Research On Macrophage-related LncRNA FOXP4-AS1 In Hepatocellular Carcinoma And Its Mechanism

Objective:Hepatocellular carcinoma(HCC)is the most common primary liver cancer.Despite the continuous improvement of treatment methods and medical resources for HCC,its mortality rate remains high.Long noncoding RNA(lnc RNA)has been proved to be a novel regulator of tumor proliferation,apoptosis and metastasis.Recent studies have found that the polarization of tumor macrophages is closely related to the occurrence and development of tumors,we analyzed tumor macrophage gene chip analysis and found that lnc RNA FOXP4-AS1 is a functional oncogene in HCC.Therefore,we will further study the prognostic significance of FOXPA-AS1 in HCC,its biological impact and regulatory network.Methods:1.The human lymphoma cell U937 was induced to differentiate into macrophages of M1 and M2 phenotypes.The macrophages of the two phenotypes were analyzed by gene chip to screen out the lnc RNA FOXP4-AS1 with significant differential expression.Statistical analysis of the expression differences of FOXP4-AS1 in HCC tissues and normal liver tissues.The clinicopathological data and prognostic information of patients with HCC were collected to determine the correlation between the expression level and pathological indicators and patient prognosis.2.Design small interfering si RNA fragments,down-regulate FOXP4-AS1 with si RNA,transfect hepatocellular carcinoma cells with lentivirus,construct stable cell lines,perform CCK8 proliferation experiments,scratch experiments,clone formation experiments,Transwell experiments,flow cytometry to detect apoptosis,and cycle experiments,Tunel experiments,angiogenesis experiments,etc.,to examine the biological effects of FOXP4-AS1 on hepatocellular carcinoma cells.3.Predicted differentially expressed mirna(DEmi RNAs)and DEmirna-mrna network of lnc RNA FOXP4-AS1.Differentially expressed micro RNAs(DEmi RNAs)were predicted.Their target genes were subjected to gene ontology(GO)annotation and KEGG(Kyoto Encyclopedia of Genes and Genome)pathway enrichment analysis.Protein-protein interaction(PPI)networks were established and hub genes identified with Cytoscape software.The GEPIA database was used to assess the prognostic roles of 20 hub genes in HCC.The c Bio Portal database was used to exhibit alterations of the genes.Construction of a potential regulatory network for lnc RNA FOXP4-mi RNA-m RNA.Results:1.FOXP4-AS1 was highly expressed in HCC tissues compared to normal liver tissues(P<0.05).Its expression was correlated with serum alpha-fetoprotein(AFP),serum aspartate aminotransferase(AST),tumor size,liver cirrhosis,BCLC staging,and patient age(all P <0.05).Kaplan-Meier test showed that patients with higher expression of FOXP4-AS1 in HCC tissues had significantly shorter disease-free survival and overall survival after liver resection(all P <0.05).Cox regression analysis further showed that FOXP4-AS1 is an independent risk factor for disease-free survival and overall survival of HCC patients after liver resection(all P <0.05).2.The FOXP4-AS1 was silenced by designing si RNA and verified functional experiments.It was confirmed that down-regulating the expression of FOXP4-AS1 can significantly weaken the proliferation and cloning ability of cells,affect the cell cycle,inhibit the invasion and migration of liver cancer cells and promote the ability of angiogenesis(all P <0.05).At the same time,it has no effect on the apoptosis effect of liver cancer cells(P> 0.05).3.Six upregulated and four downregulated DEmi RNAs were identified.Over-expressed and under-expressed predicted target genes(183 and 147,respectively)were selected for GO annotation and KEGG pathway enrichment analysis.The downregulated genes were significantly prominent in the PI3K-Akt signaling pathway;the upregulated genes in the cell cycle.The PPI networks indicated IGFBP3 and PRC1 as hub genes with the highest node degrees.Higher expressions of 9(6)genes were associated with worse(better)prognosis in HCC.Conclusion:Our results suggest that lnc RNA FOXP4-AS1 is a tumor-promoting gene in HCC and may be a key regulator of HCC proliferation,invasion,migration,cell cycle,and angiogenesis.At the same time,we constructed HCC-related lnc RNA FOXP4-AS1-mi RNA-m RNA regulatory network,elucidates the molecular mechanisms involved in the development of HCC.It provides a new direction for finding new HCC treatment targets and potential prognostic biomarkers.