N⁶-methyladenosine reader YTHDF1 regulates the proliferation and migration of airway smooth muscle cells through m⁶A/cyclin D1 in asthma

Asthmatic cellular model

As previously described (Ba et al., 2018), the primary cultured human ASM cells were obtained from 2nd–4th generation mainstem bronchi of patients undergoing lung resection surgery in accordance with procedures. Written informed consent was obtained from every human participant. The assay had been approved by the Ethics Committee of Shanxi Medical University (No. SXMU201905047). In brief, the mainstem bronchi segments were cut into pieces and ASMCs were isolated from it. After digestion, ASMCs were placed in DMEM medium containing 10% fetal bovine serum (Gibco, NY, USA) in humidified incubator at 5% CO₂ 37 °C with. Cells from passages 4–7 were used for following assays, ASMCs were treated with PDGF-BB (25 ng/ml) to mimic the asthma.

Transfection

For the transfection of YTHDF1, ASMCs were transfected with sequences following the manufacturer’s instructions. For silencing of YTHDF1, the shRNA sequences of YTHDF1 were synthesized by OBiOc (Shanghai, China) and the vectors containing shRNAs were inserted into PLKO.1. The transfection of plasmids was performed using the Lipofectamine 3000 kit (Invitrogen) according to the manufacturer’s instructions. For the overexpression of YTHDF1, the full-length cDNA sequences of human YTHDF1 (gene ID: 54915) were cloned into a pLentiEF1a-EGFP-Puro-CMV-MCS-3Flag lentivirus vector. The transfection efficiency was evaluated with qRT-PCR or western blot.

Reverse transcription quantitative polymerase chain reaction (RT- qPCR)

Total RNA was extracted according to the instruction of HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). Then, cDNA was synthesized using an PrimeScript RT reagent kit (Takara, Dalian, China). Real-time PCR was performed on the 7900 Real-time PCR System using Taqman RNA assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) acted as a control. The primer sequences were listed in Table S1. The results of transcript levels were analyzed by the 2^−ΔΔCt method.

Western blot analysis

Total protein in ASMC cells was extracted using radio immunoprecipitation assay (RIPA) lysis buffer with phenylmethanesulfonyl fluoride (PMSF) (Solarbio, Beijing, No. R0010). After incubation on ice and then concentration, the protein concentration was measured by bicinchoninic acid (BCA) kit and adjusted by deionized water. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% SDS-PAGE) and transferred to polyvinylidene fluoride membranes (PVDF) (Millipore, Billerica, MA, USA). The transferred PVDF membranes (No. ISEQ00010; Millipore, Billerica, MA, USA) was added with Tris-buffered saline tween (TBST) containing 5% dried dskimmed milk. Then, PVDF membranes were incubated with the primary antibodies (anti-Cyclin D1, ab16663; Abcam; anti-YTHDF1, 1:1000, ab220162; Abcam). Beta-actin (1:1,000; Abcam) was used as an internal control. After washing with phosphate buffer five times containing Tween-20 (PBST), PVDF membranes were incubated for 1 h at room temperature. Finally, pierce ECL western blotting substrate was employed to develop the protein bands and quantification was conducted by Image Lab software (Bio-Rad, Hercules, CA, USA).

Proliferation assays and cycle analysis

The proliferation of ASMCs was detected using CCK-8 assays using Cell counting kit-8 (8 µl of CCK-8; Dojindo, Kumamoto, Japan) with 100 µl serum free medium and incubated for 90 min. In brief, the transfected ASMCs (5 ×10³ cells/well) was inoculated at into 96-well plates at 24, 48, and 72 h of culture at 37 °C overnight. Eventually, the absorbance at 450 nm was detected by a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). For the cycle analysis, Coulter EPICS XL flow cytometer (Beckman Coulter, Inc., Fullerton, CA, USA) was performed by flow cytometry on flow cytometry with Modifit software (BD Biosciences).

Transwell migration assays and wound healing assay

The migration of ASMCs was detected by transwell assays. In brief, the transfected ASMCs (1 ×10⁵ cells/well) were suspended in serum-free medium upper transwell chamber (pore size: 8 µm; Corning, Inc., Corning, NY, USA). In the bottom chamber, medium was supplemented with 600 µl of 10% FBS. After being incubated for 24 h at 37 °C, the cells in the top compartment were wiped off by cotton swabs and the migrated cells were fixed for 20 min with 4% paraformaldehyde and stained for 10 min with 0.1% crystal violet staining solution (Sigma-Aldrich, Louis, MO, USA). Lastly, images were taken under the optical microscope (Olympus, Tokyo, Japan). For the wound healing assay, the monolayer was manually scraped by sterile pipette tip. After being 24 h incubation at 37 °C, the Images of wound closure were evaluated by inverted microscope (Olympus, Tokyo, Japan). The migration rate was calculated by the formula: migration rate = migration distance/original distance.

RNA immunoprecipitation (RIP)-PCR

The interaction within RNA binding proteins and mRNA was identified using RIP-PCR. In brief, the RIP experiment was carried out by EZ-Magna RIP Kit (Millipore) according to the manufacturer’s protocol. ASMCs were lysed in complete RIP lysis buffer, and the cell extract was incubated with protein A/G agarose beads conjugated by anti-YTHDF1 (ab220162, 1:30; Abcam) or control IgG (ab172730; Abcam) for 2 h at 4 °C. After being washed, beads were incubated with Proteinase K to remove protein in complex. Lastly, the purified RNAs were subjected to qRT-PCR analysis.

RNA stability assay

To detect the cyclin D1 mRNA stability, the ASMCs were treated with actinomycin (Act D, 2 µg/mL) treatment for 0, 3 and 6 h. The relative remaining RNA level was detected by qRT-PCR and the half-life of cyclin D1 mRNA was examined by transcript levels at indicated time points relative to those before Act D treatment.

Luciferase reporter assay

The wild-type or mutant Cyclin D1 3′-UTR was synthesized and inserted into pmirGLO reporter vector (Promega, Madison, WI, USA), and then co-transfected with WT- Cyclin D1 or Mut- Cyclin D1 pcDNA 3.0 expressing plasmid into cells using Lipofectamine 3000. Cells were transfected with pGL3-Luc (1 µg) as a control for transfection efficiency (Promega), according to the manufacturer’s instructions. The luciferase activity was tested using a luciferase reporter commercial kit (Promega, Madison, WI, USA).

Statistical analysis

Experiments were repeated three times and data was presented as means ± standard deviation (SD) in this study. Variables between was compared by student’s t-test and

Statistical analyses were performed using the SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 8.0 software (GraphPad, San Diego, CA, USA). A two-sided p-value of less than 0.05 was considered statistically significant.

YTHDF1 was up-regulated in the PDGF-induced ASMCs

To mimic the cellular asthma model, PDGF-induced ASMCs and blank control cells were constructed. Firstly, we found that YTHDF1 mRNA was up-regulated in the PDGF-induced ASMCs (Fig. 1A). Besides, the YTHDF1 protein level also up-regulated in the PDGF-induced ASMCs (Fig. 1B). Moreover, we found cyclin D1 (CCND1), an essential cell cycle control gene closely correlated to the development of asthma (Thun et al., 2013), was up-regulated in the PDGF-induced ASMCs (Fig. 1C). Besides, the cyclin D1 protein level also up-regulated in the PDGF-induced ASMCs (Fig. 1D). Overall, these findings revealed that YTHDF1 was up-regulated in the PDGF-induced ASMCs.

YTHDF1 positively regulated the proliferation and migration of ASMCs

The bio-functional roles of YTHDF1 were explored in the PDGF-induced ASMCs with YTHDF1 knockdown and overexpression. The transfection efficiency was detected using RT-PCR (Fig. 2A) and western blot (Fig. 2B). Proliferation analysis by CCK-8 assay unveiled that YTHDF1 silencing repressed the proliferative ability of ASMCs, while the YTHDF1 enforced overexpression up-regulated the proliferative ability (Fig. 2C). Furthermore, the migrative ability of ASMCs was determined by transwell assay, and results illustrated that YTHDF1 silencing repressed the migrative ability of ASMCs, while the YTHDF1 enforced overexpression up-regulated the migrative ability (Figs. 2D, 2E). Overall, these findings revealed that YTHDF1 positively regulated the proliferation and migration of ASMCs.

YTHDF1 positively facilitated the cell cycle progression and migration

To detect the role of YTHDF1 on PDGF-induced ASMCs, flow cytometry cell cycle analysis was performed. The results showed that YTHDF1 knockdown induced the cycle arrest at G1/S phase, while YTHDF1 overexpression promoted the cycle progression of ASMCs (Fig. 3A). Furthermore, the migrative ability of ASMCs was determined by wound healing assay, and results illustrated that YTHDF1 knockdown repressed the migrative ability of ASMCs, while the YTHDF1 overexpression up-regulated the migrative ability (Fig. 3B). Therefore, these data showed that YTHDF1 positively facilitated the cell cycle progression and migration.

Cyclin D1 acted as the target of YTHDF1

To discovery the potential downstream target of YTHDF1 in asthma, we utilized the predictive tool (SRAMP, http://www.cuilab.cn/sramp) to analyze the m⁶A site in these targets (Fig. 4A). Moreover, we found that the exact site of m⁶A modification (ATGGAC) on the Cyclin D1 gene (Fig. 4B). The m⁶A motif on the Cyclin D1 mRNA was predicted (https://rna.sysu.edu.cn/rmbase/) (Fig. 4C). The molecular interaction within Cyclin D1 and YTHDF1 was determined by RIP-PCR, and results indicated that YTHDF1 closely combined with Cyclin D1 (Fig. 4D). Moreover, further RIP-PCR analysis found that YTHDF1 silencing repressed the combination within Cyclin D1 and YTHDF1, and YTHDF1 overexpression enhanced the combination (Fig. 4E). Taken together, these findings revealed that Cyclin D1 acted as the target of YTHDF1.

YTHDF1 enhanced the RNA stability of cyclin D1 mRNA via m6A-dependent manner

Previous researches had revealed that YTHDF1 could recognize the m⁶A site on mRNA and then enhance its stability (Chen et al., 2021; Xu et al., 2021). In our study, we found that YTHDF1 might also target cyclin D1 mRNA to enhance its stability. Firstly, we detected the cyclin D1 mRNA level in ASMCs with YTHDF1 silencing or overexpression, and results showed that YTHDF1 silencing or overexpression didn’t significantly regulate the cyclin D1 mRNA (Figs. 5A, 5B). RNA stability analysis revealed that YTHDF1 silencing reduced the half life time (t_1/2) of cyclin D1 mRNA upon Act D treatment, while YTHDF1 overexpression up-regulated the half life time (t_1/2) of cyclin D1 mRNA (Figs. 5C, 5D). Luciferase reporter assay using wild-type (WT) cyclin D1 3′UTR or mutant (Mut) was performed and results indicated that YTHDF1 accelerated the luciferase activity within cyclin D1 wild type (Figs. 5E, 5F). In conclusion, we found that YTHDF1 enhanced the RNA stability of cyclin D1 mRNA via m⁶A-dependent manner (Fig. 6).