Background Osteosarcoma (OS) is a common aggressive primary sarcoma of bone. Drug resistance is a huge obstacle to chemotherapy for cancer. This study aimed to investigate the role and mechanism of circ_0002060 in OS resistance to doxorubicin (DOX). Methods The levels of circ_0002060, miR-198 and ATP binding cassette subfamily B member 1 (ABCB1) were measured by quantitative real-time polymerase chain reaction or western blot assay. Kaplan-Meier analysis was performed to determine the relationship between circ_0002060 expression and overall survival. The half inhibition concentration (IC50) of doxorubicin was calculated by Cell Counting Kit-8 (CCK-8) assay. Cell proliferation was assessed by colony formation assay. Cell apoptosis was monitored by flow cytometry. The levels of apoptosis-related proteins were measured by western blot assay. Xenograft assay was utilized to analyze the effect of circ_0002060 on DOX resistance in vivo . The interaction among circ_0002060, miR-198 and ABCB1 were confirmed by dual-luciferase reporter assay, RNA immunoprecipitation assay or RNA pull-down assay. Results Circ_0002060 and ABCB1 were upregulated, while miR-198 was down-regulated in OS tissues and DOX-resistant OS cells. Circ_0002060 silence reduced DOX resistance in vitro and in vivo . Moreover, circ_0002060 enhanced DOX resistance via sponging miR-198. Besides, miR-198 decreased DOX resistance by binding to ABCB1. In addition, circ_0002060 sponged miR-198 to up-regulate ABCB1 expression. Conclusion Circ_0002060 enhanced doxorubicin resistance of OS by regulating miR-198/ABCB1 axis, which provides potential therapeutic targets for OS therapy.
Background
Helicobacterpylori (H. pylori) infection causes aberrant DNA methylation and contributes to the risk of gastric cancer (GC). Guanine nucleotide-binding protein subunit beta-4 (GNB4) is involved in various tumorigenic processes. We found an aberrant methylation level of GNB4 in H. pylori-induced GC in our previous bioinformatic analysis; however, its expression and underlying molecular mechanisms are poorly understood.
Methods
The expression, underlying signaling pathways, and clinical significance of GNB4 were analyzed in a local cohort of 107 patients with GC and several public databases. H. pylori infection was induced in in vitro and in vivo models. Methylation-specific PCR, pyrosequencing, and mass spectrometry analysis were used to detect changes in methylation levels. GNB4, TET1, and YAP1 were overexpressed or knocked down in GC cell lines. We performed gain- and loss-of-function experiments, including CCK-8, EdU, colony formation, transwell migration, and invasion assays. Nude mice were injected with genetically manipulated GC cells, and the growth of xenograft tumors and metastases was measured. Real-time quantitative PCR, western blotting, immunofluorescence, immunohistochemistry, chromatin immunoprecipitation, and co-immunoprecipitation experiments were performed to elucidate the underlying molecular mechanisms.
Results
GNB4 expression was significantly upregulated in GC and correlated with aggressive clinical characteristics and poor prognosis. Increased levels of GNB4 were associated with shorter survival times. Infection with H. pylori strains 26695 and SS1 induced GNB4 mRNA and protein expression in GC cell lines and mice. Additionally, silencing of GNB4 blocked the pro-proliferative, metastatic, and invasive ability of H. pylori in GC cells. H. pylori infection remarkably decreased the methylation level of the GNB4 promoter region, particularly at the CpG#5 site (chr3:179451746–179451745). H. pylori infection upregulated TET1 expression via activation of the NF-κB. TET binds to the GNB4 promoter region which undergoes demethylation modification. Functionally, we identified that GNB4 induced oncogenic behaviors of tumors via the Hippo–YAP1 pathway in both in vitro and in vivo models.
Conclusions
Our findings demonstrate that H. pylori infection activates the NF-κB-TET1-GNB4 demethylation-YAP1 axis, which may be a potential therapeutic target for GC.
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