LncRNA MAGI2-AS3 is down-regulated in intervertebral disc degeneration and participates in the regulation of FasL expression in nucleus pulposus cells

A total of 66 patients with IDD (IDD group, 36 males and 30 females, with the age of 42 to 76 years old and 58.6 ± 4.8 years old, respectively) and 58 healthy volunteers (control group, 31 males and 27 females, with the age of 40 to 77 years and 58.0 ± 5.4 years old, respectively), who were admitted by the Liaocheng Second People’s Hospital from January 2016 to October 2017, were included in this study to serve as research subjects. Inclusion criteria of patients are as follows: 1) patients were diagnosed and treated for the first time; 2) complete medical records; 3) patients who completed treatment in Liaocheng Second People’s Hospital; 4) patients were willing to participate with signed consent form. Exclusion criteria of patients are as follows: 1) patients were diagnosed with multiple diseases; 2) patients were treated before admission. The 58 healthy volunteers received systemic physiological examinations in Liaocheng Second People’s Hospital and all physiological parameters were within the normal range. No significant differences in age and gender were found between the patients and the control groups. This study was approved by the Ethics Committee of Liaocheng Second People’s Hospital.

Fasting blood (5 ml) was extracted from each patient and healthy control 1 day after admission and on the day of discharge. Blood samples were used to prepare plasma samples using the conventional method. NP cells were isolated and cultivated according to the methods described by Wang et al. [13]. Cells were collected from passage 3–5 for subsequent experiments.

To detect the expression of lncRNA MAGI2-AS3, total RNAs were extracted using the TRIzol reagent (Thermo Fisher Scientific). Reverse transcription was performed using the AMV Reverse Rranscriptase (Gibco; Thermo Fisher Scientific). The PCR reaction systems were prepared using the PowerUp™ SYBR™ Green Master Mix (Applied Biosystems™). Primers of lncRNA MAGI2-AS3, FasL and the endogenous control GAPDH were synthesized by GenePharma (Shanghai, China). The expression levels of lncRNA MAGI2-AS3 and FasL were normalized to GAPDH and were calculated according to the 2^-ΔΔCq method [14]. .

Vectors expressing lncRNA MAGI2-AS3 and empty vectors were designed and constructed by Sangon (Shanghai, China). The Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific) was used to perform cell transfections with 10 nM vectors. Cells that were treated with Lipofectamine 2000 reagent only was used as control (C). Transfection with empty vectors was used as negative control (NC).

To detect the protein expression of FasL, total proteins were extracted using the RIPA solution (Thermo Fisher Scientific). Protein samples were denatured and 10% SDS-PAGE electrophoresis was performed. Proteins were then transferred to PVDF membranes. After blocking in 5% non-fat milk for 1 h, incubation with primary antibodies of rabbit anti-human FasL (1:1000; cat. no. ab15285; Abcam) and anti-GAPDH (1,1000; cat. no. ab8245; Abcam) was performed. Membranes were further incubated with IgG-HRP secondary antibody (goat anti-rabbit, cat. no. sc-2004; Santa Cruz Biotechnology, Dallas, TX, USA). Signals were produced using ECL Western Blotting Substrate (Pierce; Thermo Fisher Scientific). Membranes were scanned using the iBright Imaging Systems Thermo Fisher Scientific). Image J v1.48 was used for data analysis.

Plasma levels of lncRNA MAGI2-AS were assessed by qRT-PCR in both IDD patients and healthy people. Compared to the control, plasma levels of lncRNA MAGI2-AS3 were significantly lower in IDD patients (Fig. 1, p < 0.01).

Diagnostic value of lncRNA MAGI2-AS3 for IDD was evaluated by ROC curve analysis with IDD patients as true positive cases and healthy people as true negative cases. The area under the curve was 0.90, with the standard error of 0.027 and 95% confident interval of 0.84–0.95 (Fig. 2).

Results of qRT-PCR showed that the plasma levels of lncRNA MAGI2-AS3 were significantly increased after treatment compared with the pre-treatment levels (p < 0.05, Fig. 3,). Therefore, monitoring the levels of plasma circulating lncRNA MAGI2-AS3 may reflect the treatment outcomes.

Over-expression and knockdown of MAGI2-AS3 were achieved in NP cells, and expression of FasL was detected by qRT-PCR and western blot. Compared to the control (C) and negative control (NC) groups, over-expression of lncRNA MAGI2-AS3 resulted in inhibited expression of FasL in NP cells at both mRNA and protein levels (Fig. 4a, p < 0.05). In the contrary, knockdown of lncRNA MAGI2-AS3 played an opposite role (Fig. 4b, p < 0.05).