| Objective To analysis the serum expression of long non-coding RNA(lncRNA)FEZF1-AS1(FEZF1 antisense RNA1)in patients with gastric cancer(GC)and discuss its clinical value of diagnosis and prognosis of GC.To investigate the expression and clinical significance of FEZF1-AS1 in GC tissues and study the effects of FEZF1-AS1 on GC cell proliferation,apoptosis,migration and invation.Moreover,to explore the effect of FEZF1-AS1 on cell autophagy,and to investigate the mechanism of autophagy regulation on the malignant progression of gastric cancer.Methods Collect serum of GC patients diagnosed by Affiliated Hospital of Nantong University(89 pre-operated patients,63 post-operated patients,55 benign gastric lesion groups and 73 normal controls),qRT-PCR(quantitative Real-time PCR)was used to detect expression of FEZF1-AS1 and evaluate the methodology.The expression of FEZF1-AS1 in 50 GC tissues and adjacent tissues were detected.The relationship between FEZF1-AS1 and clinical pathological parameters were analyzed.FEZF1-AS1 interference vector and overexpression vector were constructed and then select the higher transfection efficiency fragment.CCK-8 and colony formation assay was used to detect the effect of FEZF1-AS1 on cell proliferation.Cell cycle and apoptopsis was detected by flow cytometry.Transwell assay was used to test the migration and invation of GC cells.FISH(fluorescence in situ hybridization)confirmed subcellular location of FEZF1-AS1.Using bioinformatics software to predict the binding protein and RIP(RNA immunoprecipitation)assay confirmed their relationship.qRT-PCR and Western blot were used to observe the effect on mRNA and protein levels of autophagy related genes.Cell cycle and apoptopsis dealed with autophagy inhibitor 3-MA were tested by flow cytometry.Results The qRT-PCR assay to detect serum FEZF1-AS1 has a single melting curve peak,well liner and repeatability.The expression of FEZF1-AS1 was much higher in GC patients than in patients with benign gastric lesion and normal controls(p<0.0001),and reduced after the surgery(p<0.01).High serum expression of FEZF1-AS1 was significantly correlated with tumor size,TNM stage,lymph node metastasis and distant metastasis(p<0.05).AUC(area under the curve)was 0.810 higher than CEA and CA199.The combined diagnostic sensitivity of the three markers was the highest of 92.1%.qRT-PCR confirmed that compared with adjacent tissues,FEZF1-AS1 increased significantly in GC tissues(p<0.01),and its expression was much higher than gastric epithelial cell line(p<0.05).Functional studies showed cell proliferation,migration and invasion ability reduced after RNAi vectors transfected into GC cell lines.Moreover shFEZF1-AS1 could induce G0/G1 phase cell cycle arrest and promoted cell apoptosis.However FEZF1-AS1 overexpression could accelerate cell proliferation,migration and invasion,S phase cell cycle increased and apoptosic cell decreased.FISH assay and nuclear plasma separation experiment confirmed FEZF1-AS1 mainly located in GC cell nuclear.Bioinformatics software predicted that sirt1(Silent information regulation 1)was a binding protein of FEZF1-AS1 and RIP assay confirmed their bingding relationship.qRT-PCR and Western blot confirmed mRNA and protein level of sirt1 up-regulated when FEZF1-AS1 was knocked down.Previous studies had demonstrated that sirt1 was highly related to cell autophagy,overexpression of sirt1 could promote autophagy.Using fluorescence microscope we found shFEZF1-AS1 can increase cell autophagosome.qRT-PCR and Western blot revealed that shFEZF1-AS1 could increase expression of sirt1 and mRNA and protein level of ATG changed.CCK-8 assay showed cell proliferation accelerated and flow cytometry showed the percentage of S phase decreased while cell apoptosis increased when 3-MA and shFEZF1-AS1 cotransfected into GC cells.Conclusions Serum expression of FEZF1-AS1 is higher in GC patients and decreases significantly after the surgery,it could be a potential GC diagnosis and treatment biomarker.FEZF1-AS1 is highly up-regulated in GC tissues and cells,it inhibits cell autophagy through sirt1 protein and then acts as an oncogene to promote malignant progression of GC by regulating cell proliferation and apoptosis.Therefore this study suggests that FEZF1-AS1 may serve as a novel biomarker for GC diagnosis,treatment and prognosis. |
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