Circular RNA WDR77 target FGF-2 to regulate vascular smooth muscle cells proliferation and migration by sponging miR-124

Accepted Manuscript Circular RNA WDR77 target FGF-2 to regulate vascular smooth muscle cells proliferation and migration by sponging miR-124 Junjiang Chen, Lianqun Cui, Jingliang Yuan, Yuqing Zhang, Hongjun Sang PII:

S0006-291X(17)32046-6

DOI:

10.1016/j.bbrc.2017.10.068

Reference:

YBBRC 38685

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 3 September 2017 Revised Date:

3 October 2017

Accepted Date: 14 October 2017

Please cite this article as: J. Chen, L. Cui, J. Yuan, Y. Zhang, H. Sang, Circular RNA WDR77 target FGF-2 to regulate vascular smooth muscle cells proliferation and migration by sponging miR-124, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.10.068. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Circular RNA WDR77 target FGF-2 to regulate vascular smooth muscle cells proliferation and migration by sponging miR-124

Author:

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Junjiang Chen1, Lianqun Cui2* Jingliang Yuan3, Yuqing Zhang3，

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Hongjun Sang3

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Affiliation:

1. Shandong University, School of Medicine, Jinan, Shandong, 250100, China. 2. Department of Cardiology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, 250021, China.

3. Department of Cardiology, Shouguang People’ Hospital, Shouguang, Shandong,

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262700, China.

*Corresponding author:

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Lianqun Cui2, Department of Cardiology, Shandong Provincial Hospital Affiliated to Shandong University, Jinwu Road, No. 324, Huaiyin District, Jinan, Shandong,

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250021, China. E-mail: [email protected]

ACCEPTED MANUSCRIPT Abstract: Increasing evidences have revealed the important role of circular RNAs (circRNAs) in cardiovascular system disease. Whereas, the expression profiles and in-depth regulation of circRNAs on vascular smooth muscle cells (VSMCs) is still

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undetermined. In present study, our research team performed circRNAs microarray analysis to present the circRNAs expression profiles in high glucose induced VSMCs in vitro. Results showed that total of 983 circRNAs were discovered to be differentially expressed, and of these, 458 were upregulated and 525 were

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downregulated. Moreover, 31 circRNAs were up-regulated and 22 circRNAs were down-regulated with 2 fold change (P<0.05). One of an up-regulated circRNA,

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circWDR77, was identified. In vitro cell assay, circWDR77 silencing significantly inhibited the proliferation and migration. Bioinformatics methods discovered that miR-124 and fibroblast growth factor 2 (FGF-2) were downstream targets of circWDR77. The RNA sequence complementary binding was validated by RNA immunoprecipitation (RIP) and/or luciferase reporter assay. Further function

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validation experiments revealed that circWDR77 regulated VSMCs proliferation and migration via targeting miR-124/FGF2. Taken together, present study firstly reveals the circRNAs expression profiles in high glucose induced VSMCs and identifies the

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role of circWDR77-miR-124-FGF2 regulatory pathway in VSMCs proliferation and migration, which might provide a new theoretical basis for diabetes mellitus

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correlated vasculopathy.

Keyword:

Circular RNA; vascular smooth muscle cells; miR-124; FGF2; proliferation; migration.

ACCEPTED MANUSCRIPT 1. Introduction In recent years, the morbidity of diabetes mellitus (DM) is gradually increased with millions of newly diagnosed patients all over the world[1, 2]. DM could cause numerous complications, such as atherosclerosis, diabetic nephropathy, and diabetic

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foot gangrene[3, 4]. For atherosclerosis, disordered proliferation of vascular smooth muscle cells (VSMCs) plays a vital role in the atherosclerotic procession[5]. In DM patients, the high concentration of blood glucose could trigger the aberrant proliferation and migration of VSMCs, aggravating atherosclerotic process[6].

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Most recently, circular RNAs (circRNAs) have become a research hotspot in series of fields, involving cardiovascular system disease, diabetes mellitus, and

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tumors[7]. CircRNAs are a type of noncoding RNA (ncRNA) and characterized by covalent closed loops without 3’- and 5’- end[8]. Next generation sequencing and bioinformatics analysis have discovered huge quantity of circRNAs without definite biological function. In the peripheral blood of type 2 diabetes mellitus (T2DM) patients, hsa_circ_0054633 has the largest area under the curve and presents a certain

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diagnostic capability for pre-diabetes and T2DM[9]. CircRNAs, accompanied by miRNA and long noncoding RNAs (lncRNAs), play important role in different stages of heart failure progression and maladaptive remodeling[10]. However, the circRNA

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expression profiles and biological function correlated to VSMCs is still unclear. To investigate the circRNA expression profiles in VSMCs, our team performed high glucose treatment to simulate diabetic condition in vitro, and utilized human

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circRNAs microarray analysis to screen the differently expressed circRNAs. After normalization and comparison, we identified an upregulated circRNA circWDR77 (ID, hsa_circ_0013509, www.circbase.org/). CircWDR77 is 305 spliced

length

and

located at chr1:111984646-111986543 in gene symbol WDR77. Bioinformatics analysis reveals the sponge role on miR-124 via targeting FGF-2. Our study aims to investigate the underlying regulation of circWDR77 on VSMCs proliferation and migration via sponging miR-124 via targeting FGF-2.

2. Materials and methods

ACCEPTED MANUSCRIPT 2.1. Cells culture and transfection Human VSMCs and HEK 293T cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). VSMCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine

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serum (FBS, Grand Island, NY, USA), 1% 100 U/ml penicillin and 1% 100 mg/ ml streptomycin sulfate and incubated in 5% CO2 at 37°C. HEK293T cells were cultured at 37°C with 5% CO2 for another 24 h. This study was approved by the Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong University and

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Shouguang People’s Hospital. All the oligonucleotides were provided from GeneChem company (Shanghai, China). The cells were transfected with

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Lipofectamine 2000 reagent (Invitrogen) according to manufacturer’s instructions.

2.2. RNA extraction and circRNA microarray analysis

Total RNA was extracted from VSMCs samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions.

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Concentration and purity of total RNA samples were detected by NanoDrop ND-1000. The criterion of total RNA was the A260/A280 ratio of 1.8-2. Before microarray, RNAs were digested with Rnase R (Epicentre, Madison, WI, USA) to remove the

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linear RNAs and enrich the circular configuration. The RNA was labelled with Arraystar Human circRNA Array (8×15 K, Arraystar, Rockville, MD, USA) and

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scanned by the Agilent Scanner G2505C (Jamul, CA, USA).

2.3. Real-time polymerase chain reaction (RT-PCR) Total RNA was extracted by using TRIzol reagent (Invitrogen, Carlsbad, Calif,

USA) according to the manufacturer’s instructions. RNA was eluted with 50 ml RNase-free water (Promega, Madison, WI, USA). Subsequently, RNA (25 nM) was reversely transcribed using the RevertAid First-Strand cDNA Synthesis kit (Fermentas, Vilnius, Lithuania). RT-PCR was performed using MiScript SYBR Green PCR Kit (Qiagen, Valencia, CA, USA). Divergent primer sequences for circWDR77 were

5’-TCCAGCAACAGGACGAAATG-3’

(sense)

and

ACCEPTED MANUSCRIPT 5’-TGGAGATCCTCGGACTGGAA-3’ (antisense). The sequences of GAPDH primers

were

5’-GTCACTTTGCGCATCTTTG-3’

(sense)

and

5’-GCGCCCACCAATAGAAATC-3’ (antisense). The expression levels were ∆∆Ct

method.

2.4. Methyl thiazolyl tetrazolium bromide (MTT) assay

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normalized to the levels of GAPDH using 2

Cell viability was assessed by the MTT assay. In brief, cells (about 1×104 cells/well) were cultured in 96-well plate added with DMEM medium. After

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transfected with interfering oligonucleotides, each group of VSMCs were treated with 50 mg MTT solution (Sigma-Aldrich, St. Louis, MO, USA). Then, being incubated

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for 4 h, the absorbance was detected at 570 nm using an ELISA plate reader (BioTek Instrument Inc., Winooski, VT, USA).

2.5. Western blot

Cells were lysed with RIPA buffer (Beyotime Biotechnology, Dalian, China).

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Then, the extracts were separated and transferred onto PVDF membranes (Millipore, USA). After being blocked with TBS-T for 1 h at room temperature, the PVDF membranes were washed with TBS-T and incubated with primary antibodies for 1 h at

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room temperature or overnight at 4 °C. Primary antibodies were all from Abcam company (Cambridge, MA, USA). Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature

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for 1 h. The PVDF membrane was e visualized using enhanced chemiluminescence (Thermo-Scientifc, Rockford, IL, USA).

2.6. Cycle analysis

Flow cytometry was performed for cycle analysis. Briefly, cells were seeded in six-well plates at density of 4×105 per well and fixed in 4 ml of cold 75% ethanol at -20°C overnight and centrifuged at 2000 bpm for 10 min. Segregated cell pellets were incubated with 1ml propidium iodide at room temperature for 30 min. Cell cycle distribution was analyzed by measuring DNA content using flow cytometry.

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2.7. Dual-Luciferase reporter assay Sequence of circWDR77 and FGF2 3’-UTR were amplified using RT-PCR and inserted into the psiCHECK-2 luciferase vector (Promega, Madison, WI, USA). The sequence

of

FGF2

mRNA

3’-UTR

was

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primer

5’-TCTCTGGCTGAGGGATGACTTACCT-3’

(forward)

5’-TTAAGGCCAAGGAATTAAGTGACTG-3’ (reverse). Recombinant mutant of circWDR77 and FGF2 3’UTR of were also constructed. For luciferase reporter assays,

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HEK293T cells were co-transfected with luciferase reporter plasmids and miR-124 mimic using Lipofectamine 2000. Firefly and Renilla luciferase activities were

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detected using GloMax-Multi Jr Single Tube Multimode Reader (Promega, Madison, WI, USA). The firefly luciferase acted as an internal control.

2.8. RNA immunoprecipitation (RIP) assay

RIP assay was performed to validate the interaction within circWDR77 and

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miR-124 using EZMagna RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. VSMCs were lysed in RNA lysis buffer and incubated with the RIP buffer containing magnetic

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beads coated with anti-human argonaute 2 (Ago2) antibodies (Millipore). IgG (Millipore, USA) was used as a negative control (input). Incubated at 4°C for 2 hours,

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RT-PCR was performed to detect the enrichment of circWDR77 and miR-124.

2.9. Transwell migration assays Transwell migration assay was performed using 24-well Transwell chamber (8

µm pores, Corning Costar, Lowell, MA, USA). VSMCS were coated with 30 µl matrigel (BD Biosciences, San Jose, CA, USA) and seeded onto the upper chamber, and in lower chamber with 600 µl complete medium. With incubated at 37 °C for 24 h, the migrated cells were stained with 0.25% crystal violet, and the other non-migrated cells were removed with cotton swabs.

ACCEPTED MANUSCRIPT 2.10. Wound-healing assay When VSMCs were cultured at 90-95% confluency, 200-µl pipette tip was used to scratch the cell layer. Then, cells were washed with warm PBS and incubated at 37°C. At 0h and 48 h, the edges of scratch were photographed under a microscope

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with a digital camera system (Olympus, Tokyo, Japan). The experiments were performed three times independently

2.11. Statistical analysis

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All statistical analysis in this study was performed using SPSS 19.0 software (IBM, Chicago, IL, USA) and GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA,

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USA). Student t test and one way analysis of variance (ANOVA) were used. P value < was considered statistically significant.

3. Results

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3.1. CircRNA expression profiles in VSMCs

To screen the circRNA expression profiles in VSMCs, our team performed high glucose (27.5mM) and normal glucose (5.5mM) treatment for 48h to simulate diabetic

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condition in vitro, and utilized human circRNAs microarray analysis to express the differently expressed circRNAs. Box plot showed the normalized intensities from the normal glucose and high glucose concentration samples (Fig. 1A). Volcano plots and

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heat map showed the dysregulated circRNAs (Fig. 1B, 1C). After normalization, total 983 circRNAs were dysregulated, including 458 up-regulated and 525 down-regulated. Among these different expression circRNAs, 31 circRNAs were up-regulated and 22 circRNAs were down-regulated with 2 fold change (P<0.05). After comparison, we randomly chose 5 up-regulated circRNAs to verify using RT-PCR. Finally, we identified

one

of

the

significantly

up-regulated

circRNA

circWDR77

(hsa_circ_0013509, www.circbase.org/) as our target (Fig. 1D). Expression of circWDR77 in high glucose treatment VSMCs was significantly up-regulated compared to normal group.

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3.2. CircWDR77 silencing inhibited high glucose induced VSMCs proliferation and migration CircWDR77 was one of most upregulated circRNAs in high glucose induced

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VSMCs, suggesting the potential physiological role on VSMCs proliferation and migration. Specific interfering sequences were designed and synthesized to knock down circWDR77 expression (Fig. 2A). Transfected with si-circWDR77, expression of circWDR77 was significantly decreased in high glucose induced VSMCs (Fig. 2B).

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MTT assay showed that circWDR77 silencing could decrease the living VSMCs number compared to control group (Fig. 2C). Cycle analysis detected by flow

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cytometry showed that circWDR77 silencing induced G0/G1 phase arrest and suppressed cycle progression (Fig. 2D, 2E). Transwell migration (Fig. 2F, 2G) and wound healing assay (Fig. 2H, 2I) showed that circWDR77 silencing decreased the migrated cells and distance compared to control group. In summary, circWDR77

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silencing could inhibit the high glucose induced VSMCs proliferation and migration.

3.3. CircWDR77 was targeted by miR-124

In high glucose treated VSMCs, our study firstly discovered the circRNA

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expression profiles. What’s more, we found that circWDR77 silencing could inhibit the high glucose induced VSMCs proliferation and migration. Bioinformatics prediction analysis revealed that there was one binding sites within circWDR77 loop

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and miR-124 (Fig. 3A). Luciferase reporter assay showed that the luciferase vitality was decreased in the combination of circWDR77 wild type vector and miR-124 mimics (Fig. 3B). RIP assay showed that circWDR77 and miR-124 were significantly enriched in Ago2-containing beads compared to input group (Fig. 3C). Expression of miR-124 detected by RT-PCR showed that miR-124 was significantly down-regulated in VSMCs treated with high glucose compared to that in normal glucose (Fig. 3D). Therefore, results strongly indicated that circWDR77 was targeted by miR-124 and represented opposite expression, suggesting the molecular sponge role of circWDR77 on miR-124.

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3.4. CircWDR77/miR-124/FGF-2 regulatory pathway in VSMCs induced by high glucose It had been illustrated circWDR77 might act as miR-124 sponge in high glucose

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induced VSMCs. Meanwhile, with the help of bioinformatics analysis, we identified that fibroblast growth factor 2 (FGF-2) was a target gene for miR-124 (Fig. 4A). Luciferase reporter assay confirmed the binding of FGF2 and miR-124 (Fig. 4B). Expression of FGF2 mRNA and protein showed that circWDR77 silencing decreased

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FGF2 expression, which was reversed by miR-124 inhibitor (Fig. 4C, 4D). MTT assay showed the reversion of miR-124 towards circWDR77 on proliferation (Fig.

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4E). Cycle analysis revealed that miR-124 inhibitor recovered the G0/G1 phase arrest induced by circWDR77 silencing (Fig. 4F). Transwell and wound healing assay showed that miR-124 inhibitor rescued the migration inhibition caused by circWDR77 silencing (Fig. 4G, 4H). Overall, results indicating the molecular sponge role of circWDR77 on miR-124 via targeting FGF2, providing an effective

4. Discussion

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downstream regulation of circWDR77 on VSMCs proliferation and migration.

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In present study, our research team performed circRNAs microarray analysis to screen the circRNAs expression profiles in high glucose induced VSMCs in vitro, and identified the regulatory pathway of circWDR77-miR-124-FGF2 in VSMCs

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proliferation and migration. CircRNAs are a type of ncRNAs without 3’- and 5’- ends[11]. It has been firstly

found in plants for decades, however, the in-depth roles of circRNAs are just recognized in last several years[12]. With the development of high-throughput sequencing, massive amount of undiscovered and aberrantly expressed circRNAs have been gradually detected[13, 14]. For example, 575 circRNA species are identified in adult murine hearts and several circRNAs can be directly attributed to host genes that associated with cardiovascular disease, and further validation studies for these candidate circRNAs may reveal new crucial findings[15].

ACCEPTED MANUSCRIPT Our study identified the overexpression of circWDR77 (hsa_circ_0013509 with gene symbol WDR77) in high glucose induced VSMCs. In loss-of-functional validation assay, circWDR77 silencing induced by especial siRNAs targeting covalent closed junction could significantly suppress the proliferation and migration of VSMCs.

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This is the first time to uncover the differently expressed circRNAs in high glucose induced VSMCs, and confirmed the biological function of circWDR77. Thus, it could be conclude that microarray sequencing is am effective methods to discover potential functional ncRNAs in cell or tissue models. For example, expression of linear and

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novel circular forms of INK4/ARF-associated ncRNA reveals the circRNA cANRIL in atherosclerosis[16, 17].

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Because circRNAs is generated from linear RNA sequences, maybe from lncRNAs, some of the characteristic of circRNAs are similar with linear RNA[18]. The most remarkable feature of circRNAs is that it contains varying number of miRNA recognizing element (MRE)[19]. For example, circRNA Cdr1as harbors near 70 miR-7 binding sites, just like a huge circular miRNAs ‘sponge’[20]. In our study,

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we also find that circWDR77 sponges miR-124 to exert the function in VSMCs. Moreover, FGF2 is the target mRNA of miR-124 confirmed by luciferase reporter assay. Therefore, the integrated regulatory pathway of circWDR77-miR-124-FGF2 is

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validated.

Up to now, the roles of circRNAs on other cardiovascular fields have been reported. For instance, circRNA Cdr1as promotes myocardial infarction by regulating

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miR-7a and the target genes expression of miR-7[21]. CircRNA MFACR (mitochondrial fission and apoptosis-related circRNA) regulates mitochondrial fission and apoptosis in the heart by directly targeting and downregulating miR-652-3p by suppressing MTP18 translation[22]. Vascular smooth muscle cells (VSMCs) phenotypic switch is one of most crucial factor in the diabetes mellitus caused vascular disease. Proliferation and migration of VSMCs could accelerate the pathological process of atherosclerosis and hemadostenosis. The pathway of circWDR77-miR-124-FGF2 identified by our study could powerfully support the underlying role of circRNAs in VSMCs, even whole blood vessel, and open a new

ACCEPTED MANUSCRIPT door for diabetes mellitus correlated cardiovascular disease. Our research team reveals the expression profiles of circRNAs in high glucose induced

VSMCs

in

vitro,

and

furtherly

identify

the

role

of

circWDR77-miR-124-FGF2 regulatory pathway in VSMCs proliferation and

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migration. However, not only limited to the function of circWDR77, there are hundreds of differently expressed and unidentified circRNAs waiting for exploration. Further functional validation studies for these candidate circRNAs may reveal disease-relevant properties or functions, providing a novel insight and therapeutic

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methods for VSMCs.

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Conflict of interest All authors declare no conflicts of interest

Acknowledgement

This work was supported by Center Medical Laboratory of Shandong University

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and Shouguang People’s Hospital and Shandong Provincial Hospital Affiliated to Shandong University. Dr. Qing Ph.D helps the authors for manuscript polishing.

Reference:

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Figure legend: Figure 1. CircRNA expression profiles in normal glucose and high glucose treated

ACCEPTED MANUSCRIPT VSMCs. (A) Box plot showed the normalized intensities from the normal glucose and high glucose concentration samples. (B) Volcano plots showed the visualizing circRNAs different expression in normal glucose and high glucose treated VSMCs. (C) Heat map showed the dysregulated circRNAs. (D) Expression of circWDR77 in high

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glucose treatment VSMCs was significantly up-regulated compared to normal group. Data were expressed as mean ± SD. **P<0.01 represents statistically difference.

Figure 2. CircWDR77 silencing inhibited high glucose induced VSMCs proliferation and migration. (A) Graphical representation of specific interfering sequences that

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designed and synthesized to knock down circWDR77 expression. (B) Expression of circWDR77 in high glucose induced VSMCs transfected with si-circWDR77. (C)

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MTT assay showed the living VSMCs number in circWDR77 silencing group compared to control group. (D, E) Cycle analysis detected by flow cytometry showed the cells distribution in each cycle phase. (F, G) Transwell migration assay showed the migrated cells. (H, I) Wound healing assay showed the migrated distance. Data were expressed as mean ± SD. *P<0.05, **P<0.01 represents statistically difference.

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Figure 3. CircWDR77 was targeted by miR-124. (A) Schematic graphics for circWDR77 loop containing one binding sites of miR-124, and sequences for circWDR77 wild type, mutant and miR-124. (B) Luciferase reporter assay showed the

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luciferase vitality within circWDR77 wild type or mutant vector and miR-124 mimics or control. (C) RIP assay showed the enrichment of circWDR77 and miR-124 Ago2-containing beads. (D) Expression of miR-124 was detected by RT-PCR in

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VSMCs treated with high glucose and normal glucose. Data were expressed as mean ± SD. *P<0.05, **P<0.01 represents statistically difference. Figure 4. CircWDR77/miR-124/FGF-2 regulatory pathway in VSMCs induced by high glucose. (A) The putative binding sequence within FGF-2 and miR-124. (B) Luciferase reporter assay assessed the binding activity of FGF-2 3’-UTR correlated to miR-124. (C) Expression of FGF2 mRNA detected by RT-PCR. (D) Expression of FGF2 protein detected by western blot. (E) MTT assay showed the proliferation of living cells. (F) Cycle analysis detected by flow cytometry. (G) Migrated cells in fields detected by Transwell assay. (H) Wound healing assay showed the migrated

ACCEPTED MANUSCRIPT distance. Data were expressed as mean ± SD. *P<0.05, **P<0.01 represents statistically difference compared to control group. #P<0.05 represents statistically

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difference compared to si-circWDR77 group.

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Highlights

1. Our research team firstly screen the circular RNA expression profiles in high glucose

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induced VSMCs using circRNA microarray analysis.

2. CircWDR77 is identified to be up-regulated in high glucose induced VSMCs.

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4. CircWDR77 acts as an endogenous sponge of miR-124.

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3. CircWDR77 silencing suppresses the proliferation and migration of VSMCs.

5. CircWDR77-miR-124-FGF2 regulatory pathway play important role in VSMCs

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proliferation and migration.