Multiomics Data Exploration-based Molecular Typing Of Intervertebral Disc Degeneration And Its Clinical Translational Value

BackgroundIntervertebral Disc Degeneration Disease(IDDD)is a prevalent musculoskeletal disorder that imposes a significant burden on public health and the social economy.The disease commences with intervertebral disc degeneration(IVDD)and advances through a sequence of structural and functional impairments.Presently,interventions and treatments for IVDD encompass conservative therapy,disc repair,spinal reconstruction,disc replacement,and decompression and fusion surgeries.Notwithstanding ongoing experimental and clinical trials,disc repair and spinal reconstruction remain in the nascent stages of development.At present,conservative and surgical modalities are the primary approaches employed in clinical practice,albeit with certain limitations.Physical therapies,such as exercise and weight loss,are only viable for patients with mild degeneration,while drug treatments,including opioids and non-steroids,may result in addiction and dependence.Disc replacement is typically reserved for patients with single-level disease unaccompanied by facet joint disease.Decompression and fusion surgeries result in the loss of segmental motion of the spine.Additionally,current treatment options for IVDD do not directly address the underlying cause of the condition,and there is a dearth of clinically recognized molecular typing and precision diagnosis and treatment strategies for IVDD.Consequently,to enhance the IVDD diagnosis and treatment system,there is a need for tools that can effectively differentiate the disease’s severity and precisely determine its prognosis,thereby facilitating clinicians in risk stratification and decision-making for IVDD patients.Objective1.To propose a IVDD molecular typing based on single-cell multi-omics sequencing techniques.2.To evaluate the clinical translational application of IVDD molecular typing.3.To examine the effects of targeted drugs on the IVDD molecular typing through in vitro experimentation.Material and Methods1.The present study acquired single-cell data and microarray data of IVDD patients from the Gene Expression Omnibus(GEO)public database.Cell differentiation trajectory analysis was performed using the Monocle software package,while the DDRTress reduce Dimension function was utilized to identify the pseudo-time of distinct cell subsets.Non-parametric testing and Venn diagram analysis were employed to cross-screen genes associated with cell differentiation and prognosis.Subsequently,the Consensus Cluster Plus software package was utilized to construct the molecular typing of IVDD based on the selected key genes.Finally,the relationship between IVDD molecular typing and clinical prognosis was assessed by utilizing the degree of disc degeneration and Thompson grade as outcome variables.2.The clinical utility of CDBIVDDC was assessed via a case-control trial that involved IVDD patients who underwent discectomy and interbody fusion at the Department of Orthopedic and Spinal Surgery,Changhai Hospital,Shanghai.During the surgical procedure,we obtained excised intervertebral discs,which were subsequently transformed into paraffin-embedded samples for the purpose of immunohistochemical staining and scoring of six marker proteins specific to the three subtypes of CDBIVDDC(ADCYAP1,PTHLH,MIA,TUBA1 C,LGALS1,and AGR2).Additionally,we gathered baseline and imaging data from patients,as well as their Oswestry Disability Index(ODI)scores.The ODI score > 50,presence of degenerative scoliosis(DS),and Pfirrmann grade of lumbar MRI were utilized as outcome variables to assess whether LSCS in CDBIVDDC was a risk factor associated with severe symptoms,poor prognosis,and outcome.3.Through the utilization of the p RRophetic package in the R programming language,we were able to predict drugs with differential sensitivity for LCSC in CDBIVDDC,including Gefitinib.In vitro,we established degenerative cell models utilizing human immortalized chondrocytes with IL-1β.The experimental design consisted of a control group,an IL-1β group,and an IL-1β + Gefitinib group.Cell proliferation was assessed through the Cell Counting Kit-8(CCK-8)assay,while chondrocyte morphology was evaluated using the toluidine blue staining assay.Real-time PCR and Western blot assays were conducted to investigate changes in m RNA and protein levels of chondrocyte synthesis and catabolism-related genes and CDBIVDDC marker genes.Results1.A multiomics integration analysis was performed utilizing scRNA-seq data from27,001 quality-controlled cells in annulus fibrosus(AF)samples,in conjunction with microarray data from IVDD patients obtained from the GEO database.The study yielded the identification of 44 genes that are associated with distinct chondrocyte differentiation fates and IVDD clinical prognosis.These genes were utilized to construct the Chondrocyte Differentiation-Based IVDD Classification(CDBIVDDC),which was categorized into three groups based on chondrocyte scores: High Chondrocyte Score Classification(HCSC),Low Chondrocyte Score Classification(LCSC),and Median Chondrocyte Score Classification(MCSC).The findings indicated that HCSC exhibited a favorable prognosis,characterized by elevated levels of ADCYAP1 and PTHLH,whereas LCSC demonstrated an unfavorable prognosis,marked by heightened expression of MIA and TUBA1 C.MCSC,on the other hand,displayed a moderate prognosis,as evidenced by increased levels of LGALS1 and AGR2.2.A total of 60 patients diagnosed with IVDD who met the study’s inclusion and exclusion criteria were included in the analysis.Of these patients,26 were male(43.33%)with a mean age of 51.92±15.16 years,while 34 were female(56.67%)with a mean age of56.35±14.90 years.Subsequently,based on the immunohistochemical staining scores of the marker genes in the three subtypes of CDBIVDDC,the 60 IVDD patients were categorized into three groups: HCSC group(14 cases,23.33%),the LCSC group(30 cases,50%),and the MCSC group(16 cases,26.67%).The findings of the univariate analysis indicated that patients belonging to the HCSC group exhibited lower ODI scale scores,lower Pfirrmann grades,and a reduced likelihood of being diagnosed with DS(P < 0.001).Conversely,patients in the LCSC group demonstrated higher ODI scores,higher Pfirrmann grades,and an increased probability of being diagnosed with DS(P < 0.001).Our investigation posited that LCSC in CDBIVDDC represented a risk factor that is associated with severe clinical symptoms,poor quality of life,and an unfavorable prognosis for IVDD patients,while the HCSC group exhibited the most favorable prognosis.Therefore,CDBIVDDC had clinical translational and application value in diagnosing and predicting the prognosis of IVDD patients.3.The results of the CCK-8 assay demonstrated that the IL-1β group exhibited a notable reduction in cell proliferation,whereas the IL-1β + Gefitinib group was able to counteract this effect and enhance cellular proliferation capacity(P < 0.001).Toluidine blue staining revealed that cells in the IL-1β group displayed an elongated and spindle-shaped morphology with mild extracellular matrix staining and indistinct nuclear borders,whereas cells in the IL-1β + Gefitinib group exhibited a polygonal shape and demonstrated more intense staining of the extracellular matrix.Real-time PCR and Western blot analyses revealed that the expression levels of COL2,SOX9,and ADCYAP1(HCSC)were significantly reduced in the IL-1β group compared to the control group,while the expression levels of MMP13 and MIA(LCSC)were significantly elevated.However,upon treatment with gefitinib,the expression levels of COL2,SOX9,and ADCYAP1(HCSC)were significantly increased,while MMP13 and MIA(LCSC)were significantly decreased.These results suggested that the expression levels of CDBIVDDC subtype marker genes were significantly altered in the IL-1β-induced degeneration model of chondrocytes in vitro.Gefitinib,a small-molecule targeted agent for LCSC,had exhibited potential for transdifferentiation,thereby rendering it a viable targeted therapy for IVDD.ConclusionThe CDBIVDDC concept was proposed for IVDD,which comprehensively accounted for diverse chondrocyte differentiation fates at the molecular level and correlated with distinct clinical outcomes of IVDD patients.The clinical applicability of CDBIVDDC in IVDD patients was validated through a case-control study.Our in vitro experimental findings indicated that Gefitinib not only effectively ameliorated IL-1β-induced chondrocyte degeneration but also exhibited the potential to transdifferentiate LCSC into HCSC.The results of our study indicated that CDBIVDDC may serve as a dependable instrument for clinicians to precisely assess the degree of IVDD severity,anticipate disease prognosis,facilitate risk evaluation,and support clinical decision-making for individuals with IVDD.