The Role And Mechanism Of PI16 In The Malignant Melanoma

Objective:To explore the role and mechanism of PI16 in Malignant Melanoma(MM).Methods:In the first part of the study,the gene variants and abnormal expression of PI16 in MM were analyzed through analyzing the public databases.IHC,RT-qPCR and WB were applied to detect the differential expression level of PI16 in MM tumor tissues,and benign melanocytic nevus and normal skin tissues were used as control.The differential expression of PI16 in multiple human MM cell lines compared with normal human epidermal melanocytes was further verified in vitro.In the second part,the specific targeted sequence of PI16 gene was synthesized,packaged by lentiviral vector,and transfected into MM cells.The stable PI16 knockdown and overexpression MM cell lines were successfully constructed,and the effect of PI16 on the malignant biological behavior of MM was studied in vivo and in vitro.CCK8 and EdU kits were used to detect cell proliferation in vitro.Cell cycle and apoptosis were detected by flow cytometry.Cell invasion and migration were detected by Transwell invasion and migration assay and scratch healing assay.The tumor formation,tumor volume and weight,and the expression levels of related biomarkers were recorded to study the effect of PI16 knockdown on the occurrence and development of MM tumors in vivo.In the third part,RNA-seq and LCMS/MS were used to identify the differentially expressed molecules after PI16 knockdown,and the potential mechanisms were analyzed by bioinformatics methods.Finally,RTqPCR,WB,IHC and Co-IP biological experiments were performed to verify the main.mechanism of PI16 in MM.Results:In the first part of the study,the expression of PI16 was found abnormally upregulated in MM,suggesting that PI16 may play an important role in the pathogenesis of MM.In the second part,the in-vitro biological behavior associated experiments were performed after PI16 knockdown and overexpression in MM cell lines.The results showed that the cell proliferation,invasion and migration ability of shPI16 group were significantly decreased,cell cycle was arrested,and cell apoptosis was increased compared with the empty vector control group.Correspondingly,the proliferation,invasion and migration of tumor cells in the O/EPI16 group were increased,the cell cycle was shortened,and the cell apoptosis percentage was decreased.The results of in-vivo experiments also showed that the weight and volume of subcutaneous transplanted tumors in the shPI16 group were lower than those in the control group;the tumors in the shPI16 grew more slowly and expressed lower level of Ki-67 marker.In the third part,based on the combined analysis of transcriptome and proteomic sequencing,the results showed that the molecular functions affected by PI16 knockdown were closely related to cell adhesion and connection,migration,extracellular matrix composition and angiogenesis.In addition,key molecules of vascular endothelial growth factor,extracellular matrix receptors,cytokines,p38MAPK,PI3K-AKT and ERK signaling pathways were also significantly different.Consistently,the results of biological experiments revealed that downregulation of PI16 inhibited the expression levels of epithelial-mesenchymal transition(EMT),anti-apoptosis and cell cycle markers,as well as the signal transduction of multiple key pathways in MM.PI16 is also involved in the regulation of remodeling extracellular matrix by binding to fibronectin 1(FN1).Conclusions:PI16 binds to FN1 in MM and regulates multiple signaling pathways,to promote tumor EMT,remodel tumor extracellular matrix,inhibit tumor apoptosis,accelerate the growth cycle,and promote tumor proliferation,invasion and migration.