Mechanosensitive Genes Screening By Multi-Omics Integrated Analysis And MYOC Functions In Osteoarthritis Cartilage

Osteoarthritis(OA),with high disability and mortality,is the most common arthritis throughout the world.The main pathological features of OA include cartilage degeneration and cartilage surface tissue damage.Many researchers suggest mechanical environment variation is the key OA initiating factor and plays a vitro role in its development.However,the mechanisms of mechanical force influences in cartilage degeneration are still uncovered.In our research,we identified and validated important mechanosensitive genes in OA and cartilage degeneration with multi-omics integrated analysis,to study the gene-mechanical interactions and its mechanobiological mechanisms.Here,we return some results and following conclusions via conducted multi-platform experiments and data analysis:1.Identification of OA-related differentially expressed genes(DEGs)in cartilage.Firstly,with keywords searching in comprehensive databases GEO,ArrayExpress and Pubmed,and data mining,we got 11 and 13 OA-related gene expression datasets from human and rat,respectively.We analyzed these datasets and got DEGs,then followed by functional analysis for DEGs with DAVID,respectively.After combination of human and rat source results,we have chosen 11 common DEGs which showed identical and significant change trend as OA-related candidate genes.Meanwhile,with data mining,we got several OA-related differentially expressed microRNAs and DNA methylation profiles,GSE76326 and GSE63695.Through comprehensive analysis between these microRNAs or methylation and OA related DEGs,we have provided the possibility of these expression variations of OA-related candidate genes from the perspective of epigenetic regulation.2.Identification of OA cartilage-related mechanosensitive gene,MYOC.With keywords screening and multi-selection in comprehensive database GEO and PubMed,we got 20 datasets of expression profiles from different mechanical models such as fluid shear,hydrostatic pressure and loading.After classified,we analyzed each dataset to find the mechanical DEGs and did functional analysis for these genes.Combined with OA related candidate genes in result(1),we identified OA cartilage related mechanosensitive gene,MYOC.3.Validation and functional analysis of mechanosensitive gene,MYOC.(1)In IL1 B induced chondrocytes model,with PCR detection,MYOC expression was decreased significantly after IL1 B induction.Meanwhile,in anterior cruciate ligament transection rat model which would induce the mechanical imbalance of cartilage,MYOC was down-regulated in cartilage compared with control groups by experimental verification.At the same time,considering the bioinformatics analysis results of IL1 B induced cell model datasets,GSE11427 and GSE75181,and mouse meniscectomy model dataset GSE53857,we found MYOC expression results were identical with the simulation results in comprehensive database and confirmed that MYOC expression is closely related with OA cartilage.(2)In hydrogel matrix model,MYOC expression was decreased followed the increase of the matrix stiffness(4.5 KPa and 37 Kpa)with PCR detection.In the normal rat,MYOC expression varies in different regions of cartilage with highest expression level in the surface,by immunofluorescence detection.Meanwhile,according to the bioinformatics analysis results of different model datasets such as matrix stiffness(GSE63615),3D culture model(GSE16464)and gene expression datasets in cartilage(GSE54216,GSE39795,GSE39796),we confirmed that MYOC expression results were identical with the results in database which suggested that MYOC expression is regulated by mechanical stimulation and its expression has spatial specificity.(3)Through analysis of MYOC transfected HEK293 cells model GSE53985 and transgenic mouse model GSE1835 datasets,we found several genes regulated by MYOC including CLO12A1,MMP15 and ADAMTS1.Meanwhile,we analyzed several cellular induction model of multiple factors,like GSE68760,GSE85254,and found Galectin inhibits MYOC expression in cartilage.Combined with protein interaction network by STRING,we identified that MYOC,as a mechanosensitive gene,is an important regulator in mechanotransduction cascade signaling in cartilage and by affects cell adhesion and extracellular matrix homeostasis.In summary,through multi-omics integrated analysis,we identified MYOC as an OA related mechanosensitive gene.Meanwhile,through various in vitro and in vivo models and bioinformatics analysis of several gene expression datasets,we did the experimental verification and function analysis of MYOC.The results showed that MYOC expression is mechanical dependent and we hypothesized that MYOC regulates mechanotransduction cascade signaling as a gene-mechanical interaction regulator and takes part in the OA degeneration.This project taking gene-mechanical coupling mechanism as a starting point,identified mechanosensitive genes and it will provide new approach and visions for OA research.