Collection of human HSCR specimens
Stenotic segments (HSCR-S) and dilated segments (HSCR-D) were obtained from the intestines of patients with HSCR who underwent surgery at Zhujiang Hospital of Southern Medical University in Guangdong province from 2020 to 2021. In all cases, HSCR diagnosis was confirmed through tissue biopsy. Study approval was obtained from the Research Ethics Committee of Zhujiang Hospital and all the patients’ guardians provided informed consent. All intestinal tissue samples were stored at − 80°C.
Quantification Of Total Ma Rna Modification
Total m6A levels were determined in tissues and cell lines (200 ng of RNA from each) using the EpiQuik m6A RNA Methylation Quantification Kit (Colorimetric) (EPIGENTEK, Farmingdale, NY, USA) following the manufacturer’s instructions.
Cell Lines And Culture
The SH-SY5Y human neuroblastoma cell line (National Collection of Authenticated Cell Culture, Shanghai, China) and the human embryonic kidney 293T (HEK-293T) cell line (Procell, Wuhan, China) were cultured in high-glucose DMEM (Gibco, New York, NY, USA) supplemented with 10% FBS (Procell, Wuhan, China) and penicillin–streptomycin (Gibco) at 37℃ in a humidified incubator with 5% CO2.
Plasmid Construction
HA-tagged METTL3 (NM_019852) and Flag-tagged YAP (NM_001130145) constructs were generated by inserting the respective sequences into the pcDNA 3.1 vector (HanYi Biosiciences Inc, Guangzhou, China). Sequences encoding the HA epitope (YPYDVPDYA) and Flag epitope (DYKDHDGDYKDHDIDYKDDDDK) were added to the respective cDNA clones by PCR.
Gene Overexpression And Knockdown
Overexpression plasmids or siRNAs were transfected into cells using GP-Transfect-Mate Reagent (GenePharma, Shanghai, China) following the manufacturer’s protocol and incubated for 24–48 h. The sequences selected for knockdown were as follows (5′–3′): siRNA-METTL3, CCUGCAAGUAUGUUCACUATT (forward) and UAGUGAACAUACUUGCAGGTT (reverse); siRNA-YAP, CUGCCACCAAGCUAGAUAATT (forward) and UUAUCUAGCUUGGUGGCAGTT (reverse).
Cck-8 And Transwell Assays
Cell growth and viability were analyzed in 96-well plates using CCK-8 assays. For this, cells were transfected with the relevant overexpression plasmids or siRNA, cultured for 24 h, and then incubated with 10 µL of CCK-8 reagent for 50 min at 37°C in a humidified incubator with 5% CO2. Absorbance was detected at 450 nm using a spectrophotometer.
For cell migration assays, approximately 5×103 SH-SY5Y cells or 5×104 HEK-293T cells were suspended in 300 µL of serum-free DMEM and transferred to the upper chamber of a Transwell plate (Corning, New York, USA) with an 8-µm-pore-size polycarbonate membrane. A total of 400 µL of complete culture medium with 10% FBS was added to the lower chamber. After culturing for 48 h, unmigrated cells in the upper chamber were removed with a cotton swab, while the migrated cells were fixed in 4% paraformaldehyde for 30 min, stained with crystal violet, and quantified under a microscope.
Rna Isolation And Reverse Transcription-quantitative Pcr (Rt-qpcr)
Total RNA was isolated from tissue samples and cells with TRIzol reagent and reverse transcribed into cDNA using the Evo-M-MLV RT Kit with gDNA Clean for qPCR II (Accurate Biology, Hunan, China). qPCR assays were performed using a SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Hunan, China) with the human GAPDH gene serving as the internal control. The qPCR amplification program consisted of denaturation at 95℃ for 30 s, followed by 40 cycles of denaturation at 95℃ for 5 s, annealing and extension at 60℃ for 30 s. The relative expression levels of target genes were detected and analyzed using Bio-Rad CFX96 Touch (BIO-RAD, California, CA, USA). The primers used for qPCR are shown in Table 1.
Table 1
Name | Sequence (5′–3′) |
METTL3 Forward Reverse | CAAGCTGCACTTCAGACGAA GCTTGGCGTGTGGTCTTT |
METTL14 Forward Reverse | ACTCTACCATTGAGGAATGCAGAT CCCATCTGGCTTGATGTAGTCT |
WTAP Forward Reverse | TTCCCAAGAAGGTTCGATTG TGCAGACTCCTGCTGTTGTT |
FTO Forward Reverse | GCCGGTTCACAACCTCAGTTTAGTTCCACCCAC GTGGGTGGAACTAAACTGAGGTTGTGAACCGGC |
ALKBH5 Forward Reverse | TCACTGCATACGGCCTCAGGACAT TTAGAGCAGGGTCCCTGTTGT |
YTHDF1 Forward Reverse | CAAGCACACAACCTCCATCTTCG GTAAGAAACTGGTTCGCCCTCA |
YTHDF2 Forward Reverse | TAGCCAACTGCGACACATTC CACGACCTTGACGTTCCTTT |
YTHDC1 Forward Reverse | AGATGGGTCTGTCAGATCTGGT TCTGAACCTGCATATGACTCTGAT |
GAPDH Forward Reverse | GGAGCGAGATCCCTCCAAAAT GGCTGTTGTCATACTTCTCATGG |
Western Blot Analysis
Cells were lysed in ice-cold RIPA buffer (CWBIO, Beijing, China) supplemented with a protease inhibitor cocktail (CWBIO) on ice for 30 min and then subjected to three rounds of ultrasound pyrolysis at 35 kHz for 10 s. After centrifuging at 12,000 rpm for 15 min at 4℃, the lysates were denatured in 5× loading buffer at 100℃ for 5 min. Total protein was separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to 0.22-µm-pore-size polyvinylidene fluoride membranes (BOSTER, Wuhan, China). After blocking with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) and then washing with TBST, the membranes were incubated first with primary antibodies targeting METTL3 (1:1,000, Cat#15073-1-AP), YAP (1:5,000, Cat#66900-1-lg), and GAPDH (1:50,000, Cat#60004-lg) (all from Proteintech, Chicago, IL, USA) overnight at 4℃, and then with the relevant secondary antibodies for 1 h at room temperature. Finally, the protein blots were detected using Immobilon ECL Ultra Western HRP Substrate (Millipore, Boston, MA, USA).
Immunohistochemistry
Tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, sliced into 3-µm-thick sections, and dried at 65℃ in an oven for 1 h. The sections were subsequently deparaffinized, rehydrated, incubated in 3% H2O2 for 10 min, boiled for 40 s in sodium citrate buffer for antigen retrieval, blocked in 5% goat serum, and cross-reacted with METTL3 (1:700) and YAP (1:2,000) antibodies for 2 h at room temperature. The samples were then incubated with the respective secondary antibodies for 30 min and stained with DAB and hematoxylin. After dehydration, the slides were sealed in neutral balsam.
Statistical analysis
All data were analyzed with GraphPad Prism 7.0. Significance was assessed with IBM SPSS 22.0 using a two-tailed Student’s paired t-test or repeated measures analysis of variance (RMANOVA). P-values < 0.05 were considered significant.