A Study Of Effect And Mechanism Of CircARMC9 On Perineural Invasion In Pancreatic Cancer

Background:Pancreatic cancer is one of the highly malignant digestive system tumors and despite the recent improvements in diagnosis and treatment techniques and updates in treatment philosophy,the 5-year survival rate remains less than 9%.Perineural invasion(PNI)is a typical pathological feature of pancreatic cancer and is considered to be the main cause of recurrent and neuropathic pain after therapeutic resection.It is also a prelude to poor patient prognosis.With the rapid development of precision medicine and molecular biology,we have gradually realized that the occurrence of PNI is not the result of a single factor.The latest studies indicate that PNI in cancer is a continuous and multi-step process that involves a specific tumor microenvironment(TME)composed of nervous cells,supportive cells,recruited inflammatory cells,altered extracellular matrix,blood vessels,and immune components.Cancer-associated fibroblasts(CAFs)as activated stromal cells are increasingly reported to promote tumor-nerve infiltration mechanisms by releasing cytokines,growth factors,and hormones.Objective:The molecular mechanisms of circRNA in regulating pancreatic cancer nerve infiltration are still to be elucidated.Therefore,exploring the mechanism of circRNA regulation of pancreatic cancer nerve infiltration provides a theoretical basis for finding targets for pancreatic cancer treatment and has important clinical and scientific significance.Methods:First,RNA-seq(GSE172096)from cancer-related fibroblasts and paired normal fibroblasts(NFs)was analyzed and 50 up-regulated circRNA in CAFs were identified.Quantitative real-time PCR(qRT-PCR)was performed to screen for the expression of CAFs-specific circRNA and the results showed that three circRNA:circFARP1,circCUL2,and circARMC9 were significantly expressed in CAFs,but not in cells or other stromal cells.The expression of circARMC9 in pancreatic cancer tissue was verified by qRT-PCR technology and fluorescence in situ hybridization,and circARMC9 was selected as the research object.Further Sanger sequencing,RNase tolerance experiments,and PCR amplification were used to identify the circularity of the CircARMC9,and the subcellular localization of the circular RNA in cancer-related fibroblasts was determined by fluorescence in situ hybridization.The effects of circARMC9 on the perineural invasion and tumor migration and invasion of pancreatic cancer cells were verified by Transwell migration assays,3D coculture models of neurospheres,and mouse xenograft models.To reveal the mechanism of circARMC9,transcriptome sequencing technology and GO and KEGG enrichment were used to identify possible pathway changes,and Western blotting and ELISA were used to further reveal the mechanism of circARMC9.Results:circARMC9 was specifically expressed in cancer-related fibroblasts and was significantly more stable than the parental gene ARMC9.Function experiments showed that high expression of circARMC9 in cancer-related fibroblasts could enhance perineural invasion of pancreatic cancer cells(Miapaca-2 cells and Panca-1 cells).Mechanism studies revealed that circARMC9 from cancer-related fibroblasts promotes the release of the chemokine CCL4,thereby promoting the progression of pancreatic cancer perineural invasion.Conclusion:circARMC9 is specifically expressed in cancer-related fibroblasts and promotes the progression of pancreatic cancer perineural invasion by promoting the release of the chemokine CCL4.In summary,this study explains the important role of circARMC9 in promoting pancreatic cancer perineural invasion,and it may serve as a potential biological marker and target for delaying pancreatic cancer infiltration and invasion.