| Objective: Pancreatic cancer is a malignant tumor originating from the exocrine glands of the pancreas,and ranks the seventh leading cause of cancer-related death worldwide.Surgery is the only way to potentially cure,and a large number of patients still have loca l recurrence or distant metastasis within one year after surgery,and the 5-year survival rate is only 9%.Micro RNAs are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level and participate in various biological processes of malignant tumors,such as proliferation,invasion,and metastasis.Currently,the role of mi R-338-5p in digestive system malignancies is controversial,and its role in pancreatic cancer has not been elucidated.This study mainly explores the expression pattern and biological function of mi R-338-5p in pancreatic cancer,and determines whether mi R-338-5p affects the malignant biological behavior of pancreatic cancer by regulating EGFR,and provides a new strategy for the targeted therapy of pancr eatic cancer.Methods: 1.First,the expression patterns of mi R-338-5p in pancreatic cancer and paired adjacent cancers were detected by q RT-PCR,and the correlation between mi R-338-5p and clinicopathological parameters was statistically analyzed.By obtai ning the m RNA and mi RNAs sequencing data of pancreatic cancer tissue from the TCGA database,combined with bioinformatics methods such as GSEA,the signaling pathways and related biological functions of mi R-338-5p were predicted.In terms of experimental v erification,the changes of cell migration and invasion ability were analyzed by Transwell experiment,and the effect of mi R-338-5p on pancreatic cancer cell metastasis was verified in vivo by liver metastasis model.2.Immunohistochemistry was used to det ect the expression of EGFR in pancreatic cancer tissues,and the correlation between mi R-338-5p and EGFR expression was statistically analyzed.Using the Target Scan database,it was predicted that mi R-338-5p might directly interact with the 3’UTR of EGFR.The wild-type and mutant reporter gene plasmids of the target gene EGFR were constructed,and the binding activity between the two was confirmed by the dual-luciferase reporter gene.EGF combined with bidirectional regulation of mi R-338-5p expression was used.Western Blot was used to detect EGFR signal and EMT marker expression,and Transwell was used to detect changes in cell invasion and migration.Finally,EGFR was overexpressed or silenced while regulating mi R-338-5p,the expression changes of EMT mar kers were detected by Western Blot method again,and the changes of cell migration and invasion ability were detected by Transwell.Finally,it was demonstrated that mi R-338-5p inhibits pancreatic cancer EMT dependent on EGFR/ERK signaling.Results: 1.The expression of mi R-338-5p was detected in 44 cases of pancreatic cancer and paired adjacent normal pancreatic tissue by q RT-PCR,and it was found that the expression of mi R-338-5p in pancreatic cancer was significantly lower than that in paired normal panc reatic tissue(P=0.004).In the correlation analysis of clinical data,the expression level of mi R-338-5p was negatively correlated with lymph node metastasis(P=0.014)and tumor pathological stage(P=0.012).GSEA based on the HALLMARK gene set showed that the EMT functional gene set was enriched at the bottom of the ranking list,that is,there was a negative regulatory effect between mi R-338-5p and EMT phenotype.Subsequently,the expression levels of mi R-338-5p in six pancreatic cancer cell lines were de tected,and two cell lines with moderate expression(Capan-2 and PANC-1)were selected for bidirectional regulation.Combined with EGF-induced EMT phenotype,we found that mi R-338-5p inhibited EGF-induced pancreatic cancer cell migration,invasion and EMT.The pancreatic cancer liver metastasis model was further constructed in vivo,and the results showed that the overexpression of mi R-338-5p inhibited the formation of pancreatic cancer liver metastasis.2.Immunohistochemical staining combined with bioinfo rmatics analysis showed that mi R-338-5p was negatively correlated with EGFR expression.The dual-luciferase reporter gene assay confirmed the direct binding between mi R-338-5p and EGFR.Western Blot results showed that overexpression of mi R-338-5p inhibited the expression of EGFR,phosphorylated EGFR(tyr1045,1068),phosphorylated ERK and the EMT mesenchymal marker MMP-9,and promoted the expression of EMT epithelial marker E-cadherin.The silencing of mi R-338-5p up-regulated the expression of EGFR,phosp horylated EGFR,phosphorylated ERK and the EMT marker MMP-9,while down-regulated the expression of E-cadherin.In the Western Blot detection of AKT/m TOR signal,overexpression of mi R-338-5p had no significant effect on the expression of signal-related proteins.Further combined with EGFR overexpression/silencing,Western Blot results showed that EGFR could reverse the regulatory effect of mi R-338-5p on EMT markers.At the same time,the results of Transwell experiment showed that EGFR could reverse the eff ects of mi R-338-5p on the invasion and migration of pancreatic cancer cells.Conclusion: In pancreatic cancer,the expression of mi R-338-5p was significantly low in pancreatic cancer tissue,and was negatively correlated with lymph node metastasis and tumo r pathological stage.mi R-338-5p inhibits pancreatic cancer cell migration,invasion and EMT through EGF-induced EGFR/ERK signaling. |
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