Overexpression Of CircRNA Chr7: 154954255-154998784+ In Cancer-related Pancreatic Stellate Cells Promotes The Progression Of Pancreatic Cancer Cells By Targeting The MiRNA4459-KIAA0513 Axis

Background and purpose Pancreatic cancer is a gastrointestinal tumor with extremely rapid malignant progression,poor prognosis,and difficult diagnosis and treatment.Its morbidity and mortality are increasing year by year.At present,targeted therapy oriented by the expression of specific genes is a research hotspot in the field of pancreatic cancer.However,because of the difference in tumor differentiation,no single targeted therapy has been proven to be very effective.However,the continuous progress of research on related biomarkers has brought dawn to the treatment of more pancreatic cancer patients.Although there are still many obstacles to the targeted therapy of pancreatic cancer,it is exciting that many studies have made good progress in deciphering the heterogeneity of pancreatic cancer.Therefore,combining conventional therapy with targeted therapy will be the key to effective treatment of this deadly disease.Recent studies have also found that circular RNA（circRNA）plays an important regulatory role in pancreatic cancer cells.The abnormal expression of circRNA in pancreatic cancer cells（PCCs）can promote the occurrence and development of pancreatic cancer.However,the role of circRNA in cancer-related pancreatic stellate cells（CaPSCs）is still unclear.Therefore,the purpose of this study is to explore how CaPSCs cells promote the proliferation,invasion and metastasis of pancreatic cancer cells through the circRNA/micro RNA（miRNA）/target protein axis,and provide new strategies for the treatment of pancreatic cancer.Methods In this study,we used tissue block explantation and modified enzyme digestion density gradient centrifugation to isolate and purify CaPSCs from the cancer tissues of 5 patients with pancreatic cancer.In the same way,normal pancreas-associated pancreatic stellate cells（NaPSCs）were isolated and purified from the normal pancreatic tissues of 5 patients with benign pancreatic diseases.After the above-mentioned pancreatic stellate cells（PSCs）and pancreatic cancer cells Panc-1 were co-cultured with the Transwell system,the CCK-8 experiment was used to detect the proliferation ability of Panc-1 cells co-cultured with different PSCs.The CaPSCs₁cells with the strongest promotion effect on Panc-1cell proliferation and NaPSCs₁cells with the weakest promotion effect on Panc-1 cell proliferation were screened out.Then,the expression profiles of circRNA,miRNA and m RNA between CaPSCs₁cells and NaPSCs₁cells were compared by RNA high-throughput sequencing.Select candidate circRNA/miRNA/target protein m RNA regulatory axis through bioinformatics analysis.Then the target circRNA and overexpression target miRNA were silenced in CaPSCs₁cells,and the expression of circRNA,miRNA,target protein m RNA and target protein were detected by RT-q PCR and Western blot respectively.Finally,CCK-8 experiment,wound healing assay and Transwell migration/invasion experiment were used to detect the proliferation,migration and invasion ability of Panc-1 cells in different co-culture groups.Results In the bioinformatics analysis,the top 3 circRNAs with the highest differentialexpressionwerechr7:154954255-154998784+,chr10:12081472-12120267+and chr10:76909966-77019099+.The circRNA with the highest differential expression was verified by RT-q PCR again.The expression trend of each circRNA was consistent with the sequencing results,but chr7:154954255-154998784+was up-regulated most significantly in CaPSCs₁cells.Afterwards,bioinformatics analysis was used to screen out the chr7:154954255-154998784+/miRNA4459/KIAA0513 axis from the candidate targets.After silencing chr7:154954255-154998784+,the expression of miRNA4459 in CaPSCs₁cells was up-regulated,and the expression of KIAA0513 was down-regulated.In addition,after co-cultured with chr7:154954255-154998784+silent CaPSCs₁cells,the CCK-8 test,wound healing assay and Transwell migration/invasion test of Panc-1 cells showed that their proliferation,invasion and metastasis ability decreased.When miRNA4459was overexpressed in CaPSCs₁cells,the expression of chr7:154954255-154998784+and KIAA0513 was down-regulated.In addition,after co-cultivation with CaPSCs₁cells overexpressing miRNA4459,the CCK-8test,wound healing assay and Transwell migration/invasion test of Panc-1 cells showed that their proliferation,invasion and metastasis ability decreased.Conclusion Chr7:154954255-154998784+may promote the proliferation,migration and invasion of pancreatic cancer cells through the miRNA4459/KIAA0513 axis in CaPSCs,which may become an important therapeutic target for pancreatic cancer patients in the future.