To Study The Effects And Mechanisms Of MiR-27a On Cellular Malignant Biological Behaviors In Gastric Cancer

Objective:Gastric cancer is one of the most common malignant diseases and the second leading cause of cancer-related mortalities.Despite advances in treatment modalities,the cure rates for gastric cancer patients have not been significantly improved.So a new therapeutic target in gastric cancer is expected.Accumulated studises suggested that miRNAs play functional roles in cell proliferation,apoptosis,differentiation and other processes.MiRNAs wrapped in exosomes are stable in body fluids.Our previous study has found that miR-27 a is stably over-expressed in the sera of gastric cancer patients and related to poor prognosis.It is suggested that miR-27 a may play an important role in carcinogenesis and progression of gastric cancer.Now,effects and mechanisms of miR-27 a are still not clear.Based on previous study results,our study aims to explore expression of miR-27 a in exosomes relased from gastric cancer cells firstly,then investigate the effects and mechanisms of miR-27 a on cellular malignant biological behaviors in gastric cancer.Methods:1.Expression of miR-27 a in exosomes released from gatric cancer cells:exosomes was isolated from gastric cancer cells by Exoquick TC.Exosomes were characterized by TEM,and the expression of the exosome marker CD63 was estimated by western blotting.The concentration of exosomal proteins was determined using a BCA kit.Realtime PCR was employed to show diverse levels of miRNAs in GC cells and determine miR-27 a expression in different concentrations of exosomes.2.Gastric cancer cell lines SGC-7901 of over-expressed miR-27 a was established.CCK-8 was used to primarily detect the proliferation.Flow cytometry(FC)was used to measure cell apoptosis.The protein expressions of p53,BCL-2,LC3 I and LC3 II were detected by Western blot(WB).3.The target gene of miR-27 a was predicted by bioinformatics prediction website(http://www.targetscan.org),and the target gene with high predictive value was achieved.Dual luciferase reporter assay was used to verify the target gene of miR-27 a.The expression of target gene in cells of up-regulated and down-regulated miR-27 awas also examined by WB.Real time PCR was employed to determine the mRNA levels of target gene.4.Gastric cancer cell lines SGC-7901 of less-expressed FOXO1 was established.CCK-8 was used to primarily detect the growth vitality.FC was used to measure cell apoptosis.The protein expression of p53,BCL-2,LC3 I and LC3 II were detected by WB.Results:1.Expression of miR-27 a in exosomes released from gatric cancer cells:Exosomes were characterized by TEM.The significant expression of CD63 was observed in the exosomes.The concentration of exosomal proteins was determined using BCA kit,and there was a remarkable difference between exosomes and PBS.By analyzing the purified exosomes released from gastric cancer cells using Realtime PCR,we observed that miR-27 a,miR-20 and miR-34 a were present at a high level,while miR-1 and miR-423-5p showed the opposite trend.Moreover,the level of miR-27 a was significantly high in these exosomes,suggesting that exosomes transport miR-27 a.2.CCK-8 assay indicated that compared with normal control cells,miR-27 a over-expressed cells proliferation ability was improved(P<0.05).FC showed that apoptosis rates of miR-27 a up-regulated cells was decreased compared with normal control cells(P<0.05).WB suggested that expression of apoptosis-related proteins BCL-2 was up-regulated while p53 showed the opposite trend.,autophage-ralated protein LC3 I and LC3 II expression were down-regulated as compared to control groups.3.The target gene FOXO1 of high predictive values was achieved by bioinformatics prediction website.Dual luciferase reporter assay showed that pre-miR-27 a remarkably decreased the fluorescence activity of wild type FOXO1 3'UTR-pRL-TK recombinant plasmids(P<0.05),while pre-miR-27 a did not change the fluorescence activity of mutant type FOXO1 3'UTR-pRL-TK recombinant plasmid(P>0.05).FOXO1 mRNA levels had no significant change in cells transfected by miR-27 a mimics and inhibitors,while FOXO1 proteins expression were down-regulated and up-regulated respectively.4.CCK-8 assay indicated that compared with normal control cells,FOXO1low-expressed cells proliferation ability was improved(P < 0.05).FC showed that apoptosis rates of FOXO1 low-expressed cells were decreased compared with normal control cells(P <0.05).WB suggested that expression of apoptosis-related proteins BCL-2 was up-regulated while p53 showed the opposite trend.,autophage-ralated protein LC3 I and LC3 II expression were down-regulated as compared to control groups.Conclusion:1.The level of miR-27 a was significantly high in exosomes realsed from gastric cancer cell.It was suggested that miR-27 a plays an important role in carcinogenesis and progression of gastric cancer combined with previous study results.Further,miR-27 a can promote cell proliferation,inhibit apoptosis and autophage,and thus lead the advance of gastric cancer.MiR-27 a maybe a new target in gastric cancer treatment.It indicates miR-27 a may be a new therapeutic target in gastric cancer.2.The results of dual luciferase reporter assay showed that FOXO1 was target gene of miR-27 a.MiR-27 a inhibited the expression of FOXO1 protein.Moreover,low-expressed FOXO1 can promote cell proliferation,inhibit apoptosis and autophage,and thus lead advances in gastric cancer.Taken together,miR-27 a promotes proliferation,inhibits apoptosis and autophage by targeting FOXO1 in gastric cancer.MiR-27a/FOXO1 axis may offer a new therapeutic strategy in gastric cancer.