Aberrantly expressed messenger RNAs and long noncoding RNAs in degenerative nucleus pulposus cells co-cultured with adipose-derived mesenchymal stem cells

Intervertebral disc degeneration (IDD) due to aging and other reasons is still a major clinical challenge for spine surgeons. Numerous strategies have been studied to prevent the deterioration and progressing of IDD, which will cause various pathologies, such as disc herniation, spinal stenosis and segmental instability [1]. However, disc degeneration is difficult to reverse in the natural course due to the avascular and aneural nature of the nucleus pulposus (NP). Biochemically, the degeneration is characterized by the decrease of the extracellular matrix and loss of water content within the NP, which are believed to play a crucial role in the progress of IDD.

Recently, stem cell therapy, introducing mesenchymal stem cells (MSCs) or cytokines into target discs, appears as a promising strategy for the IDD treatment. This emerging modality was proved to be effective both in vitro [2‐4] and in vivo [5, 6]. Co-culturing with nucleus pulposus cells (NPCs), MSCs could differentiate towards the NP-like cells with increased expression of the NP marker genes [7]. In particular, adipose-derived mesenchymal stem cells (ASCs) co-cultured with NPCs expressed significantly upregulated expression of NP-related genes including sex determining region Y box 9 (SOX9), type II collagen (COL2A1), and aggrecan (ACAN) [8]. Interestingly, our previous study demonstrated that degenerative NPCs could be activated by MSCs in the co-culture system with significantly upregulated gene expression of SOX9, COL2A1, and ACAN [4]. It is notable that degenerative NPCs could be induced towards healthy NPCs by specific cytokines and cellular factors yielded from MSCs [9]. Also, it underlines that the gene expression pattern of not only MSCs but also degenerative NPCs could be altered through the intracellular cross-talking [10, 11]. However, the underlying mechanism of these phenomena is still not fully understood.

Gene microarray technology can simultaneously measure differences in the expression level of thousands of genes of predefined groups of samples [12] and allows highly effective evaluation of genome-wide expression changes [13]. This novel technology enables researchers to develop a more comprehensive understanding of the cross-talking between MSCs and degenerative NPCs, including differentially expressed genes and long non-coding RNAs (lncRNAs).

Recently, lncRNAs have received critical attention with their regulatory effect on gene expression [14, 15]. They have been characterized with the high tissue-specificity and low sequence-conservation [16, 17] and have been demonstrated to be involved in various physiological and pathological processes as regulators, such as imprinting, X-inactivation, and development [18]. Numerous previous studies have attempted to map the phenotype of NPCs [19‐22], to the best of our knowledge, yet few studies have emphasized the function of lncRNAs related to disc degeneration, particularly their regulatory roles in the process of degenerative NPCs co-cultured with MSCs [23].

Similarly, micro RNAs (miRNAs), a group of small and non-coding RNAs, have been proved to participate in the expression regulation of coding genes and to influence various biological processes, including cell differentiation, proliferation, and metabolism [15, 24]. Many miRNAs have been demonstrated to be involved in natural disc degeneration [25, 26], and specifically miR-27b [27] and miR-93 [28] have been shown to promote matrix degradation within the discs and accelerate the disc degeneration process. Yet, there was no study focusing on the role of these miRNAs in the cell-cell cross-talk between MSCs and degenerative NPCs.

The current study aimed to use gene expression microarray analysis and bioinformatics methods to investigate the effect of MSCs on degenerative NPCs in terms of deferentially expressed lncRNAs and mRNAs, signaling pathways, and gene regulation networks involving mRNAs, lncRNAs, and miRNAs (Additional file 1).

All human tissues were obtained and used with informed consent from the patients and under the approval of the Institutional Review Broad of the Shanghai General Hospital, Shanghai Jiaotong University.

In the present study, we identified a significant number of mRNAs and lncRNAs differentially expressed by degenerative NPCs co-cultured with ASCs, using the microarray analysis and in-depth data profiling. We also identified the signaling pathways that were altered during the co-culture and outlined the co-expression relationship between mRNAs and lncRNAs. The disc-degeneration-related miRNAs, differentially expressed mRNAs, and lncRNAs were further evaluated to identify the regulatory interaction, highlighting the biological pathways and cellular events of gene expression and regulation during the stem-cell therapy process. In addition, our research validates the previous studies about NPC phenotypes [19‐22] and further investigated the regulatory role of lncRNAs. However, more in-depth understanding of these gene expression and regulation profiles will provide valuable clues for gene therapy approaches for disc degeneration.

In the present study, we revealed the altered mRNAs and lncRNAs between NPCs before and after co-culture with ASCs. Real-time PCR results of seven randomly selected mRNAs (Fig. 3) were consistent with the microarray data, further confirming the high credibility of the microarray analysis. Furthermore, we identified that the NP marker gene expression, SOX-9 (fold change = 7, P < 0.001) and COL2A1 (fold change = 6, P < 0.001), was increased in NPCs after the co-culture, indicating the upregulated synthesis and secretory activities of NPCs under the influence of MSCs [37, 38]. These findings were consistent with the previous studies reporting the significantly raised expression of SOX-9 and COL2A1 in NPCs when co-cultured with MSCs [39, 40].

In particular, we demonstrated that SPP1 was the most significantly altered gene and might be indicated as a marker of NPC (Additional file 6). SPP1, also known as osteopontin (OPN), is an extracellular structural protein secreted by various types of cells. Our data from the signal-net analysis suggested that SPP1 could activate the integrin proteins, e.g. integrin alpha (ITGA) 1, 3, 4, 6, 7, 8, and 10, and has close connections with COL11A1, COL3A1, and COL2A1 (Fig. 7). Since integrins are a class of cell adhesion molecules that regulate interactions between a cell and its surrounding matrix [41], activation of ITGA will assist NPCs to interact with the extracellular matrix, specifically collagen molecules, and then to potentially restore the intervertebral disc function. Marfia and colleagues previously reported greater expression of SPP1/OPN and CD44 in degenerative IVD comparing to herniated IVD, and SPP1/OPN was only detected in degenerative IVD tissue [42]. They therefore assumed that SPP1/OPN might mark the severity of disc degeneration. However, in disagreement with Marfia’s study, our data revealed that SPP1/OPN was upregulated with downregulated CD44 when degenerative NPCs were co-cultured with ASCs (Additional file 7). Such a considerable divergence warrants further in-depth investigations into the sophisticated underlying mechanism.

We also demonstrated that CD24 were upregulated (Additional file 6) and these data were in accord with previous studies, signifying CD24 as an important marker of NPCs and notochordal cells [22, 43‐48]. Particularly, Ricardo Rodrigues-Pinto et al. demonstrated that CD24, a glycosylphosphatidylinositol anchor protein, is one of the notochord-specific markers during the early development of human IVD [47]. Similarly, Nobuyuki Fujita et al. [46] identified CD24 as a surface marker for NPC with its high expression in the healthy and herniated NP tissue rather than in the annulus fibrosus. Furthermore, CD24 is proved to be a key marker of the irreversible cellular hierarchy during the differentiation process of the NP-progenitor cells towards NP-committed cells in mice and humans [49]. Therefore, in our study, the upregulated expression of CD24 also confirmed the positive effect of ASCs on NPC regeneration.

Functional annotation of these differentially expressed mRNAs and lncRNAs was investigated by GO and KEGG pathway analysis. The GO analysis identified significant enrichments in over 197 GO terms, including cell adhesion (GO:0007155), positive regulation of transcription from RNA polymerase II (Pol II) promoter (GO:0045944), and extracellular matrix organization (GO:0030198). These data indicate that the metabolic activities of NPCs might be enhanced by ASCs. Specifically, the cellular adhesion is crucial for stable connections between cells and tissue structure maintenance, also involved in diverse signal transduction [50]. The upregulated RNA Pol II promoter of co-cultured NPCs in the present study reflects the increased expression of protein-encoding genes. The RNA Pol II together with other factors, mediating the transcription initiation of protein-encoding genes, is an essential control point for gene expression in the eukaryotes [51]. Similarly, upregulated extracellular matrix organization would counteract the loss of extracellular matrix and further favor the regeneration of NP tissue [52].

Additionally, the most important signaling pathways altered during the co-culture were the MAPK pathway, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B) signaling pathway, and the PI3K-Akt signaling pathway (Fig. 5). Specifically, the NF-kappa B and MAPK signaling pathways, the principal regulators of inflammation and catabolism [53], are important in the symptomatic disc-degenerative diseases [54‐56]. The MAPK signaling pathway was generally increased in the aged and degenerative discs [57, 58]. Our analysis displayed the downregulation of the MAPK signaling pathway, indicating degenerative NPCs could be stimulated towards normal NPCs by ASCs. In addition, NF-kappa B targets several pro-inflammatory cytokines [59‐61] that are highly expressed in degenerative discs rather than normal discs. Therefore, the downregulated NF-kappa B signaling pathway of co-cultured NPCs in our study may indicate that ASCs protect NPCs from inflammatory response. Furthermore, the PI3K-Akt signaling pathway was identified as the most significantly upregulated pathway. Activated PI3K-Akt can protect against the disc degeneration [62, 63] with increased extracellular matrix synthesis [64], promoted cell proliferation [65], counteraction of cell apoptosis [66], and alleviated oxidative damage [67].

Moreover, the signal-net analysis displayed that glycogen synthase kinase 3 beta (GSK3B), which was downregulated, plays the most critical role in the network with the highest betweenness centrality. As a serine/threonine kinase, GSK3 is involved in the phosphorylation of numerous substrates, including signaling proteins, transcription factors and structural proteins [68, 69]. As a crucial mediator of PI3K-Akt, PKA, PKC, and Wnt/ß-catenin, GSK3 is proved to play an important role in chondrocyte differentiation [70‐72]. Miclea et al. demonstrated that GSK3B could inhibit the chondrocyte proliferation and increase the cartilage apoptosis via activating the canonical Wnt signaling pathway in the ex vivo mouse embryos [73]. Also, Itoh et al. demonstrated that GSK3 proteins are involved in early stages of chondrocyte differentiation by driving the differentiation in a cell-autonomous manner [74]. In the present study, we also found that after co-culture, the degenerative NPCs expressed significantly higher SOX9 and COL1A2, confirming the restored function of the NPCs.

Regarding the regulatory functions of lncRNAs, in the present study we investigated the co-expressional connection between mRNAs and lncRNAs and found that XIST acquired the greatest number of interactions (degree = 77) among all RNAs. XIST, a 17–20 kb RNA, binds the X chromosome [75, 76] to initiate X chromosome inactivation [77] and is required for whole-chromosome silencing [78]. Also, XIST provides one of a few tangible readouts for the stem cell quality [79] and also influences the pluripotent stem cell population, as proved in induced pluripotent stem cell treatments in regenerative medicine [80]. However, such significant roles have not studied in the context of disc degeneration and further investigations are warranted.

A competing endogenous RNAs network was constructed for the regulatory function of various lncRNAs and many miRNAs associated with disc degeneration. miR-98-5p is the most important in the network (interaction degree 57) followed by the miR-27a-34 (interaction degree, 40) (Fig. 9). miR-98 is significantly downregulated in the degenerated NP tissue and has been proved to promote type II collagen expression in NPCs [81]. Also, Li et al. reported that the downregulation of miRNA-27b would yield loss of type II collagen and lead to the development of IDD [27]. Therefore, the upregulation of these two miRNAs, favoring type II collagen synthesis of the NPCs, demonstrated the positive effect of ASCs on degenerative NPCs and might serve as potential therapeutic targets in IDD.

This study has some limitations. First, numerous RNA probes were investigated in the microarray analysis and this limited the validation of the gene chip results. Therefore, we only interpreted the results based on previous studies and our interests. Also, to avoid losing information, genes and lncRNAs were not further classified into specific subsets according to their functions and only the regulatory roles of differentially expressed RNAs of general interest were interpreted, serving as potential targets of the further in-depth investigation. Nevertheless, the results from our bioinformatics analysis only elucidate relevant relationships associated with these genes and lncRNAs; more in vitro or in vivo studies are necessary to comprehensively understand the specific involvement of the differentially expressed lncRNAs and mRNAs during the co-culturing process.

To sum up, co-culturing human ASCs with degenerative NPCs restored the biological status of the degenerative NPCs. Our study identified the interplay between ASCs and degenerative NPCs during the co-culturing and provided valuable information for the development and application of gene therapy for IDD. More studies are required to explore the functions and mechanisms of the key RNAs involved in the regeneration of the human intervertebral disc tissue and further benefit the translation of gene therapy in IDD from bench to bedside.