Mechanism Of TRF-Pro-CGG Targeting CSF1 Gene To Inhibit Pancreatic Cancer Proliferation,Invasion And Metastasis

Background:Pancreatic cancer(PC)is a highly malignant gastrointestinal tumor with difficulty in diagnosis and treatment.Although great progress has been made in chemotherapy,radiotherapy and surgery,its 5-year survival rate of PC patients is still very low.Therefore,it is important to explore biomarkers for early diagnosis of PC and new therapeutic targets to PC.tRFs(t RNA-derived-fragments)and tiRNAs(t RNA halves)are derived fragments of t RNA,which have been reported in acquired metabolic disorders,stress injury,neurodegenerative diseases,myeloma,breast cancer,B-cell lymphoma,lung cancer,prostate cancer,colorectal cancer,etc.,but have not been reported in pancreatic cancer.This paper aims to investigate the expression and mechanism of tRFs and tiRNAs in PC.tRF-Pro-CGG was selected by high-throughput second-generation sequencing as the research subject.Macrophage colony stimulating factor 1(CSF1)is a kind of classic promoting tumor gene.According to bioinformatics prediction,CSF1 is one of the target genes of tRF-Pro-CGG,and PI3K-AKT is the downstream signal pathway of tRF-Pro-CGG and CSF1.The purpose of this study was to investigate the mechanism of tRF-Pro-CGG targeting CSF1 gene mediated PI3K-AKT pathway to inhibit the proliferation,invasion and metastasis of PC.Objective:1.To study the expression profile of tRFs and tiRNAs in human PC tissues and para-carcinoma tissues.2.The expression of tRF-Pro-CGG in human PC tissues and cell lines was studied to explore the regulatory mechanism of differential expression.To study the expression of tRF-Pro-CGG in human PC tissues and cell lines,and to explore the biological functions of tRF-Pro-CGG in PC cells by overexpression or silencing tRF-Pro-CGG in a series of experiments in vitro.3.To investigate the effects of tRF-Pro-CGG down-regulating the expression of CSF1 gene mediated PI3K-AKT pathway on the biological behaviors of PC cells.Methods:1.The expression profile of tRFs and tiRNAs in PC tissues were detected by next-generation sequencing,and tRF-Pro-CGG was selected as the research subject.2.The expression of tRF-Pro-CGG in PC cells and tissues was detected by q PCR.tRF-Pro-CGG mimic and inhibitor were transfected with PC cells in vitro to detect their effects on proliferation,cloning,migration,invasion and apoptosis of PC cells.3.According to bioinformatics prediction,CSF1 gene is one of the target genes of tRF-Pro-CGG;The 3 ’-UTR luciferase reporter gene assay and westernblot assay confirmed that CSF1 was the target gene of tRF-Pro-CGG.4.Bioinformatics predicted that the PI3K-AKT signaling pathway was the downstream pathway of tRF-Pro-CGG and CSF1 gene,and westernblot verified that tRF-Pro-CGG targeted CSF1 to mediate the regulation of PI3K-AKT pathway.Results:1.In the next-generation sequencing of tRFs and tiRNAs in PC,a total of 48 differentially expressed tRFs and tiRNAs were screened,with 46 upregulated and 2down-regulated(see table 4).q PCR verification of tRFs and tiRNAs sequencing results(see table 5)showed that tRF-Pro-CGG was significantly different,so tRF-Pro-CGG was selected as the research subject.2.q PCR results showed that the expression of tRF-Pro-CGG in PC tumor tissues was lower than that in para-carcinoma tissues,and the expression was down-regulated in PC cell lines.In PC cells SW1990,enhanced tRF-Pro-CGG expression inhibited cell proliferation,cloning,migration and invasion,and promoted apoptosis.In PC cell PANC-1,knockdown of tRF-Pro-CGG expression promoted cell proliferation,cloning,migration and invasion,and inhibited apoptosis.3.Bioinformatics predicted that CSF1 gene was one of the target genes of tRF-Pro-CGG,and 3 ’-UTR double luciferase reporter gene experiment and western blot confirmed that CSF1 gene was one of the target genes of tRF-Pro-CGG.Enhanced tRF-Pro-CGG expression inhibited the expression of target gene CSF1,and vice versa.tRF-Pro-CGG can bind to the 3 ’-UTR of CSF1,inhibit the expression of CSF1 after transcription,and influence the biological behaviors of PC cells.4.Bioinformatics predicted that the PI3K-AKT signaling pathway was the downstream pathway of tRF-Pro-CGG and CSF1 gene,and westernblot verified that tRF-Pro-CGG targeted CSF1 to mediate the PI3K-AKT pathway to regulate the biological behaviors of PC.Conclusion:Therefore,this study revealed that the expression of tRF-Pro-CGG was low in both PC tissues and PC cells,which inhibited the proliferation,cloning,migration and invasion of PC cells and promoted apoptosis.CSF1 was one of its target genes,and tRF-Pro-CGG targeted CSF1 gene regulated the PI3K-AKT pathway to inhibit the proliferation,invasion and metastasis of PC.tRF-Pro-CGG may be a new diagnostic marker for PC and a potential therapeutic target for PC.