DNA-Methylation-Mediated lncRNA HOXB-AS4 Promotes Gastric Cancer Progression Through Regulating miR-130a-5p/PKP4

Abstract

Background：Gastric cancer (GC) is one of the most common cancer in the world, possessing the second leading cause of cancer-related mortality. Long noncoding RNAs (lncRNAs) have been shown to play important roles in tumorigenesis. However, the effect of lncRNA HOXB-AS4 in GC progression and the underlying mechanisms remain unknown. Methods：Firstly, the expression of lncRNA HOXB-AS4 in gastric cancer tissues and cancer cells was investigated according to GEPIA database and Real time fluorescence quantitative PCR(qRT-PCR). Then, MTT, clone formation, Transwell and Western blot were used to study the effects of overexpression or down-regulation of HOXB-AS4 on the proliferation, invasion and epithelial mesenchymal transformation of cancer cells. We further studied the molecular mechanism of HOXB-AS4 by fluorescence in situ hybridization, bioinformatics analysis, luciferase reporting, methylation specific PCR (MSP) and chromatin immunoprecipitation (chip).Results:In the study, the GEPIA database and quantitative Real-Time PCR (qRT-PCR) assay showed that HOXB-AS4 was upregulated in GC tissues and cells. Then, MTT, clone formation, transwell, and western blot assays suggested that overexpression of HOXB-AS4 increased cell proliferation, migration, and invasion, and regulated epithelial-mesenchymal transition (EMT) markers expression, while knockdown of HOXB-AS4 showed the opposite effect. Fluorescence in situ hybridization (FISH) assay found that HOXB-AS4 localized in the cytoplasm of the GSE-1 and AGS cells. Further mechanism experiments, including bioinformatics, luciferase reporter, qRT-PCR, and western blot assays showed that HOXB-AS4 sponged to miR-130a-5p to regulate the PKP4 expression. Knockdown of miR-130a-5p obliterated the effect of HOXB-AS4, which was further abolished by knockdown of PKP4 in vitro and in vivo. Methylation-specific PCR (MSP) and chromatin immunoprecipitation (CHIP) assay showed that overexpression of HOXB-AS4 in GC was mediated by SP1-dependent DNA methylation. Abnormal upregulation of lncRNA HOXB-AS4 contributed to GC progression, which was mediated by DNA methylation. The study clarified that DNA-methylation-mediated HOXB-AS4 played its role through miR-130a-5p/PKP4 axis.Conclusions: Our study provides new insights for the understanding of epigenetic regulation on lncRNA expression in GC, and indicates that HOXB-AS4 could be a biomarker of GC prognosis. Moreover, targeting HOXB-AS4 /miR-130a-5p/PKP4 axis might be a promising strategy to treat GC.