| BackgroundPancreatic cancer is a common malignant tumor of digestive tract.The incidence rate shows an obvious upward trend at home and abroad.Its malignant degree is very high and there is no clear target for the treatment of advanced pancreatic cancer,as wells as terrible prognosis.Anlotinib Hydrochloride is a multi-target oral tyrosine kinase inhibitor.Its main function is to inhibit tumor angiogenesis with a wide range of anti-tumor effects.However,there are relatively few studies on the treatment of pancreatic cancer with Anlotinib Hydrochloride.Previous studies of our team have confirmed that Anlotinib Hydrochloride can significantly inhibit the survival of pancreatic cancer cells in vitro and tumorigenesis in vivo,but the specific mechanism is not clear.ObjectiveThe purpose of this study is to preliminarily explore the mechanism of the anti-pancreatic cancer effect of Anlotinib Hydrochloride through bioinformatics methods such as data mining etc.and to screen its possible key targets.Methods（1）The GEO Database was used to mine the data set containing the m RNA SEQ data of “Anlotinib and control treated Pancreatic Cancer cells” and observing the homogeneity of each sample in the data set to ensure the credibility of the analysis results with GEO2 R tool online.（2）Clusterprofiler package and EXCEL software was used to screen significantly different genes.（3）Clusterprofiler and ggplot2 package was utilized to do GO and KEGG pathway analysis.（4）The PPI（protein-protein interaction network）map of significantly different genes was constructed by STRING database,and the key modules were analyzed by Cytoscape software.（5）The changes of m RNA abundance of BCL2A1,CXCL8,CCL20,TIMP2 and other key module genes in pancreatic cancer cell line PANC-1treated with Anlotinib Hydrochloride were detected by Real Time RT-PCR.（6）The changes of proteins levels of BCL2A1,CXCL8,CCL20,TIMP2 in Pancreatic cancer cell lines As PC-1 and PANC-1 were determined by Western blotting.（7）Autodocktools-1.5.6software and Pub Chem database was used to construct the docking models between Anlotinib Hydrochloride and key module proteins,and the binding energy was analyzed.Results（1）A data set GSE163574 was excavated containing the data set of Pancreatic cancer cell line PANC-1 treated with Anlotinib Hydrochloride.The data uniformity among samples in the data set was good.（2）189 significantly differentially expressed genes genes（DEGs）such as BCL2A1,CXCL8,CCL20 and TIMP2 were screened under the condition of "|log Fc| ≥ 1.4 and adj.P ≤ 0.05".（3）GO analysis showed that 189 DEGs concentrated in Biological Processes such as epithelial cell migration,extracellular matrix and transcriptional regulation.KEGG pathway analysis showed that 189 DEGs were mainly enriched in more than 10 signal pathways,such as lipid and atherosclerosis,m TOR signal pathway and animal autophagy.（4）6 key PPIs were screened by constructing 189 DEGs protein-protein interaction network,and proteins such as BCL2A1,CXCL8,CCL20 and TIMP2 were selected as key module proteins.（5）The results of Real Time RT-PCR showed that Anlotinib Hydrochloride could upregulate the m RNA transcriptional abundance of BCL2A1,CXCL8 and CCL20 in PANC-1 cells（P≤ 0.05）,but there was no significant change for m RNA transcriptional abundance of TIMP2.（6）Anlotinib Hydrochloride downregulated the proteins expression of BCL2A1,CXCL8,CCL20 and TIMP2 in PANC-1 cells and As PC-1cells.（7）The molecular docking 3D model of Anlotinib Hydrochloride with proteins such as BCL2A1,CXCL8,CCL20 and TIMP2 was constructed.The binding energy analysis showed that anlotinib hydrochloride bound well to proteins including BCL2A1,CXCL8,CCL20 and TIMP2.ConclusionAnlotinib Hydrochloride exerts its anti-pancreatic cancer effect through a variety of biological processes,such as epithelial cell migration,extracellular matrix formation and m RNA transcription.The anti-pancreatic cancer effect of Anlotinib Hydrochloride is related to the proteins encoded by CCL20,TIMP2,BCL2A1 and TIMP2 genes. |
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