All procedures were approved by the Animal Ethical and Welfare Ethics Committee of Hangzhou Normal University and were performed in accordance with relevant guidelines and regulations. All our animal experiments methods were in accordance with ARRIVE guidelines.
1. Construction of hearing loss mice
In this study, a combination of kanamycin sulfate and furosemide was used to establish a model of acute and severe sensorineural hearing loss. Two-month-old CBA mice (without gender distinction) were selected, and each mouse was injected subcutaneously with kanamycin sulfate (Biosharp, BS152) 1,000 mg·kg−1, and after waiting for half an hour, the mice was injected with furosemide (Sigma, PHR1057) 500 mg·kg−1 to establish a hearing loss model. In this experiment, the mice were divided into 3 groups, the first group was the control group, the second group was the drug-induced injury group for 12 hours, and the third group was the drug-induced injury group for 24 hours. The animals used in this experiment were CBA mice (SPF grade), and all the experimental animals were purchased from the Experimental Animal Center of Hangzhou Normal University.
2. Auditory Brainstem Response (ABR)
Each CBA mouse was intraperitoneally injected with sodium pentobarbital (50 mg·kg−1) and placed in a standard shielded soundproof room after anesthesia. The detection electrodes were placed behind the left and right auricles and under the scalp of the mouse to perform the auditory brainstem response of the mouse. The stimulus sound is a short stimulus, the scanning duration is 10 ms, the stimulus repetition rate is 11 times·s−1, the filter bandpass is 150-1 500 Hz, and the superimposition is 500-1 000 times. Each mouse was tested for binaural hearing, and the second wave threshold was used as the mouse hearing threshold.
3. Preparation of Corti RNA
The cochlea was removed immediately after the mouse was sacrificed by cervical dislocation, and was quickly transferred to a dissecting microscope to strip the outer cochlear bone structure and remove the Corti organ. Combine 6 Corti apparatuses into a set, grind with liquid nitrogen and transfer the powder to a 1.5 mL EP tube. Add 1ML TRNzol Universal ageant to the EP tube, mix well and let stand for 10 minutes. Add 200ul chloroform to the EP tube, shake vigorously for 30s and then let it stand for 10 minutes. Centrifuge at 12000 rpm for 15 minutes. Take the upper liquid to a new 1.5ML EP tube, add 500 mL of isopropanol, mix well and let it stand at room temperature for 10 minutes. Centrifuge at 12000 rpm for 10 minutes. Discard the supernatant, add 1ML of 75% ethanol, and let stand at room temperature for 5 minutes. Centrifuge at 8000rpm for 5 minutes, discard the supernatant, add 20ul DEPC-treated water, and store at -80°C.
4. miRNA sequencing and bioinformatics analysis
The RNA-seq method was used to perform miRNA sequencing on sample RNA, and the sequencing depth of each sample was 20 M. The DEG-seq method was used to analyze the miRNA sequencing results and screen the miRNAs with different expressions. The screening criteria are log2FC>1 and Qvalue<0.001. The miRNA that was up-regulated in the 12-hour group and the 24-hour group was selected as a candidate miRNA. "NRN1" is used as a keyword to predict the miRNA targeting Neuritin gene from 7 different websites (TargetScan, miRDB, DIANA, miRNAMap, miRWalK, miRmap, trabase), and then take the intersection with the candidate miRNA to obtain the possible targeted regulation of Neuritin and Candidate miRNAs that are up-regulated during hearing loss.
The stem-loop method was used to synthesize candidate miRNA primers, and U6 was used as an internal reference. According to Takara Reverse Transcription Kit (Takara RR 037A) 10 µL system (5×PrimeScript Buffer 2 µL, PrimeScript RT Enzyme Mix I 0.5 µL, PCR Reverse Primer 0.5µL, Total RNA 2 µL, ddH2O 5µL) the extracted RNA was reverse transcribed into cDNA. The cycle conditions were: 95℃ 30s (1 cycle), 95℃ 5s, 60℃ 30s (40 cycles) ). According to the 20 µL system of Takara qPCR kit( TB Green Premix Ex Taq II 10 µL, PCR Forward Primer 0. 8 µL, PCR Reverse Primer 0. 8 µL, cDNA 2µL, ddH2O 6.4 µL), the obtained cDNA was subjected to real-time fluorescence quantitative PCR, and the cycling conditions were 95℃ 30s (1 cycle), 95℃ 5s, and 60℃ 30s (40 cycles). Use the fluorescence quantitative PCR instrument (Germany analytik-jena 870wer 3G) to measure the fluorescence of the sample, and calculate the CT value and TM value. The △△t method was used to calculate the differential expression folds of candidate miRNAs between the 12 h group, 24 h group and the control group, and the target miRNAs were screened according to the differential fold.
The primers were designed by the Primer Premier 5.0 Software and as follows:
miR-224-5p-F: 5’- CGCGCGTAAGTCACTAGTGGT -3’
6. Western blot
The synthetic mimics of candidate miRNAs were transfected into 293T cells with Lipofectamine 3000 (ThermoFisher, L3000015), and after 48 hours of culture, the cells were lysed with RIPA (Beyotime, P0013B) to extract total protein. Configure a 15% concentration SDS-PAGE gel, separate about 60-100ug of protein on the SDS-PAGE gel under the conditions of 80 V for 30 min and 110 V for 90 min, and use the semi-dry transfer method to transfer the protein under the conditions of 23 V for 43 min. Onto PVDF membrane (Immobilon, ISEQ00010). Use 5% skimmed milk powder (Biosharp, BS 102) to seal the PVDF membrane for 2 h, then use 5% skimmed milk powder to dilute Neuritin antibody (Abcam, 64186) at a ratio of 1:1000, and incubate overnight at 4°C. On the next day, the goat anti-rabbit secondary antibody (Nakayama Jinqiao) was diluted at a ratio of 1:10000, incubated at room temperature for 2 hours, and an enhanced chemiluminescence system (BIO-RAD, 1705060) was used to detect the immune response zone.
Neuritn and miR-224-5P binding sites were predicted through miRmap (https://mirmap.ezlab.org/). Neuritn sequences with a length of 60 bp were cloned into the GP-miRGLO vector. GP-miRGLO vector and miR-224-5P mimics rely on Lipofectamine 3000 (Thermofisher, L3000015) to be transfected into 293T cells. After 48 hours of transfection, the lysate was collected, and the phochillase report system (Dual-Luciferase®Reporter (DLR™) Assay System,E1980) was used to measure the photinus pyralis luciferase and Renilla ReniFormis luciferase activity. Computational fluorescence activity ratio of firefly and sea kidney fluorescence activity. Three biological repeats were performed.
The Neuritn sequences were cloned into the GP-miRGLO vector as follows:
NRN1 -miR-224-5p WT: GAGAGGGAAAAGGAGAAGGCCAGGGGAATGACTTCAAGAGTGGTGTCCACGTGGGAATCA
NRN1 -miR-224-5p MUT: GAGAGGGAAAAGGAGAAGGCCAGGGGAAACTGAACAAGAGTGGTGTCCACGTGGGAATCA