| Pancreatic cancer(PC)is highly malignant with a very poor prognosis which is difficult to diagnose at an early stage.About 80%-85% of patients are diagnosed and treated at an advanced stage of the disease or after developing symptoms associated with metastasis.Among them,the vast majority of patients had liver metastases at the time of diagnosis and lost the best time for surgical resection.In addition,more than 60% of patients have recurrence with liver metastases within two years after tumor resection.Liver metastases are common in pancreatic cancer patients and are associated with poor prognosis.However,there is still a lack of effective methods for early diagnosis of pancreatic cancer with liver metastases,and existing imaging methods cannot detect liver micrometastases.Imaging examinations(such as MRI and contrast-enhanced CT)are the most commonly used methods for preoperative diagnosis of pancreatic cancer with liver metastases,but their sensitivity to micrometastases is still poor.Many patients with liver micrometastases are misdiagnosed.The effect is still not ideal,so there is an urgent need for some more sensitive methods to detect patients with pancreatic cancer with liver metastases at an early stage.For example,through high-throughput sequencing methods to detect genomes in tissues,developing potential molecular biomarkers to overcome the defects of imaging-based diagnostic methods,predict the risk of liver metastasis in pancreatic cancer patients in the early stage of the disease and improve the early diagnosis rate of liver metastasis,so as to intervene as soon as possible and improve the survival prognosis of patients.Several studies have shown that the prognosis of pancreatic cancer is related to epigenetic changes regulated by microRNA(miRNA),which are abnormally expressed in pancreatic cancer cell initiation,proliferation,induction of epithelial-mesenchymal transition(EMT),metastasis,and chemoresistance.miRNA plays an important role in pancreatic cancer,suggesting that miRNA may be a diagnostic tool for pancreatic cancer with liver metastases.miRNA is a small non-codingRNA involved in cancer development and progression that can silence specific target genes by repressing translation,One miRNA can bind up to 1000 different target genes,thereby exerting a strong regulatory function on the cell metabolism.Moreover,depending on the target gene and the cell type,miRNA can have varying effects on tumor progression by altering the expression of oncogenes and tumor-suppressive genes.Since miRNA is very resilient against degradation and can be detected in tissues,plasma,and body fluids(such as serum,urine,and breast milk),it is considered a powerful diagnostic tool,but its role in PC with liver metastasis has not been extensively investigated.The research of miRNA-related genomics may provide a more convenient and efficient new method for the diagnosis of pancreatic cancer patients with liver metastases.Part 1 Analysis of differentially expressed miRNAs in pancreatic cancer tissues between liver metastases and non-liver metastases by small RNA sequencingObjective : To explore the differentially expressed miRNAs in pancreatic cancer tissues with liver metastases and non-liver metastasis.Methods : Twenty cases of pancreatic cancer tissue obtained by endoscopic ultrasound-guided fine-needle aspiration(EUS-FNA)were selected as the discovery cohort of this study,including 10 cases with liver metastasis and 10 cases without liver metastasis.High-throughput small RNA sequencing was performed to evaluate The differential expression profiles of miRNAs in tissue samples of patients between the two groups.GO function enrichment analysis and KEGG signaling pathway enrichment analysis were performed on the target genes.Results:Sixty-one differentially expressed miRNAs were detected by small RNA sequencing analysis,among which 32 miRNAs were significantly up-regulated and 29 miRNAs were significantly down-regulated in the tissues of pancreatic cancer with liver metastasis group.12 miRNAs with up-regulated expression are annotated miRNAs in the mi RBase database,including hsa-mi R-122-5p,hsa-mi R-3122,hsa-mi R-4797-5p,hsa-mi R-483-5p,hsa-mi R-516b-5p,hsa-mi R-5189-3p,hsa-mi R-548ar-3p,hsa-mi R-548d-5p,hsa-mi R-548 q,hsa-mi R-6734-5p,hsa-mi R-7113-5p,hsa-mi R-766-5p.GO functional enrichment analysis and KEGG signaling pathway enrichment analysis were performed on the target genes.we found that these differentially expressed miRNAs were enriched in many important biological processes,including "positive and negative regulation of transcription","signal transduction","actin binding","DNA binding","angiogenesis" and "actin binding".Some biological signaling pathways,such as G protein-coupled receptor signaling pathway,autophagy signaling pathway,longevity signaling pathway,Erb B signaling pathway,Rap1 signaling pathway,and pathways in cancer were also significantly enriched.These pathways are related to cell proliferation,differentiation,metastasis and apoptosis,which are all important processes of tumor invasion and metastasis.Cnclusions:MiRNAs are differentially expressed in pancreatic tissues of pancreatic cancer with liver metastases and non-liver metastases groups,and are closely related to liver metastasis of pancreatic cancer,which may provide a novel idea for the diagnosis and treatment of pancreatic cancer with liver metastasis.Part 2 Validation of differential expression of miRNAs by real-time PCR and exploration of their clinical significanceObjective:To validate the expression levels of 12 annotated miRNAs obtained by sequencing in the training cohort,and analyze their relationship with clinicopathology and prognosis.Methods : Twelve annotated microRNAs that were up-regulated in the liver metastases group and annotated in the mi RBase database were selected for quantitative validation.The expression of the 12 miRNAs in the tumor tissues obtained by EUS-FNA in the training cohort was analyzed by real-time PCR.The training cohort includes 48 cases in the pancreatic cancer liver metastasis group and 48 cases in the non-liver metastasis group.Combined with the clinical follow-up data of the patients,the correlation of miRNA expression,clinicopathological parameters and prognosis in pancreatic cancer tissues of 96 patients was further statistically analyzed,and a prediction model of pancreatic cancer liver metastasis based on miRNA-related genomics was constructed.Results : Compared with the non-liver metastasis group,the expression of hsa-mi R-6734-5p,hsa-mi R-483-5p,hsa-mi R-548 q,hsa-mi R-548ar-3p in pancreatic cancer tissues with liver metastasisthe is significantly higher,which was consistent with the sequencing results.The expression of hsa-mi R-483-5p,CA125 and history of diabetes were closely related to liver metastasis of pancreatic cancer.The overall survival of the pancreatic cancer with liver metastasis group was significantly shorter than that of the non-liver metastasis group,and the overall survival of the hsa-mi R-483-5p high expression group was significantly shorter than that of the non-liver metastasis group.Cnclusions:The expression of hsa-mi R-6734-5p,hsa-mi R-483-5p,hsa-mi R-548 q,hsa-mi R-548ar-3p was significantly higher in pancreatic cancer tissues with liver metastasis.The expression of hsa-mi R-483-5p,CA125 and diabetes were closely related to liver metastasis of pancreatic cancer,and the overall survival of patients with high expression of hsa-mi R-483-5p was significantly reduced.In addition,we propose a novel prediction model of pancreatic cancer with liver metastasis based on miRNA-related genomics,which has high diagnostic performance and can be used as a potentially effective auxiliary tool for the diagnosis of liver metastasis in pancreatic cancer patients. |
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