Cell culture and treatment
American Type Culture Collection (ATCC, Manassas, VA, USA) offered NP cells, and cells were cultivated within Dulbecco's Modified Eagle Media-Nutrient Mixture F-12 (DMEM/F12) that contained 15% FBS and 1% P/S (Gibico, NY, USA) were used to culture cells, and cells were incubated within the humid incubator under 37℃ and 5% CO2 conditions. For drug treatment, IL-1β (10 ng/mL, RiboBio, Guangzhou, China) treated NP cells for 24 h to induce IDD models in vitro.
Cell transfection
MiR-199a-5p mimics and miR-199a-5p inhibitors and their negative controls (NC mimics and NC inhibitors) were designed and synthesized by RiboBio (Guangzhou, China). GenePharma (Shanghai, China) synthesis small interfering RNA (si-RNA) of KCNQ1OT1 and ACTG1 (si-KCNQ1OT1 and si-ACTG1), and si-NC served as negative control were acquired from. The full-length sequence of KCNQ1OT1 was inserted into pcDNA3.1 (Invitrogen) for KCNQ1OT1 overexpression. Subsequently, the corresponding plasmid was transfected into NP cells cultured to 80% confluence with Lipofectamine™ 3000 Transfection Reagent (Invitrogen, California, USA).
Western blot
NP cell lytic was prepared with RIPA cleavage buffer (Beyotime, Nanjing, China). Protein was separated by SDS-PAGE gel and transferred to PVDF membrane. The PVDF membrane was sealed with TBST and 5% skim milk powder and incubated with specific antibodies overnight, with primary antibodies (Abcam, Cambridge, UK): anti-Bcl-2 (ab32124, 1:1000), anti-Bax (ab32503, 1:1000), anti-MMP3 (ab52915, 1:1000), anti-Collagen II (ab34712, 1:1000), ACTG1 (ab200046, 1:1000), and anti-GAPDH (ab8245, 1:1000) at 4°C. The second day, incubated with secondary antibody and bands were detected by a gel imaging system (Bio-RAD), and the protein was quantitatively analyzed by Image J software.
qRT-PCR
TRIzol reagent (Invitrogen) isolated total RNA. Then, reverse transcription was carried out by PrimeScript RT kit (Dalian TaKaRa, China) and microRNA first strand cDNA synthesis kit (Sanggong Biotechnology, China). KCNQ1OT1, Bcl-2, Bax, MMP3, Collagen II, ACTG1 and miR-199a-5p genes levels were quantified by qRT-PCR. The relative quantitative comparison cycle threshold method was used to determine the target mRNA. The whole process was repeated three times. The primers used in this study were shown in Table 1.
Table 1
Primer name | Primer sequences |
F- KCNQ1OT1 | 5′- -3′ ACTCACTCAAGGTGTAGCAGG |
R- KCNQ1OT1 | 5′- -3′ CAGCGACTCAGTTTCCATGTC |
F- Bcl-2 | 5′- -3′ GACTGAGTACCTGAACCGGC |
R- Bcl-2 | 5′- -3′ GGGCCAAACTGAGCAGAGTC |
F- Bax | 5′- -3′ GCCCTTTTCTACTTTGCCAGC |
R- Bax | 5′- -3′ CTGGAGACAGGGACATCAGT |
F- MMP3 | 5′- -3′ TGAGGACACCAGCATGAACC |
R- MMP3 | 5′- -3′ ACTTCGGGATGCCAGGAAAG |
F- Collagen II | 5′- -3′ CTGGAAAGCCTGGTGATGAT |
R- Collagen II | 5′- -3′ GGAACCACTCTCACCCTTCA |
F- miR-199a-5p | 5′- -3′ CGGCCCAGTGTTCAGACTAC |
R- miR-199a-5p | 5′- -3′ GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCA CTGGATACGACGAACAG |
F- ACTG1 | 5′- -3′ AAAAGGCGGGGTCGCAAT |
R-ACTG1 | 5′- -3′ CCCGACGATGGAAGGAAACA |
F-U6 | 5′- -3′ CTCGCTTCGGCAGCACA |
R-U6 | 5′- -3′ AACGCTTCACGAATTTGCGT |
F-GAPDH | 5′- -3′ AGGTCGGAGTCAACGGATTT |
R-GAPDH | 5′- -3′ TGACAAGCTTCCCGTTCTCA |
Subcellular fractionation analysis
According to the instructions, cytoplasmic and nuclear grades were isolated from NP cells with nuclear and cytoplasmic extraction reagents (Beyotime, Nanjing, China). Cytoplasmic and nuclear RNA extracts were analyzed by qRT-PCR, and GAPDH and U6 were used as normal controls, respectively.
Dual-luciferase reporter assay
The wild type and mutant sequences of KCNQ1OT1 or ACTG1 were cloned into pGL3 reporter vectors (Promega, Madison, WI, USA), respectively. Lipofectamine™ 3000 Transfection Reagent (Invitrogen, California, USA) was used to co-transfect these vectors and miR-199a-5p mimics or NC mimics into NP cells. Following 48 h, luciferase activity was detetced.
RIP assay
Magna RIP RNA binding protein immunoprecipitation kit (Millipore, Bedford, MA, USA) used to performed RIP assay, based on previous research [18]. In short, NP cell lysates were incubated with magnetic beads coupled with negative control IgG and anti-AGO2 antibodies (Abcam, Cambridge, UK) overnight at 4℃. After incubation, the immunoprecipitated RNA was extracted and detected by qRT-PCR to confirm the enrichment of binding targets.
CCK-8 assay
CCK-8 assay kit (Solarbio, Beijing, China, CA1210) detected cell viability. In short, CCK-8 reagent (50 µL/well) was added to the NP cells after washing 3 times with sterile PBS and incubated for 2 h. Subsequently, 100 µL of the supernatant was transferred to a 96-well plate (n = 3). The absorbance of the supernatant at 450 nm was measured with a microplate reader, and a cell proliferation curve was drawn.
Flow cytometry
Annexin V-FITC/PI apoptosis detection kit (Beyotime, Nanjing, China, C1062S) detected cell apoptosis. In short, treated NP cells were washed with sterile PBS for three times and stained with anti-Annexin V-FITC/PI. Finally, the apoptosis rate was measured by flow cytometry.
Statistical analysis
The data of the three replicates were expressed as mean ± standard deviation (SD). Comparisons between the two groups were performed by Student’s t-test, while comparisons between multiple groups were performed by one-way analysis of variance (ANOVA). A value of P < 0.05 was considered statistically significant.