Abstract
BackgroundThe role of circular RNAs (circRNAs) in the occurrence and development of gastric cancer (GC) has recently attracted increasing interest. The following study investigates the role of a newly discovered hsa_circ_0008434, which has been confirmed to be highly expressed in GC tissues, in regulating GC biological behaviour.MethodsHigh-throughput RNA sequencing was used to identify differentially expressed genes between normal gastric tissues and GC tissues; actinomycin D and RNase R assays were used to determine the stability and loop structure of hsa_circ_0008434; and the miRanda database was used to predict the target genes of hsa_circ_0008434. The role of hsa_circ_0008434 in cell proliferation, migration, and invasion was examined using CCK-8, wound healing, Transwell and colony formation assays. The regulatory relationships among hsa_circ_0008434, microRNA-6838 (miR-6838), and ubiquitin-specific peptidase 9X (USP9X) were determined by dual-luciferase activity assays. The expression of hsa_circ_0008434 and miR-6838 was measured by qPCR; the expression of USP9X was detected by immunohistochemistry and Western blotting. The effects of hsa_circ_0008434 on in vivo tumour growth were assessed in xenograft models.ResultsWe found that hsa_circ_0008434 was one of the most upregulated circRNAs in GC tissue versus normal tissue. Further in vitro testing indicated that by acting as a miRNA sponge for miR-6838-5p, hsa_circ_0008434 promotes the expression of USP9X and further increases the proliferation, migration, and invasion of GC cells. In addition, animal studies indicated that hsa_circ_0008434 could promote tumour growth in vivo.ConclusionsHsa_circ_0008434 may promote GC proliferation, invasion and migration by regulating the expression of miR-6838 and USP9X.
Highlights
The role of circular RNAs in the occurrence and development of gastric cancer (GC) has recently attracted increasing interest
Hsa_circ_0008434 is expressed at a high level in GC and maintains a highly stable loop structure To determine the involvement of circRNAs in the progression of GC, we explored the expression profile of circRNAs in GC tissues by using high-throughput RNA sequencing (RNA-seq)
CircRNA analysis was performed on tumour tissues and paired normal gastric tissues that were collected from three GC patients
Summary
High-throughput RNA sequencing was used to identify differentially expressed genes between normal gastric tissues and GC tissues; actinomycin D and RNase R assays were used to determine the stability and loop structure of hsa_circ_0008434; and the miRanda database was used to predict the target genes of hsa_circ_0008434. The regulatory relationships among hsa_circ_0008434, microRNA-6838 (miR6838), and ubiquitin-specific peptidase 9X (USP9X) were determined by dual-luciferase activity assays. Clinical samples A total of 13 paired tumour samples and adjacent healthy tissue samples were collected from GC patients who received surgical treatment in 2018 at Shanghai Ninth People’s Hospital. Written consent was obtained from all patients involved, and the study was approved by the Institutional Ethical Review Board of Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University. Cell culture and transfection Normal human gastric epithelial cell lines GES-1 and GC (SGC7901, MKN-45, AGS) were obtained from the cell bank of Fudan University. SGC7901 cells were transfected with three hsa_ circ_0008434 siRNA fragments, one USP9X siRNA fragment and one antagomiR-6838 fragment using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. All siRNAs and antagomiRs sequences were provided by GenePharma (Shanghai, China)
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