ABSTRACT
Objective@#To investigate the role of microRNA-96-5p in the proliferation and invasion of gastric cancer cells and its molecular mechanism.@*Methods@#From June 2015 to January 2017, 53 resected specimens were collected. The transcriptional levels of microRNA-96-5p and forkhead box Q1 (FoxQ1) in gastric cancer tissues and the matched para-cancerous tissues were quantified by quantitative real-time PCR (qRT-PCR). The expression of FoxQ1 protein was also detected by immunohistochemistry (IHC). The relationship between microRNA-96-5p expression and the clinicopathological features of gastric cancer and its correlation with FoxQ1 expression were analyzed. The expressions of miRNA-96-5p in gastric cancer tissue and adjacent normal tissue were detected by qRT-PCR. miRNA-96-5p mimics was transfected to BGC-823 gastric cancer cells. The effects of miRNA-96-5p on cell proliferation and invasion were detected by cell counting kit-8 (CCK-8) assay and Transwell assay, respectively. The protein expressions of FoxQ1, E-cadherin and vimentin were determined by western blot. The relationship between FoxQ1 and miRNA-96-5p expressed in BGC-823 cells was detected by dual-luciferase reporter assay.@*Results@#The median expression of miRNA-96-5p in gastric cancer tissue was 1.05, significantly lower than 3.23 of para-cancerous tissues (P<0.05). The positive rate of FoxQ1 expression in gastric cancer tissue was 71.7%, significantly higher than 28.3% of para-cancerous tissues (P<0.05). The expression of FoxQ1 was negatively corelated with the level of miRNA-96-5p (r=-0.613, P=0.006). The expression of miRNA-96-5p in gastric cancer cell BGC-823 was significantly decreased compared with normal gastric epithelial cell (0.96±0.08 vs 2.84±0.15, P<0.05). The results of CCK-8 assay and Transwell assay showed that overexpression of miRNA-96-5p significantly reduced the proliferation and invasion abilities of gastric cancer cells (P<0.05). Overexpression of miRNA-96-5p decreased the protein level of FoxQ1. Moreover, it upregulated the expression of E-cadherin and downregulated the expression of vimentin. The result of dual-luciferase-3′-UTR reporter assay confirmed that miRNA-96-5p binds to the 3′UTR of FoxQ1.@*Conclusion@#miRNA-96-5p may suppress the proliferation, migration and epithelial-mesenchymal transition (EMT) of gastric cancer cell by down-regulation of FoxQ1.
ABSTRACT
Objective To investigate the feasibility of labeling iris pigment epithelial(IPE)cells of rabbits with 5-(and 6-)carboxyfluorescein diacetate succinimidyl ester(CFSE). Methods Enzyme-assisted microdissection was used to isolate the cultured rabbit′s IPE cells.The third or forth subcultured IPE cells were incubated with 2.5,5,10,20,and 40 ?mol/L of CFSE for 1,5,and10min respectively.The fluorescence intensity was detected by flow cytometry,and the leakage of CFSE and its dyeing were observed by fluorescence antibody labeling. Results Incubation with 20 ?mol/L CFSE under 37℃for1minute was the most optimal condition for IPE cells labeling.The coloration of IPE cells stained by CFSE lasted 4 weeks.There was no leakage of dye from labeled rabbit IPE cells to non-labeled human IPE cells in mixed culture process. Conclusion With the advantages of high rate of dyeing,long time of tracing,safety and convenience,CFSE can be used as a new method to label the rabbit′s IPE cells.