| Objective: To investigate the expression levels of ZFAS1 in the tumor tissues,serum samples and serum exosomes of gastric cancer patients and analyze the correlation between ZFAS1 expression and the clinicopathological features.To explore the potential roles of ZFAS1 in gastric cancer and offer a new molecular target for the diagnosis and therapy of gastric cancer.Methods: 1.We selected 10 candidate cancer related lnc RNAs from previous literatures and bioinformatics analysis,including CCAT1,MALAT1,PVT1,HULC,GAS5,MYCLO-2,ZFAS1,lnc RNA-ATB,HOTTIP and linc RNA-p21.We identified that lnc RNA ZFAS1 was significantly upregulated(fold change ≥ 2)in gastric cancer cells by using quantitative real-time polymerase chain reaction(qRT-PCR).2.ZFAS1 expression was detected in tumor tissues,serum samples and serum exosomes by using qRT-PCR.Combined with clinicopathological features of gastric cancer patients such as gender,age,tumor size,lymphatic metastasis,and TNM stage,the correlation between ZFAS1 expression and clinicopathological features was analyzed.And the stability of serum ZFAS1 was analyzed.3.Gastric cancer cells MGC-803 and BGC-823 were transfected with theshRNA that directed against ZFAS1 and MKN-28 cells were transfected with overexpressed ZFAS1.And the interference and overexpressed efficiency were determined by qRT-PCR.Cell proliferation ability of the transfected cells was measured by cell counting and cell colony formation assay.Transwell migration assay was used to investigated the mobility of the transfected cells.The cell cycle distribution and cell apoptosis were analyzed by flow cytometry.The expression of cell proliferation and migration related genes and ERK signaling pathway influenced by ZFAS1 knockdown and overexpression were quantified by qRT-PCR and western blot.4.The exosomes were extracted from BGC-823 cells.The characteristics of exosomes were analyzed by transmission electron microscope(TEM),Nanoparticle Tracking Analysis(NTA)and western blot.MKN-28 cells were treated with exosomes and the expression of ZFAS1 in MKN-28 cells after treatment was detected by qRT-PCR.Cell proliferation ability of the exosomes-treated MKN-28 cells was measured by cell counting and cell colony formation assay.Transwell migration assay was used to investigated the mobility of the cells treated with exosomes.The cell cycle distribution was analyzed by flow cytometry.The expression of cell proliferation and migration related genes and ERK signaling pathway influenced by exosomes treatment were quantified by qRT-PCR and western blot.Results: 1.The results of qRT-PCR showed that ZFAS1 expression was upregulated in tumor tissues,serum samples and serum exosomes of gastric cancer patients.And ZFAS1 was relatively stable in gastric cancer patients’ serum.2.In this study,we found that ZFAS1 expression was associated with lymphatic metastasis and TNM stage(P(27)0.05).3.ZFAS1 expression was downregulated after ZFAS1 knockdown,while that was upregulated after ZFAS1 overexpressed.And our results showed that ZFAS1 could promote gastric cancer cells proliferation and migration by promoting cell cycle progression,inhibiting cell apoptosis and promoting epithelial-mesenchymal transition(EMT).4.The exosomes were small vesicles with the diameter around 100 nm under TEM and the results of western blot showed that CD9 and CD81 were abundantly expressed in the exosomes.NTA analysis showed that the exosomes had a predominant size of 50-150 nm and a concentration ranging from 1.0 × 1011 to 2.5 × 1011 particles/ml.The results of flow cytometry imaging analysis showed that MKN-28 cells could uptake exosomes.5.The results of qRT-PCR showed that ZFAS1 expression was increased in MKN-28 cells treated with exosomes.After exosomes treatment,the cell proliferation ability and mobility were increased.And the Flowcytometry imaging analysis showed that cells in S stage were significantly increased.The expressions of Cyclin D1,Bcl-2,N-cadherin,Slug,Snail,Twist,ZEB1 and p-ERK were increased,while that of Bax and E-cadherin were decreased.Conclusions: ZFAS1 expression levels were highly expressed in cancer tissues,serum and serum exosomes of gastric cancer patients.And its expression was associated with lymphatic metastasis and TNM stage.ZFAS1 could promote gastric cancer cells proliferation and migration through promoting cell cycle progression,inhibiting cell apoptosis and inducing EMT.ZFAS1 could be transmitted by exosomes to promote gastric cancer progression.Taken together,ZFAS1 may serve as a new biomarker for the diagnosis and therapy of gastric cancer. |
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