The Function And Mechanism Of COMP In The Development Of Salivary Gland Pleomorphic Adenoma

ObjectivePleomorphic adenoma(PA)is the most prevalent benign salivary gland tumor type,and accumulating evidence shows that PA exhibits a unique capsular variation,such as pseudopodia,incomplete capsules,capsule infiltration,and satellite nodules,with a crucial role in its recurrence.Although numerous studies have been extensively conducted on the pathological and genetic characteristics of PA to elucidate the pathogenesis of PA,the mechanisms underlying the development of PA are still unclear.Thus,this study was designed to explore m RNA expression profiles in salivary gland PA in an attempt to further analyze genes associated with capsule invasion,and further explore its regulatory mechanism in the development of PA.This finding may help provide a new perspective and theoretical basis for our further understanding of PA,meanwhile,may help in the diagnosis and surgical decision-making of salivary gland PA.Methods1.We evaluated the expression profiles of 4 salivary gland PA patients by RNAsequencing.The principal functions of the differentially expressed m RNAs(DEGs)were explored using GO and KEGG analysis.2.Then,RT-q PCR and correlation analyses were performed to verify the candidate DEGs in 59 PA patients,and immunohistochemical examinations were conducted to validate candidate DEGs.3.The P1 and P2 phase cells were separated and purified,and a novel PA cell line was constructed.The growth characteristics of the P3 phase cells were detected using CCK8 and the phenotype of the P3 phase cells was identified using immunofluorescence(IF).4.Construction of the PA cell line with COMP knockdown,and construct a PA cell line that knocks down COMP stably using lentiviral gene knockdown technology.Construction of a 293 T cell line with COMP overexpression and collect COMP-conditioned medium(COMP-CM).5.Used CCK-8,flow cytometry,Transwell invasion and migration assay,and spheroidization assay to detect the effect of COMP on the biological function of PA.Such as cell proliferation ability,cell cycle progression,cell apoptosis,migration,invasion,and stem cell characteristics.6.Verified that COMP regulates the epithelial-mesenchymal transition mechanism(EMT)of tumor through the PI3K/AKT pathway using western blot and other experiments.Then explored the mechanism that affects the proliferation,invasion and apoptosis of PA cells.Results1.A total of 974 DEGs were significantly upregulated and 1464 were downregulated.2.Based on GO and KEGG analyses,extracellular matrix organization and the PI3K-AKT signaling pathway were found might play pivotal roles in the tumorigenesis of PA.3.40 DEGs were screened and validated by RT-q PCR,11 upregulated and 5downregulated DEGs were consistent with the sequencing results.Cartilage oligomeric matrix protein(COMP)was identified to have a significant correlation with the capsular invasion of PA and the expression of COMP in patients with capsular invasive PA was significantly stronger than PA and adjacent salivary gland tissues.4.Obtained two novel PA primary cells PA25 and PA75 successfully,both of which have the self-renewal ability.5.Successfully constructed PA25 and PA75 cell line that knocks down COMP stably and successfully collected and concentrated COMP-conditional medium.6.COMP was verified can promote the proliferation,migration and invasion while inhibit the apoptosis of PA cells in vitro,and promote the cell cycle of PA cells to the G2/M phase;7.COMP was revealed can activate the PI3K/AKT signaling pathway in salivary gland PA cells;8.COMP can affect the biological functions of PA cells,such as proliferation,migration,invasion,apoptosis and self-renewal ability through the PI3K/AKT signaling pathway.9.COMP promotes the occurrence and development of PA by regulating the EMT and stem cell characteristics of tumor cells.ConclusionsIn this study,through RNA sequencing and verification of PA tissue samples and primary cells,it is the first time to report that COMP plays a crucial role in capsular invasion of PA.Otherwise,it is also the first time to verify that COMP could promote the proliferation,migration and invasion of PA cells,while inhibit it’s apoptosis.It also confirmed that COMP could activate the stem cell characteristics and EMT mechanism of PA cells through the PI3K/AKT signaling pathway,which affects the proliferation,migration,invasion,apoptosis and self-renewal capacity of PA cells,and then regulate the occurrence,development and invasion of PA cells.These results provide a new research perspective on the invasive properties of PA,which may represent a novel potential diagnosis target,too.