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. 2021 Feb;54(2):e12975.
doi: 10.1111/cpr.12975. Epub 2020 Dec 28.

RNA-binding protein HuR suppresses senescence through Atg7 mediated autophagy activation in diabetic intervertebral disc degeneration

Zhenxuan Shao  1   2   3 , Libin Ni  1   2   3 , Sunli Hu  1   2   3 , Tianzhen Xu  1   4 , Zaher Meftah  1   2   3 , Zupo Yu  1   2   3 , Naifeng Tian  1   2   3 , Yaosen Wu  1   2   3 , Liaojun Sun  1   2   3 , Aimin Wu  1   2   3 , Zongyou Pan  5 , Linwei Chen  5 , Weiyang Gao  1   2   3 , Yifei Zhou  1   2   3 , Xiaolei Zhang  1   2   3 , Xiangyang Wang  1   2   3
Affiliations

RNA-binding protein HuR suppresses senescence through Atg7 mediated autophagy activation in diabetic intervertebral disc degeneration

Zhenxuan Shao et al. Cell Prolif. 2021 Feb.

Abstract

Objectives: Diabetes is a risk factor for intervertebral disc degeneration (IVDD). Studies have demonstrated that diabetes may affect IVDD through transcriptional regulation; however, whether post-transcriptional regulation is involved in diabetic IVDD (DB-IVDD) is still unknown. This study was performed to illustrate the role of HuR, an RNA-binding protein, in DB-IVDD development and its mechanism.

Materials and methods: The expression of HuR was evaluated in nucleus pulposus (NP) tissues from diabetic IVDD patients and in high glucose-treated NP cells. Senescence and autophagy were assessed in HuR over-expressing and downregulation NP cells. The mRNAs that were regulated by HuR were screened, and immunoprecipitation was applied to confirm the regulation of HuR on targeted mRNAs.

Results: The results showed that the expression of HuR was decreased in diabetic NP tissues and high glucose-treated NP cells. Downregulation of HuR may lead to increased senescence in high glucose-treated NP cells, while autophagy activation attenuates senescence in HuR deficient NP cells. Mechanistic study showed that HuR prompted Atg7 mRNA stability via binding to the AU-rich elements. Furthermore, overexpression of Atg7, but not HuR, may ameliorate DB-IVDD in rats in vivo.

Conclusions: In conclusion, HuR may suppress senescence through autophagy activation via stabilizing Atg7 in diabetic NP cells; while Atg7, but not HuR, may serve as a potential therapeutic target for DB-IVDD.

Keywords: Atg7; HuR; autophagy; diabetes; intervertebral disc degeneration; senescence.

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Conflict of interest statement

There is no conflict of interest between the authors.

Figures

FIGURE 1
FIGURE 1
The expression of HuR is decreased in diabetic NP tissues and high glucose‐treated NP cells. A, Venn diagram of all differentially down‐regulated genes, and data of three diabetic tissues from GEO DataSets (Series Accession: GSE55100, GSE29231 and GSE19420). B, The expression of HuR gene of degenerated NP tissues (Series Accession: GS23130). C, The expression of HuR gene of high‐glucose treatment cells (Series Accession: GSE60038). D, Representative immunofluorescence staining of HuR in NP tissue from normal and diabetic patients (scale bar: 40 μm). E, F, The protein expression of HuR in NP tissue from normal and diabetic patients, detected by Western blot and quantified by Image J software. G, Representative immunofluorescence staining of HuR in NP tissues from normal and diabetes rats (scale bar: 800 μm). H, I, The protein expression of HuR in rat NP cells after various concentrations of glucose treatment for 6 hours, detected by Western blot. All data were shown as mean ± SD. *P < .05, **P < .01
FIGURE 2
FIGURE 2
HuR downregulation prompts senescence in high glucose‐treated NP cells. The cells were transfected with LV‐NC, LV‐shHuR or LV‐HuR before high‐glucose treatment (100 mM). A, The protein expression of HuR in rat NP cells transfected with lentivirus, detected by Western blot. B, C, EdU staining for NP cells with or without high‐glucose treatment for 6 hours and transfected with lentivirus (scale bar: 50 μm). D, E, SA‐β‐gal staining for NP cells (scale bar: 50 μm). F, G, The protein expression of p21 and p16INK4a, and the ratio of p‐p53/p53 were detected by Western blot and quantified by Image J software. All data were shown as mean ± SD. *P < .05, **P < .01, &P < .001
FIGURE 3
FIGURE 3
HuR regulates autophagy activation in high glucose‐treated NP cells. The cells were transfected with LV‐NC or LV‐shHuR or LV‐HuR before high‐glucose treatment (100 mM). A, TEM images of autophagic vesicles in rat NP cells (bar: 0.5 μm). B, The LC3‐II was detected by immunofluorescence combined with DAPI staining for nuclei (scale bar: 20 μm). C‐E, The protein expression of p62 and the ratio of LC3‐II/ LC3‐I in NP cells treated with or without high‐glucose treatment (100 mM) for 6 hours and transfected with lentivirus, detected by Western blot and quantified by Image J software. F, EdU staining for NP cells (scale bar: 50 μm). G, SA‐β‐gal staining for NP cells (scale bar: 50 μm). H, I, The protein expression of p21 and p16INK4a in NP cells with or without rapamycin (the classical autophagy activator, 10 μM, 24 hours), detected by Western blot. All data were shown as mean ± SD. *P < .05, **P < .01
FIGURE 4
FIGURE 4
HuR regulates Atg7 expression through binding to Atg7 AU‐rich element and upregulating its mRNA level. The cells were transfected with LV‐shHuR or LV‐HuR before high‐glucose treatment (100 mM). A, Multiple autophagy‐related mRNA expressions in NP cells with high‐glucose treatment, detected by qPCR. B‐D, The protein expression of Atg7 in NP cells with or without high‐glucose treatment (100 μM) for 6 hours, detected by Western blot and quantified by Image J software. E, Potential HuR‐binding element at 3′UTR of Atg7 mRNA on multiple species. F, Schematic diagram showing the workflow of the RNA‐binding protein immunoprecipitation (RIP) assay. G, The mRNA expression of Atg7 in rat NP cells, detected by qPCR, and normalized to IgG isotype controls. All data were shown as mean ± SD. *P < .05, **P < .01
FIGURE 5
FIGURE 5
Atg7 reverses HuR‐down‐regulated autophagy in high glucose‐treated NP cells. The cells were transfected with LV‐NC or LV‐Atg7 before high‐glucose treatment (100 mM) or LV‐shHuR. A, B, The protein expression of Atg7 in rat NP cells with LV‐Atg7 transfection, detected by Western blot and quantified by Image J software. C, TEM images of autophagic vesicles in rat NP cells, with LV‐NC or LV‐Atg7 (scale bar: 0.5 μm). D, LC3‐II was detected by immunofluorescence combined with DAPI staining for nuclei (scale bar: 20 μm) E, F, The protein expression of p62 and the ratio of LC3‐II/ LC3‐I were detected by Western blot and quantified by Image J software. All data were shown as mean ± SD. *P < .05, **P < .01, &P < .001
FIGURE 6
FIGURE 6
Atg7 inhibits HuR‐regulated senescence in high glucose‐treated NP cells. A, C, E, The protein expression of p21, p16INK4a and the ratio of p‐p53/p53, detected by Western blot. B, F, EdU staining for NP cells (scale bar: 50 μm). D, G, SA‐β‐gal staining for NP cells (scale bar: 50 μm). All data were shown as mean ± SD. *P < .05, **P < .01, ***P < .001
FIGURE 7
FIGURE 7
Overexpression of Atg7, instead of HuR, ameliorates the progress of DB‐IVDD in vivo. A, T2 weighted MRI of a rat‐tail disc at 3 weeks after disc puncture surgery, with or without lentivirus transfection (white arrows). B, The respective Pfirrmann grade scores of a rat‐tail disc. C, X‐ray of a rat‐tail disc at 4 weeks after disc puncture surgery in normal and diabetic rats, with or without lentivirus transfection (white arrows). D, The disc height index (DHI) of a rat‐tail disc. E, Representative H‐E, S‐O and Alcian Blue staining of NP tissues (scale bar: 800 μm). F, The histological grades evaluated in three groups. G‐I, The immunohistochemical staining of p16 and LC3‐II in intervertebral disc sections; the levels were determined using Image‐Pro Plus software. All data were shown as mean ± SD. *P < .05, **P < .01
FIGURE 8
FIGURE 8
Schematic illustration. With high‐glucose treatment, the expression of HuR is decreased, which induces autophagy inactivation and increased senescence. Mechanistic study reveals that HuR regulates Atg7 expression through binding to Atg7 AU‐rich element and adjusting its mRNA stability
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