N6-methyladenosine METTL3-induced circMYO1C accelerates the pancreatic ductal adenocarcinoma tumorigenesis through IGF2BP2/VEGFA manner

1. Introduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors with high mortality and tens of thousands of newly diagnosed cases are reported by global statistics^{1, 2}. Worldwide, the widespread application of screening has reduced the global morbidity and mortality associated with PDAC³. Although great progresses have done in PDAC treatment, the five-year survivals are still fabulously less than 6%^{4, 5}. Until now, the molecular mechanism underlying the progression of PDAC is still unclear, thus it would be highly ponderable for the identification of early diagnosis and novel therapeutic targets establishment.

Circular RNAs (circRNAs) is newly identified special class of non-coding RNA (ncRNA) that plays critical regulatory roles in the progression of cancer^{6, 7}. In the tumorigenesis of PDAC, it has been demonstrated that circRNAs regulate the pathophysiological process through diverse molecular mechanism. For instance, circRNA circCUL2 specifically expresses in cancer-associated fibroblasts and is significantly correlated with the PDAC patients’ poor prognosis. Mechanistically, circCUL2 modulates the miR-203a-3p/MyD88/NF-κB/IL6 axis⁸. Thus, we could conclude that circRNAs significantly regulate the PDAC progression, however, the relationship between the oncogenic role of the circRNA and pathogenetic mechanism remains unclear.

N⁶-methyladenosine (m⁶A) is the most abundant internal modification of RNAs, including coding and non-coding RNA transcripts. Physiologically, m⁶A plays great moderating impact on RNA transcripts’ dynamic regulation, referring to splicing, transcribing and translation. M⁶A modification is deposited by the methyltransferase complex composed by methyltransferase-like-3 (METTL3), METTL14, and WTAP. Inversely, the m⁶A modification is uninstalled by m⁶A demethylases, mainly consisting by FTO and ALKBH5. Existing evidence has indicated that m⁶A modification wildly regulated the PDAC progression. For instance, ALKBH5 overexpresses in pancreatic cancer cell line and regulates RNA stabilities of LC25A28 and SLC25A37 through modulating regulators of iron metabolism and underscore the multifaceted role of m6A in pancreatic cancer⁹. Moreover, m⁶A regulator HNRNPC knockdown significantly reduces PDAC cell invasion in vitro and metastasis in vivo, while, HNRNPC overexpression provokes malignant phenotypes of PDAC cells¹⁰. Although a number of literatures have reported on m⁶A, the profound mechanisms involved the posttranscriptional RNA modification are elusive.

Given that circRNAs are a group of specific back-spliced RNA characterized by circular construction, the m⁶A modification could deservedly occur on the circRNAs. Previous research revealed that thousands of m⁶A-circRNAs were screened by high-throughput sequencing with cell-type-specific expression¹¹. Subsequent studies further demonstrated that m⁶A-modified circRNAs regulate the cancer progression, including liver cancer¹² and non-small cell lung cancer¹³. However, the role of m⁶A on the biogenesis and function of circular RNAs (circRNAs) in PDAC has yet to be addressed. Here, our research found that numerous circRNAs were dysregulated upon METTL3 overexpressed and finally identified a novel circRNA circMYO1C (hsa_circ_0041234, 186 bp) in PDAC. Subsequently, our research performed functional assays to test the role of circMYO1C on PDAC progression and further explore the potential mechanism by which m⁶A modification altered circMYO1C fate.

2. Materials And Methods

2.1. Tissue samples collection

Total sixty fresh frozen PDAC tissues and paired normal adjacent tissues were collected from surgery at University of Electronic Science and Technology of China. The clinical parameters of the included patients were shown in Table 1. None of patients received any chemoradiotherapy before operation. The study had been approved by the Ethics Review Board of University of Electronic Science and Technology of China. All patients were duly informed before the samples collection, and written informed consent was received from each patient.

Table 1

The correlations within circMYO1C levels and PDAC patients’ clinicopathological characteristics

		circMYO1C		p
		Low (30)	High (30)	p
Age
<60 year	25	14	11	0.432
≥60 year	35	16	19
Gender
Male	34	18	16	0.794
Female	26	12	14
TNM stage
I-II	23	17	6	0.003
III-IV	37	13	24
Differentiation
Well	11	5	6	0.942
Moderate	37	19	18
Poor	12	6	6
Lymph node metastasis
No	28	18	10	0.038
Yes	32	12	20

2.2. Cell culture, vector construction and cell transfection

PDAC cells (PANC-1, Capan-2) and normal human pancreatic ductal epithelial cell line (HPDE6) were obtained from the American Type Culture Collection (ATPDAC, Manassas, VA, USA) and Chinese Academy of Sciences Cell Bank (Shanghai, China). Cells were cultured with were cultured in DMEM medium (Gibco, USA) supplemented with 10% of fetal bovine serum (Gibco), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco). Cells were cultured in 5% CO₂ incubator (Thermo Fisher, USA) with the humidified environment at 37 °C. For the overexpression of circMYO1C, the full length sequences were amplified and then cloned to pCD5-ciR vector (Greenseed Biotech, Guangzhou, China). For the silencing of circMYO1C, the lentiviral based specific short-hairpin RNA (shRNA) targeting circMYO1C were designed and synthesized by GenePharm (Shanghai, China). The transfection of plasmids was performed using the Lipofectamine 3000 kit (Invitrogen) according to the manufacturer’s instructions.

2.3. High-throughput circRNA microarray

To investigate the circRNA expression profile upon METTL3 overexpression, circRNA microarray (ArrayStar, Aksomics) were performed according to the instruction. Total RNA was extracted from the PANC-1 cells transfected with METTL3 overexpression plasmids or controls. The RNA was digested with RNase R to remove linear RNAs. Enriched circRNA was amplified and labeled with Arraystar Super RNA Labeling Kit (Arraystar, Rockville, the USA). The labeled cRNA was hybridized onto Arraystar Human circRNA Array (8*15 K, ArrayStar, Aksomics). CircRNAs with 2-fold changes and p values <0.05 were regarded as significantly different.

2.4. Quantitative real-time polymerase chain reaction (RT-qPCR)

For PCR of circRNA and mRNA, RNA was extracted from PDAC cells and reversely transcribed using Transcriptor First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN). Quantitative PCR was performed using YBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). The relative expression levels were calculated using the 2⁻^△△^CT method. The relative level was normalized by GAPDH. Primers were listed in Table S1.

2.5. Actinomycin D assays and RNase R treatment

PDAC cells were seeded in 6-well plates (1×10⁵ cells per well) for 24-four hours. Then, cells were exposed to Actinomycin D (Act D, 2 μg/ml, Sigma) and collected at indicated time points. RNA stability was identified by analyzing the remaining level using qRT-PCR normalized to blank control. For the RNase R treatment, total RNA (2 μg) was incubated with 5 U/μg RNase R (5 U/μg, Epicentre Technologies) and linear RNA or circular RNA transcripts were subsequently analyzed by qRT-PCR.

2.6. Proliferative assays

Proliferative assays were performed to detect the proliferation ability of PDAC cells, including CCK-8 and Ethynyl-2-deoxyuridine (EdU) assays. For CCK-8, PDAC cells were seeded in 96-well plates. Then, 24 hours later, cells were administrated with 10 μl of CCK-8 assay kit (Dojindo Japan). The absorbance was measured at 450 nm at indicated time. For EdU, cells were cultured in 24-well plates and 10 mM EdU was added to each well. After being fixed with 4% formaldehyde and washing, cells were stained with DAPI and visualized using fluorescent microscope.

2.7. Migration assays

Migration assays were performed to detect the migration of PDAC cells, including wound healing assay and transwell assay. In brief, for wound healing assay, PDAC cells were seeded in 6-well plates at 90% conﬂuence. Cell monolayers were manually wounded by 200 ul pipette tip scraping. After 48 h culture, the migration rate was quantified with measurements of the distance by the following formula: migration distance/original distance. For transwell migration assay, the transfected cells were suspended in serum-free medium and seeded into the upper chambers of transwell (8 mm pore size). While, full medium with 20% FBS was added to the lower chambers. After incubation, cells on the top chamber were removed and invaded cells on the lower surface were fixed with methanol and stained with 0.1% crystal violet. Photographs were captured by microscope (Olympus).

2.8. MeRIP-qPCR

Total RNA was extracted from PDAC cells using Trizol (Thermo Fisher) and RNA (100 μg) was subjected to 500 μl MeRIP buffer (500 μl). RNA was incubated with anti-N⁶-methyladenosine antibody (ab151230) and rabbit IgG (1 μl) to pull down m⁶A modified circMYO1C. Lastly, the m6A-bound RNA was identified by RT-qPCR.

2.9. RNA immunoprecipitation (RIP)

RIP assay was performed to identify the molecular interaction as previously described¹⁴. In brief, cells were lysed in complete RIP lysis buffer (Magna RIP Kit, Millipore, MA), and the isolated RNAs were fragmented by sonication and immunoprecipitated with protein A/G magnetic beads conjugated with specific antibodies (anti-METTL3, no. ab195352, Abcam; IGF2BP2, no. 11601–1-AP, Proteintech; anti-Flag, no. 8146, Cell Signaling) or control IgG in RIP Immunoprecipitation buffer (Magna RIP Kit, Millipore, MA) for 2h at 4 °C. Beads were washed and incubated with Proteinase K to remove proteins. RNAs was extracted and subjected to qRT-PCR using primersand normalizing to input.

2.11. RNA pull-down assay

The pull-down assay was performed as previously described¹⁵. In brief, biotinylated circMYO1C probe and oligo probe were designed and synthesized by Genepharm (Shanghai, China). Probes (3 μg) were incubated with Streptavidin magnetic beads (50 μL, Invitrogen) at room temperature for 2 h to generate probe-coated beads. RIP buffer supplemented with cocktail and agarose beads were used to lyse the cells. Then, lysate was incubated by biotinylated probe with streptavidin-coated magnetic beads for 6 h at 4 °C. Finally, the pulled-down protein or RNA was collected and then detected by western blot or qPCR.

2.12. RNA fluorescence in situ hybridization (RNA-FISH)

FISH was performed according to the manufacturer's instructions. In brief, FAM-labeled circMYO1C probe, cy3-labeled probe IGF2BP2 probe and DAPI-labeled U6 probes were synthesized by GenePharm (Shanghai, China). In brief, PDAC cells (Capan-2) were fixed and washed in PBS and the suspension was pipetted onto glass slides. After dehydration with ethanol, the hybridization was performed in dark at 37 °C overnight. After being washed twice, the RNA FISH was performed using fluorescent in situ hybridization kit Genepharma (Shanghai, China) according to the manufacturer’s protocol. The images were acquired using a confocal microscope (Olympus).

2.13. Xenograft in vivo mice assay

Male BALB/c nude mice (5-6 weeks) were obtained from Slac Laboratory Animal Center (Shanghai, China) and maintained under pathogen-free condition. PANC-1 cells (2×10⁶ cells suspended in 100 μl PBS) transfected with circMYO1C knockdown (sh-circMYO1C) or controls (sh-NC) were subcutaneously injected into the flank of nude mice. One week later, the tumour size was measured every three days. All procedures were in accordance with the ethical standards and the care of animal and licensing guidelines. The assay had been approved by the Ethics Committee of University of Electronic Science and Technology of China.

2.14. Statistical analysis

Data were analyzed using GraphPad Prism 8.0 (GraphPad, San Diego, CA, USA) and SPSS 19.0 (Chicago, IL, USA), which were expressed as mean ± SD (standard deviation). The statistical approach comprised Student's t-test for two independent groups and one-way analysis of variance (ANOVA) for multiple group comparisons. Correlation analysis was analyzed by Pearson's correlation coefficient and two-tailed p value. P<0.05 was considered significant. All experiments were performed in triplicate.

3. Results

3.1. circMYO1C was a METTL3-induced circRNA and up-regulated in PDAC

To investigate the potential circRNAs regulated by m⁶A modification, we chose METTL3, a crucial m⁶A methyltransferase, to construct the overexpression of METTL3 and its control in PANC-1 cells. CircRNA microarray analysis revealed that numerous circRNAs were dys-regulated and we focused on an up-regulated circRNA (circMYO1C). CircMYO1C was a 186 bp length transcripts derived from the exon9-exon8 of MYO1C gene. Its ID was hsa_circ_0041234 (circBase) and hsa_circRNA_101936 (ArrayStar, Aksomics) (Figure 1A). Moreover, several candidate circRNAs were quantificationally validated by RT-PCR, and results demonstrated that circMYO1C showed a higher expression as compared to control group (Figure 1B). CircMYO1C was an exonic circRNA spliced from MYO1C gene exon9-exon8 via back splicing, thus named as circMYO1C, which was confirmed by Sanger sequencing (Figure 1C). To detect the stability of circMYO1C, PANC-1 cells were treated with RNase R (RNA synthesis inhibitor) and actinomycin D (DNA repair inhibitor). The half-life time of circMYO1C was longer than MYO1C mRNA (Figure 1D). qRT-PCR results showed that circMYO1C was more capable of resistance to RNase R digestion (Figure 1E). RNA fluorescence in situ hybridization (RNA-FISH) displayed that the circMYO1C distributed in the cytoplasm of PDAC cells (Figure 1F). In the clinical specimens of PDAC patients, quantitative analysis found that the expression of circMYO1C up-regulated in PDAC as compared to normal controls (Figure 1G). Taken together, these findings indicated that circMYO1C was a METTL3-induced circRNA and up-regulated in PDAC.

3.2. circMYO1C promoted the proliferation and migration of PDAC cells

In PDAC cells, we found that the circMYO1C expression was up-regulated as compared to normal cells (Figure 2A). To investigate the functions of circMYO1C on PDAC cells, the enforced overexpression and silencing of circMYO1C were respectively transfected into Capan-2 cells (vector, circMYO1C overexpression) and PANC-1 cells (sh-NC, sh-circMYO1C) (Figure 2B). CCK-8 proliferation assay indicated that circMYO1C overexpression promoted the proliferative ability of PDAC cells and circMYO1C silencing repressed the proliferation (Figure 2C). Migration assay indicated that circMYO1C overexpression promoted the migrative ability of PDAC cells and circMYO1C silencing repressed the migration (Figure 2D). Wound healing assay unveiled that enhanced ircMYO1C accelerated the migrative ability, while the knockdown of circMYO1C inhibited the migration (Figure 2E). Ethynyl-2-deoxyuridine (EdU) incorporation assay illustrated that circMYO1C overexpression promoted the DNA synthesis, while the knockdown of circMYO1C repressed the DNA synthesis (Figure 2F). Overall, these data demonstrated that circMYO1C promoted the proliferation and migration of PDAC cells.

3.3. METTL3 induced the expression of circMYO1C via m⁶A-modified manner

Latest research shows that METTL3 could regulate the biogenesis of circRNAs, thus we investigate the interaction within METTL3 and circRNAs. Given that circMYO1C was up-regulated in the sequencing upon METTL3 overexpression, we proposed a hypothesis that METTL3 induced the circularization of circMYO1C. Firstly, the up-regulated or down-regulated METTL3 construction was performed, whose transfection efficiency was identified by western blot (Figure 3A). Then, RT-qPCR analysis fund that circMYO1C level was upregulated upon METTL3 overexpression transfection, while circMYO1C level was repressed upon METTL3 silencing (Figure 3B). MeRIP-qPCR analysis found that the m⁶A-modified enrichment was higher in the PDAC cells (Capan-2, PANC-1) (Figure 3C). RNA pull-down following western blot assays further verified that METTL3 could interact with circMYO1C in Capan-2 cells (Figure 3D). Overall, these findings unveiled METTL3 induced the expression of circMYO1C via m⁶A-modified manner.

3.4. circMYO1C promoted the VEGFA mRNA stability

MeRIP-Seq revealed that several candidate genes (MYC, VEGFA, HOXA10, Notch1) demonstrated the m⁶A modification in their genomic location (Figure 4A). Then, RT-qPCR analysis revealed that circMYO1C overexpression significantly up-regulated the VEGFA mRNA level, while circMYO1C knockdown reduced the expression of VEGFA mRNA (Figure 4B, 4C). Meanwhile, the other candidates showed no significant change. Moreover, RNA stability assay using Act D administration showed that circMYO1C overexpression remarkably increased the VEGFA mRNA stability and circMYO1C knockdown repressed the VEGFA mRNA stability (Figure 4D). Taken together, these findings found that VEGFA exhibited the m⁶A modification and circMYO1C promoted the VEGFA mRNA stability.

3.5. circMYO1C interacted with VEGFA through m⁶A reader IGF2BP2

Previous research has reported that circRNA could regulate its target mRNA stability, which is mediated by m⁶A reader IGF2BP2¹⁶. For the further mechanism by which circMYO1C regulates the stability of VEGFA mRNA, our research put forward a hypothesis that circMYO1C might interact with VEGFA through m⁶A reader IGF2BP2. Analytical investigation revealed that IGF2BP2 shared the potential m⁶A modified sites with both circMYO1C junction sites and VEGFA 3’-UTR (Figure 5A). RNA binding protein immunoprecipitation (RIP) analysis proved that, comparing with the control IgG, circMYO1C expression was enriched in the anti-IGF2BP2 antibody precipitation (Figure 5B). RNA pull-down assay using circMYO1C probe confirmed that circMYO1C specifically combined with VEGFA (Figure 5C). As we known, IGF2BP2 possesses 2 RNA-recognition-motif (RRM) domains and 4 K homology (KH) domains^{17, 18}. Thus, the next work for our team was to explore which domain might combine with circMYO1C. FLAG tagged full-length and truncated IGF2BP3 mutants were constructed (Figure 5D). RIP analysis proved KH3-KH4 domains of IGF2BP2 specifically interacted with circMYO1C, which was required for its interaction with circMYO1C and VEGFA mRNA (Figure 5E). RIP analysis demonstrated that circMYO1C overexpression enhanced the IGF2BP2-VEGFA interaction, while circMYO1C knockdown reduced the protein-RNA interaction (Figure 5F). By performing RNA fluorescence in situ hybridization (RNA-FISH) assays, we confirmed the colocalization of endogenously expressed circMYO1C and VEGFA in the cytoplasm (Figure 5G). Overall, these findings unveiled that circMYO1C interacted with VEGFA through m⁶A reader IGF2BP2.

3.6. circMYO1C knockdown repressed the tumor growth in vivo

To investigate the role of circMYO1C in vivo, the xenograft in vivo mice assay was performed. Results indicated that circMYO1C knockdown reduced the tumor weight (Figure 6A) and volume (Figure 6B). Immumohistochemical staining (IHC) showed that the VEGFA protein was decreased upon circMYO1C knockdown (Figure 6C). Bioluminescence in vivo imaging showed that circMYO1C knockdown repressed the tumor metastasis (Figure 6D). Overall, these finding illustrated that circMYO1C knockdown repressed the tumor growth in vivo.

4. Discussion

Emerging evidence has indicated the critical role of epigenetic modification on PDAC tumorigenesis^{19, 20}. As an important branch of epigenetics regulation, circRNAs play essential roles covering series of areas, including energy metabolism, proliferation and metastasis. Moreover, N⁶-methyladenosine (m⁶A) mRNA modifications regulate the PDAC phenotypic characteristic through controlling RNA fate. On the other hand, as a kind of RNA, circRNA itself is also regulated by m⁶A. Here, our present study preliminarily investigates the function of circRNA circMYO1C and m⁶A, and further unveils their associated mechanism in PDAC.

Although a huge number of circRNAs have been reported and identified to availably modulate the PDAC tumour progression, these circRNAs were only significantly overexpressed/downexpressed in PDAC and did not show association with specific tumor features²¹. The circRNAs with remarkable overexpression or down-expression are usually screened by high-throughput sequencing, and the inclusion criteria for study is their differential expression levels. Although we all know the fact that higher/lower circRNAs could regulate PDAC, the molecular functions of circRNAs appear to be rather diverse and are in most cases only poorly understood.

Our research didn't just look at this traditional area that circRNA modified PDAC via miRNAs sponges, moreover, we turned the focus of research into its biogenesis. We found that m⁶A methyltransferase METTL3 could interact with circMYO1C via a m⁶A-dependent manner. More importantly, METTL3 increased the enrichment of circMYO1C. These interesting finding give us an important insight that the investigation about circRNA biogenesis could provide additional knowledge regarding PDAC tumorigenesis rather than downstream cascade reaction.

To date, emerging novel findings about m⁶A and PDAC have been authenticated by researchers. Given the identified role of METTL3 in PDAC, we relate the METTL3 functions with circRNA biogenesis to explore the possibility of m⁶A-circRNA interaction. To test the interaction within METTL3 and circRNAs, we performed the circRNA screening upon METTL3 overexpression. Results found that numerous circRNAs were screened out, and we focused on a novel circRNA circMYO1C. Functional experiments revealed that circMYO1C promoted the proliferation and migration of PDAC cells, indicating the oncogenic roles of circMYO1C.

During the progression of splicing reaction, pre-mRNAs could produce covalently closed circRNAs. The highlight that attracted us most was the cyclization-promoting of METTL3 for circMYO1C. Given that circMYO1C was a METTL3-releated circRNA with m⁶A modification site, we aimed to investigate the potential role of METTL3 on circMYO1C back splicing. Results indicated that METTL3 overexpression indeed up-regulated the level of circMYO1C, suggesting the cyclization-promoting role of METTL3 for circMYO1C. Moreover, MeRIP-Seq revealed that there was remarkable m⁶A modified site on the 3’-UTR of VEGFA and circMYO1C enhanced the stability of VEGFA mRNA. Further research illustrated that circMYO1C interacted with VEGFA through m⁶A reader IGF2BP2, and circMYO1C specifically interacted with the KH3-KH4 domains of IGF2BP to interact with VEGFA mRNA.

The latest researches about m⁶A and circRNA have partially revealed the interesting filed²². Dan Xie¹⁶ (2019) reported that a m⁶A-modified circRNA circNSUN2 frequently upregulated in tumor tissues from colorectal carcinoma patients with liver metastasis. The m⁶A modification of circNSUN2 promotes its export to the cytoplasm mediated by YTHDC1. Moreover, m⁶A-modified circNSUN2 enhances the stability of HMGA2 mRNA through m⁶A site interaction, forming circNSUN2/IGF2BP2/HMGA2 RNA-protein ternary complex. Besides, a novel METTL3-induced circRNA, circ1662, exhibits significant higher-expression in colorectal carcinoma and circ1662 promotes cell invasion and migration in vitro and in vivo. Mechanistically, METTL3 binds the flanking sequences of circ1662 and installing the m⁶A modification, thereby inducing circ1662 biogenesis²³. Thus, the evidence indicates that m⁶A modification of circRNA could effectively regulate the pathophysiological process, suggesting the novel m⁶A-circRNA manner.

In conclusion, our findings indicated that circMYO1C significantly upregulated in PDAC tissues and acted as oncogenic factor for PDAC. These results suggest that circMYO1C/IGF2BP2/VEGFA form an RNA-protein complex to accelerates the proliferation and migration of PDAC cells (Fig. 7). Our findings provide additional evidence for the m⁶A-circRNA regulation in the PDAC tumorigenesis, providing a novel insight for the circRNA biological effect.

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