Ghrelin Affects Ovarian Cancer SKOV3 Cell Autophagy By Targeting MiR-581

OBJECTIVE:To find the differentially presented miRNAs after Ghrelin deals with human ovarian cancer SKOV3 cells;to further reserch the mode of Ghrelin affecting the autophagy of ovarian cancer SKOV3 cells by targeting miRNAs,this can provide a new means for the treatment of ovarian cancer.METHODS:① Divided ovarian cancer SKOV3 cells into control(blank)group and Ghrelin(600 ng/ml,24 h)treatment group.Filtering unequal appearance of miRNAs in these two groups through Next-generation sequencing technology,depending on the fold difference(|log2FoldChange|>3)and significance(P-value<0.05),forecasting the target genes of the unequal appearance of miRNAs and analysing the GO function as well as the KEGG signaling pathway of the target genes by bioinformatics technology.qRT-PCR technique was applyed to sure the display of miR-581 in ovarian cancer SKOV3 cells in control group and Ghrelin-treated group(600 ng/mL,24 h),and the expression of miR-581 after miR-581 NC(miR-581 negative control)and miR5 81 mimics(miR-581 overexpression)were transfected by liposome method quantity;the expression of target gene FOXO1 in control group,miR-581 NC group and miR-581 mimics group was confirmed by western blot.② The show of autophagy-related proteins LC3Ⅱ and Beclinl in control group,miR-581 NC group and miR-581 mimics group were verified by Western blot.③Discovering the control group,Ghrelin-treated group,Ghrelin+miR-581 NC group and Ghrelin+miR-581 mimics group’s LC3Ⅱ and Beclinl autophagy-related proteins’pression through Western blot.④By using Western blot to confirm control group and AS 1842856 group’s(FOXO1 inhibitor)expression of LC3Ⅱ、Beclinl and FOXO1.⑤Comparing control group,AS1842856 group,miR-581 mimics group and miR-581 mimics+AS1842856 group’s amount of LC3Ⅱ and Beclinlby Western blot.RESULTS:①The second-generation sequencing results show that in ovarian cancer SKOV3 cells,contrast with the control group,22 miRNAs were differentially delivered in the Ghrelin treatment group,among them,12 were up-regulated,10 were down-regulated,and there were about 13,919 target genes can be effected by these miRNAs;these results of qRT-PCR experiment mean that comparing with the control group,Ghrelin treatment group’s quantity of miR-581 was down-regulated(P<0.05),and juxtaposing with the control group and miR-581NC group,the amount of miR-581 in the group of miR-581 mimics was rised(P<0.05);under the results of Western blot experiments,contrasted with the control and miR-581 NC groups,the expression of FOXO1 in the miR-581 mimics group was down-regulated(P<0.01).②Western blot outcomes display that compared with the control and miR-581 NC groups,the show of Beclinl and LC3Ⅱ in the miR-581 mimics group were dropped(P<0.01).③The reslutes of Western blot experiment reveal that the amount of autophagy-related proteins LC3Ⅱ and Beclinl were rised(P<0.01)in the Ghrelin group and the Ghrelin+miR-581 NC group by comparing with the control group,and the amount of autophagy-related proteins LC3Ⅱ and Beclinl were dropped(P<0.05,P<0.01)in miR-581 mimics group by comparing with Ghrelin group and the Ghrelin+miR-581NC group.④ The consequence means that comparing with the control group,AS1842856 group’s amount of autophagy-related proteins LC3Ⅱ and Beclinl were decreased(P<0.05),while FOXO1 had no significant changing(P>0.05)by using Western blot experiments.⑤The outcomes of Western blot experiments show that the amount of autophagy-related proteins LC3Ⅱ and Beclinl were declined(P<0.05,P<0.01)in the AS1842856 group and miR-581 mimics group,and were also declined(P<0.01、P<0.01)in the miR-581 mimics+AS1842856 group by comparing with the control group;the quantity of autophagy-related proteins LC3Ⅱ and Beclinl were droppted(P<0.05,P<0.01)in the group of miR-581 mimics+AS 1842856 by comparing with AS 1842856 group;the quantity of autophagy-related proteins LC3Ⅱand Beclinl were down-regulated(P<0.05,P<0.01)in the miR-581 mimics+AS 1842856 group by comparing with miR-581 mimics.CONCLUSION:Ghrelin can up-regulate FOXO1 by down-regulating the expression of miR-581 to promote autophagy in ovarian cancer SKOV3 cells.