MiR-216A-Mediated Upregulation Of TSPAN1 Contributes To Pancreatic Cancer Progression Via Transcriptional Regulation Of ITGA2

Pancreatic cancer(PC)is recognized as the most aggressive tumor,which is the number seventh highest mortality of all cancers in humans.Despite the development of diagnosis and treatment of PC,the 5-year survival rate is still less than 30%.Therefore,exploring the molecular mechanism and developing new diagnostic and therapeutic strategies are prerequisites for the effective treatment of PC.TSPAN1(Tetraspanins 1),a member of the transmembrane4-super-family(TM4SF),has been found abnormally elevated expression in patients with pancreatic cancer and the high expression of TSPAN1 is associated with poor prognosis.However,the molecular mechanism of TSPAN1 in pancreatic cancer is still unclear.Both the Target Scan and mi Randa databases predicted several possible mi RNAs that potentially target TSPAN1.To further investigate the interaction between mi R-216a-3p and the TSPAN1 3'-UTR,we performed the luciferase activity assay.In PANC-1 cells,mi R-216 a significantly decreased the firefly luciferase activity of the TSPAN1-3'UTR-WT reporter but not that of the mutant edition.Subsequently,we successfully established two stable transfected cell lines(PANC-1-mi R-216a-3p and PANC-1-NC).The functional regulatory role of mi R-216 a on TSPAN1 was investigated through various essential phenotypic experiments.We found that mi R-216a-OV cells showed significantly slower proliferation rates,reduced clone-forming potency,and attenuated migration and invasion abilities compared with parallel control cells.To explore the downstream pathways that may be regulated by TSPAN1,we performed RNA sequencing(RNA-Seq)using TSPAN1-OV and sh TSPAN1 and their corresponding control cells.Combine the screening of DEGs,KEGG pathway enrichment analysis and GO analysis,we selected ITGA2 for further study as the downstream target of TSPAN1 in PC.RNA-Seq results showed a remarkable increase in ITGA2 m RNA in TSPAN1-OV cells and decreased ITGA2 m RNA levels in sh TSPAN1 cells.Finally,we confirmed the oncogenic role of ITGA2 in PC and the tumor-promoting capacity of TSPAN1 could be markedly abolished by ITGA2 knockdown.In summary,these results reveal that the oncogenic function of TSPAN1 in pancreatic cancer is closely related to its regulatory effect on ITGA2.The RNA-seq results revealed that overexpression of TSPAN1 resulted in the elevation of TET2 enzymes,but a decrease in the levels of the methyltransferases DNMT3 B and DNMT1.Meanwhile,we found epigenetic regulation of ITGA2 expression via alterations in the methylation status of ITGA2,which was regulated by TSPAN1.In addition,we identified two predicted significant methylation sites affected by TSPAN1 by bisulfite sequencing.Our results reveal the mechanism by which TSPAN1 regulates ITGA2 expression,and also verified that the regulation of methyltransferases by TSPAN1 is one of the reasons for the hypomethylation of ITGA2 in PC.In conclusion,the oncogenic function of TSPAN1 in pancreatic cancer is closely related to its epigenetically regulation of ITGA2.Meanwhile,the upregulation of TSPAN1 in PC is the consequence of low expression of miR-216a.