin, streptomycin and B27) containing 10% FBS and cultured in a CO2 incubator at 37 °C.
Cell grouping and transfection
The PC cells PANC-1 and SW1990 at the logarithmic growth phase subsequently underwent transfection. The cells were assigned into shRNA-negative control (NC) group (PC cells transfected with shRNA-NC vector), shRNA-AFAP1-AS1–1 group (PC cells transfected with shRNA-AFAP1-AS1–1 vector), shRNA-AFAP1-AS1–2 group (PC cells transfected with shRNA-AFAP1-AS1–2 vector), empty vector group (PC cells transfected with empty vector), AFAP1-AS1 group (PC cells transfected with AFAP1-AS1 overexpression vector), miR-384 mimic-NC group (PC cells transfected with miR-384 mimic NC), miR-384 mimic group (PC cells transfected with miR-384 mimic), miR-384 inhibitor-NC group (PC cells transfected with miR-384 inhibitor NC), miR-384 inhibitor group (PC cells transfected with miR-384 inhibitor) and miR-384 inhibitor + shRNA-AFAP1-AS1–1 group (PC cells transfected with miR-384 inhibitor + shRNA-AFAP1-AS1–1 vector). All the transfection vectors or plasmids were synthetized by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China).
The PC cells were passaged one day prior to transfection, and seeded into 6-well plates (1 × 105 cells/well). Cell transfection was performed when cell confluence reached 70–80% and in accordance with the instructions of Lipofectamine® 2000 reagent (11,668,019, Invitrogen Inc., Carlsbad, CA, USA). A total of 250 μL serum-free DMEM was used to dilute 100 pmoL shRNA-AFAP1-AS1–1, shRNA-AFAP1-AS1–2, shRNA-NC, miR-384 mimic-NC, miR-384 mimic, miR-384 inhibitor-NC, miR-384 inhibitor and miR-384 inhibitor + shRNA-AFAP1-AS1–1 (a final concentration of 50 nM), mixed in a gentle manner and incubated at room temperature for 5 min. An additional 250 μL serum-free DMEM was used to dilute 5 μL of Lipofectamine 2000, gently mixed and incubated at room temperature for 5 min. Next, the two aforementioned properties were mixed well, incubated at room temperature for 20 min and then added to the cell culture wells. The transfected cells were continually cultured in a 5% CO2 incubator at 37 °C for 6–8 h. After the introduction of a complete culture medium, a further 24–48 h period of culturing was performed to further process the experiment.
The cells at the logarithmic growth phase were constructed into a single cell suspension, tallied using a hemocytometer and diluted to 103 orders of magnitude for use by 10 × gradient dilution method. A total of 3 mL of cell culture medium containing 0.1% agar was prepared in the 15 mL centrifuge tube, mixed on the whirlpool mixer and then added to 300 cells, further mixing on a whirlpool mixer was then performed. One mL of liquid (containing 100 cells) for each group was inoculated into the 6-well plates, and shaken in a prompt fashion for even distribution purposes. Three duplicates were set for each well. When the agar was confirmed to have taken shaped, 1 mL fresh medium was added on superior surface and incubated in a 5% CO2 saturated incubator at 37 °C for 7–10 days. The number of spheres was counted under a microscope.
The cells at the logarithmic growth phase were made into the single cell suspension, counted with a hemocytometer and diluted to 100 μL medium containing 1 cell by using 10 × gradient dilution method. The two kinds of cells were inoculated into the 96-well plates respectively, followed by the addition of 100 μL diluted cell suspension. The number of successfully inoculated wells was counted under a microscope, and then incubated in a 5% CO2 saturated incubator at 37 °C for 7–10 days. The number of wells with clone spheres (> 50 cells) was counted under a microscope, and the colony formation rate was calculated as follows: (number of wells with clone spheres/number of successfully inoculated wells) × 100%.
5-ethynyl-2′-deoxyuridine (EdU) staining
The cells at the logarithmic growth phase were seeded into the 96-well plates with 6000–10,000 cells per well and incubated in an incubator overnight. An EdU kit (C00031Apollo®567, RiboBio Co., Ltd., Guangzhou, Guangdong, China) was applied to detect cell proliferation. The cells were labeled by EdU: each well was added with 100 μL medium containing 50 μmol/L EdU solution and incubated for 2 h. After the cells had been fixed with 4% paraformaldehyde for 30 min and cleared with 0.5% TritonX-100 for 10 min, nuclear staining was performed with the addition of 100 μL Apollo® staining solution under conditions void of light. All the nuclei were stained blue (Hoechst staining) under a microscope, while the nuclei of the proliferated cells were stained red.
Transwell assay
A total of 50 mg/L of Matrigel (Sigma-Aldrich, SF, CA, USA) was diluted at a ratio of 1: 8. The membrane at the apical chamber was coated with 60 μL diluted Matrigel, and air-dried at room temperature. After the residual liquid in the culture plate had been aspirated, 50 μL of serum-free culture medium containing 10 g/L bovine serum albumin (BSA) was added to each well and placed at 37 °C for 30 min. Cells at the logarithmic growth phase were obtained from each group, and the cell density was adjusted to 1 × 105 cells/mL with serum-free culture medium containing 10 g/L BSA. Next, 200 μL cell suspension was added to the Transwell chamber, while the basolateral chamber of the 24-well plates was added with 500 μL culture medium containing 100 mL/L FBS. The Transwell chamber was placed in a culture plate and incubated in a 5% CO2 incubator at 37 °C for 24 h. After a 24-h period of incubation, the cells on the inner side of the chamber closer in proximity to the PVPF membrane were removed using a cotton swab, fixed with 95% alcohol for 30 min at room temperature, stained with crystal violet (Sigma-Aldrich, SF, CA, USA) for 20 min and then rinsed 3 times with water. Finally, the number of invasive cells was counted under the guidance of an inverted microscope (CKX41SF, Olympus, Tokyo, Japan).
Fluorescence in situ hybridization (FISH) assay
The subcellular localization of AFAP1-AS1 in cells was identified using a FISH assay. In accordance with the instructions of Ribo™ lncRNA FISH Probe Mix (Red) (RiboBio Co., Ltd., Guangzhou, Guangdong, China), the specific method applied was performed as follows: the coverslips were placed in the 6-well plates, with the cells at the logarithmic growth phase inoculated with the cells/well. When cell confluence had reached approximately 80% after 1 day culture, the coverslips were fixed in 4% paraformaldehyde at room temperature after PBS washing. The cells were subsequently treated with 2 μg/mL protease K, glycine and phthalide reagent, added with 250 μL prehybridization solution and incubated at 42 °C for 1 h. After that, 250 μL hybridization solution containing 300 ng/mL probe was added after the prehybridization solution removed, and further incubated at 42 °C overnight. The next day, the cells were washed 3 times with PBST, after which 4′,6-diamidino-2-phenylindole (DAPI) staining solution diluted with PBST was added at a ratio of 1: 800 to the 24-well plate for nucleus staining over a period of 5 min. After three PBST washes (3 min/time), the coverslips were sealed with an anti-fluorescence quencher. Finally, 5 different fields were selected under the guidance of a fluorescence microscope (Olympus, Tokyo, Japan), observed and photographed accordingly.
RNA pull down
Magnetic RNA-Protein Pull-Down kit (Pierce, Rockford, IL, USA) was applied, 1 μg biotin-labeled RNA AFAP1-AS1 was placed into Eppendorf (EP) tubes, added with 500 μL Structure Buffer, water bathed at 95 °C for 2 min, followed by ice bathing for 3 min. A total of 50 μL fully resuspended beads were incubated in EP tubes at 4 °C overnight. The beads were subsequently centrifuged at 1812×g for 3 min with the supernatant discarded, followed by three 500 μL RNA binding protein immunoprecipitation (RIP) wash buffer rinses. Then, 10 μL cell lysate was added and placed at room temperature for 1 h. The incubated bead-RNA-protein mixture was centrifuged at a low speed with the supernatant recycled, and washed 3 times with 500 μL RIP wash buffer. Next, 10 μL cell lysate supernatant was used as protein Input. After the protein concentration had been determined, western blot analysis methods were performed in order to measure protein expression. The experiment was repeated 3 times.
RIP assay
The AFAP1-AS1 binding ability to Argonaute 2 (AGO2) protein was detected using the RIP kit (Millipore, Bedford, MA, USA). The cells were washed with pre-cooled PBS, after which the supernatant was discarded. The cells were then lysed using an equal volume of radioimmunoprecipitation (RIPA) lysate (P0013B, Beyotime Biotechnology Co., Shanghai, China) in an ice bath for 5 min, centrifuged (4 °C) at 39452×g for 10 min, with the supernatant was extracted. A portion of the cell extract was used as an Input, and the other portion was incubated with antibodies for co-precipitation. Each co-precipitation reaction system was washed with 50 μL of beads and resuspended in 100 μL of RIP wash buffer. Each group was added with 5 μg antibodies for combination purposes. The magnetic bead-antibody complex was then washed and resuspended in 900 μL RIP wash buffer, followed by incubation with 100 μL cell extract at 4 °C overnight. The samples were then placed on a magnetic base to collect the magnetic bead-protein complexes. The samples and Inputs were then digested with proteinase K for RNA extraction, which was then used for western blot analysis. The antibodies used for RIP assay were AGO2 (ab32381, 1: 2000, Abcam Inc., Cambridge, MA, USA) diluted at room temperature for 30 min, and Immunoglobulin G (IgG) (ab172730, 1: 100, Abcam Inc., Cambridge, MA, USA) used as the NC. The experiment was repeated 3 times.
Dual-luciferase reporter gene assay
The target gene of AFAP1-AS1 and its targeting relationship to miR-384 were analyzed and predicted using the online prediction website RNA22. The AFAP1-AS1–3’UTR gene fragment was synthesized, and cloned into pmirGLO (Promega, Madison, WI, USA) via the endonuclease sites Spe I and Hind III. The mutant (Mut) sites of complementary sequence on the AFAP1-AS1 wide type (DCLK1-Wt) plasmid were designed. After restriction endonuclease digestion, the target fragment was inserted into the pMIR-reporter plasmids by using T4 DNA ligase. The luciferase PRL-TK vector expressed renilla (TaKaRa, Dalian, Liaoning, China) was used as the internal control in order to adjust the difference between the number of cells and the transfection efficiency. The miR-384 mimic as well as the NC were co-transfected in a respective manner with a luciferase reporter gene vector into PANC-1 cells for 48 h, after which the cells were collected and lysed. Finally, the luciferase activity was assessed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The experiment was repeated 3 times.
The online prediction website
microRNA.org was applied to predict and analyze the target gene of miR-384 and its targeting relationship between ACVR1. ACVR1–3’UTR gene fragment was synthesized, and cloned into pmirGLO (Promega, Madison, WI, USA) via the endonuclease sites Spe I and Hind III. The Mut sites of complementary sequence on an ACVR1-Wt plasmid were designed. After restriction endonuclease digestion, the target fragment was inserted into the pMIR-reporter plasmids using T4 DNA ligase. The luciferase PRL-TK vector expressing renilla (TaKaRa, Dalian, Liaoning, China) was used as the internal control and utilized when adjusting for the difference between the number of cells and the efficiency of transfection. The miR-384 mimic and NC were respectively co-transfected with luciferase reporter gene vector into PANC-1 cells for 48 h, after which the cells were collected and lysed accordingly. Finally, luciferase activity was examined using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The experiment was repeated 3 times.
Twenty-four specific-pathogen-free (SPF) male nude mice (4-week-old, 18–20 g) were purchased from Shanghai SLAC laboratory animal Co., Ltd. (Shanghai Laboratory Animal Center of Chinese Academy of Sciences, Shanghai, China). The mice were housed under stable temperature (25 °C–27 °C) conditions at a stable humidity of 45–50%. The mice were randomly assigned into 4 groups (shRNA-NC, shRNA-AFAP1-AS1–1, miR-384 inhibitor-NC and miR-384 inhibitor groups) with 6 mice placed in each group and inoculated with transfected cells after anesthesia administration. In brief, the cells exhibiting logarithmic growth following transfection were resuspended in 50% Matrigel (BD Biosciences, Bedford, MA, USA) with cell density adjusted 1 × 107 cells/mL. A single cell suspension of 0.5 mL containing 5 × 106 of transfected cells was subcutaneously injected into each mouse at the left under-axillary. On the 1st, 2nd, 3rd, 4th, 5th week, the tumor size of nude mice was measured with using a Vernier caliper. Tumor volume (mm3) = (L × W2)/2 (L stood for the tumor length and W stood for tumor width). The experiment was repeated 3 times.
Statistical analysis
All data were analyzed by SPSS 21.0 software (IBM, Armonk, NY, USA). Measurement data were expressed as mean ± standard deviation. Paired t-test was applied for comparison between tumor tissues and adjacent normal tissues, while unpaired t-test was used for comparisons between the other two groups. Comparisons among multiple groups were analyzed by analysis of variance (ANOVA). In the event that variance was equal, Q test was applied for pairwise comparison. When the variance was uneven, a non-parametric rank test was used. Repeated measures ANOVA was used to analyze the data at different time points during the experiment. The measurement data were expressed as a percentage (%), and the data were analyzed by the chi-square test. A value of p < 0.05 was considered to be statistically significant.
Discussion
PC represents a troublesome malignancy accompanied with high rates of mortality. A small subpopulation of pancreatic tumor cells with high carcinogenesis called pancreatic CSCs have been shown to lead to the high numbers of PC fatality [
19]. LncRNAs have been reported to act as modulators of cellular development and human diseases through the regulation of gene expression [
20]. More recently, lncRNAs were implied to exert as a ceRNA for specific miRNAs and regulate their function as well as miRNA target gene expression, affecting cancer development [
21,
22]. In the present study, we set out to investigate the underlying mechanism by which the lncRNA AFAP1-AS1 acting as a ceRNA acts to regulate ACVR1 by competitively binding to miR-384, thus playing the role of a carcinogenic factor in the progression of PC.
Initially, bioinformatics prediction suggested that AFAP1-AS1 was up-regulated in PC, both the FISH and RT-qPCR results provided evidence verifying that the up-regulated expression of AFAP1-AS1 in the PC tissues. However, in the PC cells transfected with shRNA-AFAP1-AS1, PC cell sphere formation, proliferation and invasion were all repressed. AFAP1-AS1 has been reported to be up-regulated in a large variety of cancers including that of gallbladder cancer [
23] and cholangiocarcinoma [
24], whereby the overexpression of AFAP1-AS1 has been implicated in the promotion of cell proliferation. A study previously revealed the effect of AFAP1-AS1 depletion on PDAC, indicating that PDAC cell proliferation, migration and invasion were suppressed [
9], all of which was consistent with the findings of our study.
Additionally, the mRNA and protein expression of the CSC markers (Oct4, ABCG2, Nestin, CK19 and CD133) were decreased in the PC cells when AFAP1-AS1 was knocked-down, which ultimately demonstrated that the stemness of the PC cells was attenuated when AFAP1-AS1 was reduced. Stem cell markers possess the ability to control embryonal stem cell self-renewal and differentiation, as well as acting to maintain a variety of biological characteristics [
25]. Studies have shown that CSCs isolated form PANC-1 cell line exhibited high expression of CD133/CD44/Oct4/Nestin and resistance to gemcitabine [
26]. Oct4 as a marker of CSCs played important role in maintaining CSC stemness, and knockdown of Oct4 inhibited PCSC biologic characteristics, chemoresistance and tumorigenesis, which was demonstrated previously both in vivo and in vitro [
27]. Another CSC marker in the form of overexpressed ABCG2 has been linked with the regulation of hypoxia-induced chemoresistance to gemcitabine [
28], moreover, ABCG2 expression induced by gastrin has been reported to elevate the proportion of SP cells, increasing the possibility of tumor cell metastasis potential and activity of cell invasion by activating NF-κB signaling in PC [
29]. Based on the aforementioned evidence, we subsequently asserted that the self-renewal, stemness of PC cells as well as the biological characteristics of maintenance of CSCs could be suppressed through the knockdown of AFAP1-AS1, which could also lead to the repression of CSC makers.
AFAP1-AS1 was subsequently verified to act as a ceRNA of miR-384 which was confirmed through the application of a dual-luciferase reporter gene assay. AFAP1-AS1/miR-384 coimmunoprecipitation with anti-Ago2 revealed the existence of a physical interaction between themselves among PC cells, further highlighting the activity of AFAP1-AS1 sequestering miRNA. Moreover, the overexpression of miR-384 suppressed PC cell sphere formation, proliferation, invasion and CSC markers expression, which was consistent with the response observed in relation to AFAP1-AS1 knockdown. LncRNAs that act as gene expression regulators by competing for miRNA binding can be defined as ceRNAs [
30]. Additionally, down-regulation of miR-384 was revealed in various cancers including non-small-cell lung cancer (NSCLC) [
31] and RCC [
13], and miR-384 could inhibit NSCLC cell as well as RCC cell growth and invasion through regulation of astrocyte elevated gene-1. In PC, the expression of miR-384 has been previously demonstrated to be inhibited by lncRNA colorectal neoplasia differentially expressed (CRNDE), which on the whole enhanced PC cells proliferation and metastasis by up-regulating insulin receptor substrate 1 (IRS1) through competitively binding to miR-384 [
32]. Interestingly, another study also concluded that CRNDE up-regulated pleiotrophin (PTN) by competitively binding to miR-384, while suggesting that the CRNDE/miR-384/PTN axis promoted papillary thyroid cancer cell proliferation, invasion and migration [
33]. With the aim of exploring the miR-384 related functions of AFAP1-AS1 in PC pathogenesis, ACVR1 was confirmed to be negatively regulated by miR-384. ACVR1 was further highlighted as a regulator of stem cell markers, with the hepatocellular carcinoma CSC subtype featured by the miR-148a-ACVR1-bone morphogenetic protein (BMP)-Wnt circuit, whereby the expression of ACVR1 was suppressed by miR-148a [
14]. Bearing in mind the interaction between AFAP1-AS1 and miR-384, our results obtained further suggested that AFAP1-AS1 could act to regulate the expression of ACVR1 by competitively binding to miR-384, which signifies the role of AFAP1-AS1 in the regulatory network involving PC tumorigenesis.