Mechanism Of Circ-SFMBT2 Promoting The Proliferation Of Gastric Cancer Cells Through Sponging MiR-182-5p To Enhance CREB1 Expression

Background: Circular RNAs(circ RNAs)have been reported as a diverse class of endogenous RNA that regulate gene expression in eukaryotes.Recent evidence suggested that a number of circ RNAs could act as oncogenes or tumor suppressors through sponging micro RNAs(mi RNAs).However,the function of circ RNAs in gastric cancer remains largely unknown.Materials and methods: The expression of circRNAs in gastric carcinoma cells,tissues and plasmas were detected using real-time quantitative reverse transcriptionpolymerase chain reaction(q RT-PCR).The correlation between target circ RNA expression and clinic pathological features of gastric cancer patients was analyzed.Gastric cancer cells were treated with RNase R and actinomycin D,and the target circ RNA was confirmed to be a circular closed structure with high stability.The proliferation ability of gastric cancer cells was detected by CCK-8 and colony formation assay.We performed RIP experiments to verify that target circ RNA could bind to Argonaute2(AGO2).We detected the subcellular localization of target circ RNA by nuclear cytoplasmic separation experiment and RNA-FISH.Next,we predicted the interaction networks between circ RNAs-mi RNAs and mi RNAs-m RNA by use of bioinformatics softwares.q RT-PCR and western blotting(WB)were performed to examine the expression levels of relative genes and proteins.Dual luciferase reporter gene assay was performed to verify the binding of target circ RNA with downstream mi RNA as well as mi RNA with target m RNA.Results: We characterized one circ RNA named circ-SFMBT2,which showed an increased expression level in gastric cancer tissues compared to adjacent non-cancerous tissues and was associated with higher tumor stages of gastric cancer.We found that circ-SFMBT2 was significantly higher expressed in peripheral blood from gastric cancer patients compared with healthy subjects.Our experimental results showed that silencing of circ-SFMBT2 could inhibit the proliferation of gastric cancer cells.Next,the result of RIP assay verified that circ-SFMBT2 could bind to AGO2.Furthermore,nuclear cytoplasmic separation experiment and RNA-FISH confirmed that circSFMBT2 was mainly distributed in the cytoplasm.The results of dual luciferase reporter gene assay demonstrated the binding of circ-SFMBT2 with mi R-182-5p and mi R-182-5p with target gene c AMP response element binding protein 1(CREB1).The rescue experiments further confirmed that circ-SFMBT2 and mi R-182-5p could regulate the expression of CREB1 m RNA,which in turn affected the proliferation of gastric cancer cells.Conclusion:(1)circ-SFMBT2 is derived from exons 5-8 of SFMBT2 gene in chromosome 10;(2)circ-SFMBT2 is a circular closed structure with high stability;(3)circ-SFMBT2 is highly expressed in gastric cancer cells,tissues and peripheral blood;(4)Expression of circ-SFMBT2 in gastric cancer tissues is associated with TNM staging of gastric cancer;(5)circ-SFMBT2 can regulate the proliferation of gastric cancer cells;(6)circ-SFMBT2 can bind to AGO2;(7)circ-SFMBT2 is mainly distributed in the cytoplasm;(8)circ-SFMBT2 is involved in the regulation of gastric cancer cells proliferation by competitively binding to CREB1 m RNA with mi R-182-5p.