| Objective To investigate the actual expression level of microRNA-760 in human gastric cancer tissues and cells and its effect on autophagy in human gastric cancer cells,Dextran sulfate(DS)whether affect autophagy in human gastric cancer cells by regulating microRNA-760.Method Part Ⅰ 1.Four miRNAs were screened out by Veny map which was drawn according to the results of sequencing and database,and the expression levels of these three miRNAs in gastric cancer tissues and para-cancer tissues were detected by RT-qPCR,the miRNA-760 with the greatest difference in expression was selected as the study object.2.The expression of miRNA-760 was detected by RT-qPCR in 33 gastric cancer tissues and 4 gastric cancer cell lines(HGC-27,MKN-45,MKN-28 and AGS).3.Gastric cancer cells HGC-27 and AGS were cultured in vitro and transfected with miRNA-760-inhibitor and miRNA-760-mimics.After 24 h,the transfection efficiency was detected by inverted Fluorescence microscope and RT-qPCR,and the cell model of miRNA-760 knockdown and overexpression was constructed.4.Cell phenotype test: CCK-8 and Clonogenic assay were used to detect cell proliferation,and Wound healing and Transwell test were used to observe cell migration and invasion.5.Autophagy was detected by transmission electron microscopy;The expression of autophagy-related factors was detected by immunofluorescence;The expression levels of autophagy-related markers(LC3,P62,Beclin-1)and relevant proteins downstream of the classical autophagy pathway PI3K/AKT/mTOR signaling pathway(mTOR,p-mTOR,PI3 K,p-PI3 K,Akt,p-AKT)were detected by Western Blot.Part Ⅱ 1.Human gastric cancer cell lines(HGC-27 and AGS)were treated with DS for 48h;The expression level of miRNA-760 was detected by RT-qPCR,and the levels of autophagy-related factors(LC3,P62,Beclin-1)in human gastric cancer tissue and gastric cancer cells and DS intervention posterity in gastric cancer cell were detected by Western Blot.2.Gastric cancer cells transfected with miRNA-760 mimics and miRNA-760-inhibitor were treated with DS for 48 h,respectively;The ability of proliferation,invasion and migration was detected by CCK-8,clone formation,scratch test and Transwell test.3.The expression levels of autophagy-related factors(LC3,P62,Beclin-1)and relevant proteins of PI3K/AKT/mTOR signaling pathway(mTOR,p-mTOR,PI3 K,p-PI3 K,Akt,p-AKT)were detected by electron microscopy,Western Blot and immunofluorescence staining.4.Construction of nude mice intraperitoneal implantation tumor model:The nude mice were randomly divided into control group and DS Group.After two weeks of DS intervention,the mice were killed and the expression of miRNA-760 in omentum and mesentery were detected by RT-qPCR;The expression levels of autophagy-related factors(LC3,P62,Beclin-1)were detected by Western Blot and immunohistochemistry.Result Part Ⅰ :1.Four miRNAs,miRNA-503-5p,miRNA-149-5p,miR-342-3p and miRNA-760,were screened by combining the sequencing results with the database.According to the expression results of these four miRNAs in human gastric cancer tissues,miR-760,which had the lowest expression and the greatest difference in expression,was selected as the study object(P<0.01,P<0.001).2.The results of RT-qPCR showed that miR-760 was low expressed in 33 gastric cancer tissues and 4kinds of human gastric cancer cells(P<0.01,P<0.001);The expression of miRNA-760 was the highest in HGC-27 cells and the lowest in AGS cells,so the transfection inhibitor of HGC-27 cells and mimics of AGS cells were selected for follow-up experiments;3.The optimal transfection concentration of mimics and inhibitor was30 n M by RT-qPCR(P<0.05,P<0.01,P<0.001);The results of inverted Fluorescence microscope showed that more than 90% of AGS and HGC-27 cells were transfected with mimics and inhibitor respectively.The results of RT-qPCR showed that the miRNA-760 relative expression level in the mimics group was significantly higher than that in the mi-NC group(P<0.01).In contrast,the miRNA-760 relative expression level in the inhibitor group was significantly lower than that in the in-NC group(P<0.01).The results of cell phenotype experiment showed that downregulation of miRNA-760 significantly promoted the proliferation,migration and invasion of human gastric cancer cells,while overexpression of miRNA-760 significantly inhibited the proliferation,migration and invasion of human gastric cancer cells.Knockdown of miRNA-760 up-regulated the expression of autophagy-related factors LC3Ⅱ/Ⅰ and Beclin-1,and down-regulated the expression of P62(P<0.05,P<0.01);Conversely,the expression of autophagy-related factors LC3Ⅱ/Ⅰ and Beclin-1 decreased(P<0.01),while the expression of P62 increased(P<0.01,P<0.001).Moreover,up-regulation of miRNA-760 inhibited the expression of p-mTOR,p-PI3 K,p-Akt,and P-mTOR proteins,which are relevant proteins of the classical autophagy pathway PI3K/AKT/mTOR signaling pathway(P<0.01).Knockdown of miRNA-760 increased the expression of p-mTOR,p-PI3 K and p-Akt,proteins-related of PI3K/Akt/mTOR signaling pathway(P<0.01,P<0.001).Part Ⅱ 1.The expression of miRNA-760 was up-regulated in human gastric cancer cells(HGC-27,AGS)after DS treatment(P<0.05,P<0.01);Autophagy-related factors LC3Ⅱ/Ⅰ and Beclin-1 were highly expressed in human gastric cancer tissues and gastric cancer cells,while P62 was low(P<0.05,P<0.01,P<0.001).DS could promote the expression of LC3Ⅱ/Ⅰ and Beclin-1 and inhibit the expression of P62(P<0.05,P<0.01).2.DS can obviously enhance the inhibition of miRNA-760 overexpression on the proliferation and invasion of human gastric cancer cells,and reverse the promotion of miRNA-760 knockdown on the proliferation and invasion and migration of human gastric cancer cells(P<0.05,P<0.01).3.The results of Western Blot and cell immunofluorescence staining showed that DS intervention mimics could further increase the expression of autophagy-related factors LC3 and Beclin-1,while the expression of P62 was further decreased(P<0.05,P<0.01,P<0.001),moreover,the expression of p-mTOR,p-PI3 K and p-AKT relevant proteins in the classical autophagy pathway PI3K/Akt/mTOR signaling pathway is further inhibited(P<0.05,P<0.01);DS after intervention inhibitor,can inhibit knock on reducing the miRNA-760 human gastric cancer cells in autophagy related factors and autophagy classic(P<0.05,P<0.01,P<0.001).4.In vivo experiments showed that:Compared with the control group,the expression of miRNA-760 was up-regulated in the peritoneal greater omentum and mesenteric metastases tumor of the nude mice in DS Group(P<0.05,P<0.01),and could increase the expression of autophagy-related factors LC3Ⅱ/Ⅰ and Beclin-1,the expression of P62 decreased(P<0.01).Conclusion:1.Mi RNA-760 was low expressed in human gastric cancer tissues and gastric cancer cells,and miRNA-76 inhibit human gastric cancer cell proliferation,invasion,migration and autophagy.2.DS inhibit the proliferation,invasion,migration and autophagy of human gastric cancer cells by regulating the expression of miRNA-760. |
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