Integrated transcriptome and proteome analyses identifies novel genes and regulatory networks in intervertebral disc degeneration [post]

2019 unpublished
Intervertebral disc degeneration is a major cause of symptoms like low back pain and neck pain. Many groups have tried to reveal the regulatory network using either transcriptome or proteome profiling technologies, however, the relationship between these differentially expressed proteins and mRNAs are not elucidated. Since posttranscriptional regulation and other mechanisms may affect the translation of mRNA to protein, a combined transcriptome and proteome study may give more precise data on
more » ... veiling important regulatory network and key genes of Intervertebral disc degeneration. Results: In the present study, we used the label-free quantification proteomic approach and identify 656 proteins expressed in either degenerated or normal nucleus pulposus, of which 503 proteins are differentially expressed. Taking advantage of the existing nucleus pulposus transcriptome data, we combine and reanalyze the data and find 105 differentially expressed mRNA between degenerated and normal nucleus pulposus. By comparing these data, only 9 genes show significant changes in both protein and mRNA data, while 6 genes (TNFAIP6, CHI3L1, KRT19, DPT, COL6A2 and COL11A2) show concordant changes in both protein and mRNA level. Further functional analyses show different functions of the altered mRNAs and proteins in degeneration, indicating great difference between protein network and mRNA network. Using the gene co-expression network method, we uncover novel regulatory network and potential genes that may play vital roles in intervertebral disc degeneration by combining protein and mRNA data. Conclusions: This is the first study to identify novel regulatory network of intervertebral disc degeneration using combined analysis of both transcriptome and proteome, which may give new insight into the molecular mechanism of intervertebral disc degeneration. Background 4 Low back pain and neck pain are a general muscular disorder causing severe social and economic burdens and loss of work (1). Current studies have revealed that these symptoms are mostly associated with intervertebral disc degeneration (IDD) (2, 3). IDD is characterized by a series of pathogenic processes including cellular, biochemical and structural impairment which resulted in metabolic imbalances of the extracellular matrix (ECM), which mainly take part in the nucleus pulposus (NP). The etiology however, is complex and multifactorial, in which aging, certain diseases and injuries, and genetic factors are all involved. Since the molecular mechanisms of IDD are still not completely understood, current treatment is largely depended on symptomatic relief using nonsteroidal or steroidal anti-inflammatory medication, while surgical intervention for latestage IDD with severe neurological symptoms caused by herniation of the disc. Thus, a better understanding of then etiology of IDD will definitely throw light on targeted and less invasive therapies while promoting the living quality of the patients. The first organ-level change in disc degeneration appears to be a functional compromise in the ability of the nucleus pulposus (NP) to imbibe water, leading to a decrease in intradiscal pressure (4). Biomechanically, the consequences of a swollen nucleus are to alter the transfer of load and to create hydrostatic pressure in the center of the disc (5). The distinct biomechanical functions of the annulus fibrosus (AF) and NP are confirmed by their respective constituents or extracellular matrix (ECM) (6). The NP is proteoglycan-rich with type II collagen, which provides the osmotic properties necessary for optimal disc hydration, while the AF is mostly ligamentous fibrocartilage with type I collagen optimized for resisting tensile load. When their balance is altered, the degeneration occurs. The altered ECM of NP involving a decrease in synthesis and accumulation of proteoglycans and type II collagen, increased synthesis of collagen I, and a decreased glycosaminoglycan/hydroxyproline ratio are present at the degenerated disc (7). 5 Mechanism studies have showed different factors that play vital roles in disc degeneration. The activities of matrix-degrading enzymes (matrix metalloproteinases, MMPs), their regulators (such as TIMPs and THBS), inflammatory molecules such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor α (TNFα) are shown related to IDD (8, 9) . Other anabolic factors like insulin-like growth factor (IGF-1), its receptor (IGF-1R), chondroitin sulfate synthase 1 (CHSY-1), chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACT1) are also greatly associated with IDD(10). However, these factors alone are not sufficient to reveal the whole regulatory network of IDD. High-throughput technologies have provided new ways to identify critical factors with the help of bioinformatics analysis, also the interaction and function of unknown genes or network can be predicted. Thus, many studies have dedicated to decipher the disease regulatory network using such method (11-13). Intriguingly, the results are not always consistent between studies, and such problems can be caused by sample variation or other regional factors. Moreover, recent studies purpose that posttranscription and translational regulation are important ways to affect the gene expression in IDD, add to the complexity of the mechanism network of IDD (14). Thus, more precise global expression data are needed before focusing on the detailed mechanism of each gene. mRNA translation involves many mechanism such as post-transcription and translational regulation. Disorder of such mechanisms can cause uneven translational production of the mRNAs (15). Thus, either transcriptome or proteomic data alone would not fully elucidate the network. In order to acquire a comprehensive and precise profile data of IDD, here we try to combine the proteomic data with transcriptome data. Using label-free highthroughput proteomic technique, we analyzed the global protein expression of normal and IDD NP tissues. Taking advantage of the existing NP high-throughput microarray data, we (PANTHER) database v 6.1 ( Pathway analysis were performed with the tools on the Kyoto Encyclopedia of Genes and Genome (KEGG) database Co-expression network costruction. By comparing the normal NP with degenerated NP data, we constructed a co-expression network for both groups according to the gene's mRNA or protein expression trend during IDD (fold change). Both the mRNA and the protein data were used, and if a gene's expression is reversed between mRNA and protein level, we confirm it as a noise. The relationship between the genes were calculated using KCore method, and the correlation of the two genes were calculated using the pearson correlation, and the candidate genes were selected according to the FDR and P value. The network is further constructed according to the KCore and the calculated Degree. Immunohistochemistry analysis. Immunohistochemistry was performed to localize KRT19, COL6A2, DPT, COL11A2, CLIP and CHI3L1 in NP samples of 3 Pfirrmann grade 1 (IVD group) and 3 NP samples of grade IV-V (IDD group). The procedure is according to the standard protocol, briefly, antigen retrieval was performed using trypsin for 30 min at 37°C, and the sections were blocked with 1% bovine serum albumin for 15 min at room temperature. Next, the sections were incubated at 4°C overnight with the rabbit polyclonal antibody against KRT19, COL6A2, DPT,
doi:10.21203/rs.2.15949/v1 fatcat:p5nedns7o5b2ribaitvturrsrq