Patient Tissue Samples
In this study, samples of 50 cases with HSCR, which consisted of 35 subjects of S-HSCR, 12 subjects of L-HSCR and 3 subjects of TCA and 54 normal controls patients were obtained from patients who underwent surgery in the Xinhua Hospital of Shanghai Jiao Tong University between 2019 and 2020. All HSCR cases were diagnosed by histopathological examination of biopsy/surgical resection material for the absence of ganglion cells. For HSCR tissues, we used the normoganglionic dilated segment of the HSCR colon. The normal controls were randomly gathered from patients with no history of chronic constipation or other enteric neural malformations. Clinical samples were quickly placed in liquid nitrogen after operation and then stored at − 80℃. The present study was conducted in accordance with the Helsinki Declaration and approved by the Xinhua Hospital Ethics Committee, and all patients were provided with the consent forms signed by their parents. The detailed clinical information of patients is summarized in Table 1.
Cell Culture And Transfection
The normal human neuroblastoma cell line SH-SY5Y was purchased from the Culture Collection of Chinese Academy of Sciences (Shanghai, China). SH-SY5Y cell lines were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Corning, USA) with 10% FBS (Gibco, USA), 1% penicillin (100U/ml) and 0.1 mg/ml streptomycin (Gibco, USA) in a humidified chamber with 5% CO2 and 95% air at 37◦C. The miR-181b-5p mimics, mimics nc, miR-181b-5p inhibitor, inhibitor nc, si-PROX1, si-NOTCH1, si-circTIMMDC1, si-circANKRD12 and control siRNA were designed and synthesized by GenePharma (Shanghai, China). The plasmid of circTIMMDC1, circANKRD12 and control plasmid were designed and synthesized by Genechem (Shanghai, China).Lipofectamine 2000 reagent (Thermo Fisher Scientific, USA) was used as transfection medium according to the manufacturer’s protocol. Oligonucleotide sequences were listed in Table 2.
Table 1. Clinical features of study population
|
Variable
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HSCR(n = 50)
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Normal(n = 54)
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P-valve
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Age[months; mean(SEM)]
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9.06 (0.80)
|
7.81 (1.17)
|
0.41*
|
Weight[kg; mean(SEM)]
|
8.76(0.38)
|
8.249(0.58)
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0.48*
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Sex[n(%)]
|
|
|
|
Male
|
42(84)
|
38(70.4)
|
0.09ꝉ
|
Female
|
8(16)
|
16(29.6)
|
|
Abbreviation: HSCR, Hirschsprung's disease.
*Student's unpaired t test. †Two-sided χ2 test.
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Rna Extraction And Real-time Quantitative Real-time Polymerase Reaction (Qrt-pcr)
Total RNA from tissues or cells were extracted using TRIzol Reagent (Takara, Japan) according to the manufacturer’s instructions. The concentration and purity of RNA samples were measured by Nanodrop 2000 (Thermo Fisher Scientific, USA). For miRNA, the complementary deoxyribonucleic (cDNA) was obtained using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, USA), and quantitative real-time polymerase chain reaction (qRT-PCR) was performed using TaqMan Universal Master Mix II (Thermo Fisher Scientific, USA). U6 small nuclear RNA (U6 snRNA) was used as an endogenous control for normalization. For circRNA and mRNA, the reverse transcriptions were performed using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA) with random primers, and qRT-PCR was performed using SYBR Green Real-Time PCR Master Mixes (Thermo Fisher Scientific, USA). GAPDH was used as an endogenous control for normalization. Each sample was repeated three times. Relative quantification of miRNA, circRNA and mRNA expression was compared to endogenous control and analyzed using the 2−ΔΔCT method. The primers were listed in Table 3.
Table 2
Sequence of oligonucleotide used in this study
Gene Name
|
Primer type
|
Sequence (5′−3′)
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miR-181b-5p mimics
|
Sense
|
AACAUUCAUUGCUGUCGGUGGGU
|
|
antisense
|
CCACCGACAGCAAUGAAUGUUUU
|
miR-181b-5p inhibitor
|
Sense
|
ACCCACCGACAGCAAUGAAUGUU
|
|
antisense
|
/
|
mimics NC
|
Sense
|
Provided by manufacturer
|
|
antisense
|
|
Inhibitor NC
|
Sense
|
Provided by manufacturer
|
|
antisense
|
|
si-PROX1
|
Sense
|
GAGUUGACAUUGGAGUGAATT
|
|
antisense
|
UUCACUCCAAUGUCAACUCTT
|
si-NOTCH1
|
Sense
|
GAUGCGAGAUCGACGUCAATT
|
|
antisense
|
UUGACGUCGAUCUCGCAUCTT
|
NC
|
Sense
|
Provided by manufacturer
|
antisense
|
si-hsa_circ_0003865
|
Sense
|
ACACAGAUUCAGAUCCAGGTT
|
|
antisense
|
CCUGGAUCUGAAUCUGUGUTT
|
si-hsa_circ_0006884
|
Sense
|
GCUGGGUGAACAGCAGAGATT
|
|
antisense
|
UCUCUGCUGUUCACCCAGCTT
|
Table 3
Primer sequences used in this study
Gene Name
|
Primer type
|
Sequence (5′−3′)
|
PROX1
|
Forward
|
CCGTTTCAGAGTCCGTTAGGT
|
|
Reverse
|
TGGTGGGATGACATCTTGGTC
|
NOTCH1
|
Forward
|
TGGACCAGATTGGGGAGTTC
|
|
Reverse
|
GCACACTCGTCTGTGTTGAC
|
HES1
|
Forward
|
ACGTGCGAGGGCGTTAATAC
|
|
Reverse
|
GGGGTAGGTCATGGCATTGA
|
GAPDH
|
Forward
|
GAGTCAACGGATTTGGTCGT
|
|
Reverse
|
TGTGGTCATGAGTCCTTCCA
|
miR-181b-5p
|
Forward
|
CCAGCTGGGCTCACTGAACAATGA
|
|
Reverse
|
Provided by manufacturer
|
U6
|
Forward
|
CTCGCTTCGGCAGCACA
|
|
Reverse
|
AACGCTTCACGAATTTGCGT
|
hsa_circ_0003865
|
Divergent
|
TGGCTGGTGGCATAATTGGA
|
|
Convergent
|
GTATTCCCCCATACACCCAGC
|
hsa_circ_0006884
|
Divergent
|
CGTCCAGTGGATGTAGCAGA
|
|
Convergent
|
TTTCTGGACCTTCTTTCTCTGAGT
|
Table 4. Probe sequences used in this study
|
Probe Name
|
Probe Sequence
|
hsa_circ_0003865 probe
|
TGGATCTGAATCTGTGTAACTTTCATCATC-biotin
|
Control probe
|
GTGTAATCGTATCTCTGTTGTACACATATC-biotin
|
hsa_circ_0006884 probe
|
TGCTGTTCACCCAGCAAGGCTCCAATTA-biotin
|
Control probe
|
ATTCTACGGACCTTAGGCCACACATCTG-biotin
|
Western Blot Analysis
SH-SY5Y cells were lysed in Radio Immunoprecipitation (RIPA)Assay lysis buffer (Thermo Fisher Scientific, USA) and the total proteins concentration was measured and quantified by bicinchoninic acid (BCA) protein assay (Beyotime, China). The equal amounts of protein samples were separated by SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibody anti-PROX1 (1:1000, Cell Signaling Technology, USA), anti-NOTCH1 (1:1000, Cell Signaling Technology, USA), anti-HES1(1:1000, Cell Signaling Technology, USA), anti-Argonaute2 (1:1000, Cell Signaling Technology, USA) and anti-GAPDH (1:5000, Cell Signaling Technology, USA) at 4℃ overnight. Then the membranes were washed and incubated with goat anti-rabbit secondary antibody (1:5000, Cell Signaling Technology, USA) for 1h at room temperature. Finally, the membranes were visualized by enhanced chemiluminescence (ECL) system (Thermo Fisher Scientific, USA).The images were analysed by Image Lab Softwave. GAPDH was used as an endogenous control for normalization.
Dual-luciferase Reporter Assay
The Luciferase reporter vector of circTIMMDC1, circANKRD12 and PROX1–3’UTR and their corresponding mutation were constructed by Genechem (Shanghai, China), including circTIMMDC1-WT, circTIMMDC1-MUT, circANKRD12-WT, circANKRD12-MUT, PROX1–3’UTR-WT and PROX1–3’UTR-MUT. SH-SY5Y cells were seeded in 24-well plates in triplicate and co-transfected with corresponding plasmids and miR-181b-5p mimics or mimics NC. Then, after 48h of incubation, the Firefly and Renilla luciferase activities were measured by Dual Luciferase Assay Kit (Promega, USA) according to the manufacturer’s instructions. Relative luciferase activity was normalized to the Renilla luciferase internal control.
Wound-healing Assay
SH-SY5Y cells were seeded in 6-well plates and incubated with drugs for additional 24h until they reached about 90–95% confluence. Afterwards, a straight scratch wound was created in the central area of each well with a 200ul pipette tip. Subsequently, cells were washed with 1X PBS twice and then cultured with the serum-free medium. The wound closure images were photographed at 0 and 24 h (objective lens:×10; eye piece:×10). Cell migration was quantified using the following equation: (0 hour wound area − 24 hours wound area)/0 hour wound area×100. The images were analysed by ImageJ Softwave. Each experiment was performed in triplicate.
Transwell Assay
For measure the ability of cell migration after transfection, transwell migration chambers (8 µm pore size, Corning, USA) were used. After transfection with drugs for 48h, the cells were washed by 1X PBS twice and resuspended in serum-free DMEM medium(1×106 cells/mL). Then, 100µl of the suspension was seeded into the upper chamber and 600µl DMEM with 20% FBS was added to the lower chamber. After 24h of incubation at 37℃, cells were fixed with a 4% paraformaldehyde for 30 min and stained with 1% crystal violet for 15 min. The images were analysed by ImageJ Softwave. Three visual fields were randomly selected for manual counting (objective lens:×10; eye piece:×10). Each experiment was performed in triplicate.
Rna Immunoprecipitation
The RNA immunoprecipitation (RIP) assay was performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation kit (Millipore, USA) according to the manufacturer’s protocol. 2×107 SH-SY5Y cells were lysed in RIP lysis buffer (Thermo Fisher Scientific, USA). Then, the cell extract was incubated with magnetic beads conjugated with anti-Argonaute2 (AGO2, Cell Signaling Technology, USA) or anti-IgG antibody (Millipore, USA) at 4℃ overnight. Finally, RNA was extracted by TRIzol Reagent (Takara, Japan) and analyzed by qRT-PCR. Each experiment was performed in triplicate.
Biotin-coupled Probe Rna Pull Down Assay
The Biotin-coupled probe RNA pull down assay was performed using the Pierce Magnetic RNA-Protein Pull-Down Kit(Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. The 3’-end Biotinylated circTIMMDC1 and circANKRD12 probe were constructed by Genechem (Shanghai, China). 2×107 SH-SY5Y cells were lysed in RIP lysis buffer (Thermo Fisher Scientific, USA).Then, cell lysates were incubated with the probe-coated magnetic beads mixture at 4°C overnight. Finally, the RNA-Binding protein complexes were extracted by TRIzol Reagent (Takara, Japan) and analyzed by qRT-PCR. Each experiment was performed in triplicate. The probe sequences were listed in Table 4.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 8.02 (GraphPad Software, USA). All values were presented as mean ± SEM. Two-tailed Student’s t-tests was used to calculate statistical significance. The Pearson correlation coefficient was used to analyze the correlations. P-value < 0.05 was considered statistically significant.