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      An analysis of the transcriptional landscape in hypoxia-treated primary nucleus pulposus cells

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          Abstract

          Intervertebral disc degeneration (IDD) is the main cause of lower back pain. Lower back pain places a huge burden on society, and all current treatments for IDD cannot restore the original function of the intervertebral disc. Proposing new treatments for IDD requires clarifying the mechanisms of IDD. Physiological hypoxia is an important feature of the nucleus pulposus because of its special anatomical structure. Using RNA sequencing (RNA-seq), we obtained the whole transcriptome of nucleus pulposus cells (NPCs) under hypoxia and hypoxia-inducible factor (HIF1A) deletion. Results demonstrated possible effects of oxygen concentration and HIF1A on NPCs. In addition, our results showed that hypoxia can affect lipid metabolism in NPCs. The degree of disc degeneration in the patients (Table S1) was evaluated by MRI scans, and we obtained the patients' surgically resected discs after obtaining the consent of the patients. We knocked down the expression of HIF1A in NPCs using small RNA interference technology (RNA-si), and cultured NPCs in a normoxia chamber (NXNC group) or hypoxia chamber (HXNC group) for subsequent RNA-seq. Before RNA-seq, we assessed the expression of HIF1A in different groups of NPCs by q-PCR, and the results showed that our treatment effectively reduced the expression of HIF1A in NPCs (Fig. 1A). Figure 1 Transcriptomic analysis and validation of NPCs. (A) The qPCR and RNA-seq results of treatment of NPCs with HIF1A-si under hypoxia (HXSI) or negative control (HXNC) and treatment of NPCs with HIF1A-si under normoxia (NXSI) or negative control (NXNC). (B) Venn diagram of DEG volcano plots for HXNC vs. NXNC, HXSI vs. HXNC, and HXSI vs. HXNC. (C) GO enrichment analysis of DEGs in HXNC vs. NXNC. (D) GSEA analysis of GO, including lipoprotein metabolic process and positive regulation of reactive oxygen species metabolic process. (E) GO enrichment analysis of DEGs in HXNC vs. NXSI. (F) GSEA analysis of GO, including canonical glycolysis and response to vitamin D. (G) Western blot experiments and densitometric analysis of NPCs treated with HIF1A-si and negative control at different oxygen concentrations (β-actin as internal control). (H) The sequencing results of the expression levels of each gene are roughly consistent with the Western blotting results. (I) The detection of reactive oxygen species and fluorescence intensity analysis of nucleus pulposus treated with HIF-1A and negative control at different oxygen concentrations. ns, no significant difference. ∗ P < 0.1, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. Scale bar = 200 μm. Fig. 1 Our results show that the treatment conditions described above significantly affect the transcriptomics of NPCs. Compared with the NXNC group, the HXNC group had 77 up-regulated differential genes (DEGs) and 30 down-regulated DEGs (Fig. 1B and Table S2). We also explored the biological processes and pathways in which these DEGs might be involved. Results show that, DEGs enriched in various Gene Ontology (GO) enrichment analysis terms including regulation of cell cycle, cell division, DNA replication initiation, and response to hypoxia (Fig. 1C and Table S3). Gene Set Enrichment Analysis (GSEA) of DEGs showed 63 significantly enriched pathways (Table S4), including the lipoprotein metabolic process and positive regulation of the reactive oxygen species (ROS) metabolic process (Fig. 1D). Hypoxia-inducible factor (HIF1A), a key transcription factor of cells in a hypoxic environment, regulates a series of genes for cell survival under hypoxic conditions. HIF1A has been identified as involved in all mechanisms underlying IDD. 1 Interestingly, the deletion of HIF1A under hypoxic conditions resulted in 28 DEGs (Table S5), while the deletion of HIF1A under normoxic conditions resulted in 68 DEGs (Table S6) that included the former 28 DEGs (Fig. 1B). This result suggests that HIF1A may play significant roles in NPCs under normoxic conditions. In order to fully understand the effect of HIF1A on NPCs, we compared and analyzed the DEGs of the NXSI and NXNC groups. After analyzing DEGs under HIF1A loss and normoxic conditions, we found that DEGs enriched in multiple GO terms including negative regulation of growth, response to hypoxia, negative regulation of oxidative stress-induced neuron intrinsic apoptotic signaling pathway, and hypoxia-inducible factor-1 alpha signaling pathway (Fig. 1E and Table S7). GSEA of these DEGs showed 14 significantly enriched pathways, including canonical glycolysis, response to hypoxia, negative regulation of ROS metabolic process, and response to vitamin D (Fig. 1F and Table S8). In order to confirm our sequencing results, we selected 4 genes and verified whether their expression trends were consistent with Western blotting results (Fig. 1G). Comparing Western blotting results with RNA-seq results (Fig. 1H), the change trends of gene expression are roughly the same. To further explore the contribution of HIF1A to the production of ROS in NPCs under different oxygen concentrations, we examined the production of ROS by NPCs (Fig. 1I). The results are consistent with the sequencing results that hypoxia can inhibit the production of ROS in NPCs and, when HIF1A is depleted, the production of ROS increases regardless of the oxygen concentration. Physiological hypoxia is a characteristic feature of the nucleus pulposus, 2 and the oxygen concentration of the environment in which NPCs live may change with the progression of IDD. 3 Our results suggest that when the oxygen concentration increases, the expression levels of genes involved in lipid metabolism (ABAC1, PCSK9, LDLR, and LRP1) increase. In addition, when HIF1A is disrupted, the gene expression of LRP4 is down-regulated, which requires further study. Previous studies have reported that lipid metabolism disorders may promote IDD. 4 Our results reveal that oxygen concentration can affect lipid metabolism in NPCs. On the other hand, previous studies successfully constructed IDD models using APOE knockout rabbits. 5 Taken together with our results, studies on lipid metabolism in NPCs should take hypoxia into consideration. In conclusion, our results explain the possible effects of oxygen concentration and HIF1A on NPCs, including the effect of oxygen concentration on NPC lipid metabolism. Further study is required to apply these findings to the subsequent treatment of IDD. Author contributions Zhicai Peng and Zhuo Wang: manuscript preparation; Shuaichi Guo, Bing Tan and Ruichao Cao: extraction and processing of NPCs and validation of RNA-seq results; Shengqiang Cheng, Jun Chen and Chunwang Xie: data acquisition and analysis; Dehong Mao and Zhenming Hu: concept and design of the study and revision of the manuscript. We ensure that all authors have agreed to the manuscript's content and its submission to Genes & Diseases. Conflict of interests The authors declare no conflict of interests. Funding The study was funded by the Chongqing Science and Technology Committee and Health Commission Joint Project (China) (No. 2022ZDXM040). Funders had no role in the experimental process, data collection and analysis, and manuscript writing.

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          HIF1A Alleviates compression-induced apoptosis of nucleus pulposus derived stem cells via upregulating autophagy

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            The potential role and trend of HIF-1α in intervertebral disc degeneration: Friend or foe?

            Lower back pain (LBP) is one of the most common reasons for seeking medical advice in orthopedic clinics. Increasingly, research has shown that symptomatic intervertebral disc degeneration (IDD) is mostly related to LBP. This review first outlines the research and findings of studies into IDD, from the physiological structure of the intervertebral disc (IVD) to various pathological cascades. The vicious cycles of IDD are re-described in relation to the analysis of the relationship among the pathological mechanisms involved in IDD. Interestingly, a ‘chief molecule’ was found, hypoxia-inducible factor-1α (HIF-1α), that may regulate all other mechanisms involved in IDD. When the vicious cycle is established, the low oxygen tension activates the expression of HIF-1α, which subsequently enters into the hypoxia-induced HIF pathways. The HIF pathways are dichotomized as friend and foe pathways according to the oxygen tension of the IVD microenvironment. Combined with clinical outcomes and previous research, the trend of IDD development has been predicted in this paper. Lastly, an early precautionary diagnosis and treatment method is proposed whereby nucleus pulposus tissue for biopsy can be obtained through IVD puncture guided by B-ultrasound when the patient is showing symptoms but MRI imaging shows negative results. The assessment criteria for biopsy and the feasibility, superiority and challenges of this approach have been discussed. Overall, it is clear that HIF-1α is an indispensable reference indicator for the accurate diagnosis and treatment of IDD.
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              Possible involvement of the oxLDL/LOX-1 system in the pathogenesis and progression of human intervertebral disc degeneration or herniation

              Epidemiological studies have concluded that hyperlipidemia and atherosclerosis were related to intervertebral disc degeneration (IVDD). The presence of oxidized low density lipoprotein (ox-LDL) and the expression of lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) have not been explored in this tissue. In this study, we investigated the presence of ox-LDL and the expression of its receptor LOX-1 in non-degenerated, degenerated or herniated human intervertebral discs (IVDs). The expression of LOX-1 and matrix metalloproteinase 3 (MMP3) were studied after incubating nucleus pulposus cells (NPCs) with ox-LDL. The presence of ox-LDL and LOX-1 was positively related with the extent of IVDD in nucleus pulposus (NP), end-plate cartilage and outer annulus fibrous, but not with the extent of degeneration of inter annulus fibrous. Ox-LDL significantly reduced the viability of human NPCs in a dose and time-dependent manner, and increased the expression of MMP3 induced by LOX-1. Pretreatment with anti-human LOX-1 monoclonal antibody reversed these effects. Ox-LDL, principally mediated by LOX-1, enhanced MMP3 production in NPCs through the NF-κB signaling pathway. In conclusion, increased accumulation of ox-LDL and LOX-1 in IVDs indicates a specific role of the receptor-ligand interaction in degeneration or herniation of IVDs.
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                Author and article information

                Contributors
                Journal
                Genes Dis
                Genes Dis
                Genes & Diseases
                Chongqing Medical University
                2352-4820
                2352-3042
                18 May 2023
                March 2024
                18 May 2023
                : 11
                : 2
                : 558-560
                Affiliations
                [a ]Department of Orthopaedics, Yongchuan Traditional Chinese Medicine Hospital of Chongqing, Chongqing 402160, China
                [b ]Department of Orthopaedic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China
                [c ]Department of Spine Surgery, The Third Hospital of Mianyang, Sichuan Mental Health Center, Mianyang, Sichuan 621000, China
                Author notes
                [1]

                These authors contributed equally to this work.

                Article
                S2352-3042(23)00200-3
                10.1016/j.gendis.2023.04.009
                10491900
                37692476
                c8fc1f4b-3fbb-45b3-b3ff-d722afa38d54
                © 2023 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 26 October 2022
                : 24 March 2023
                : 17 April 2023
                Categories
                Rapid Communication

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