| Background:Gastric cancer is the fourth most common cancer in the world.It is still one of the most common and deadly tumors in the world due to the difficulty of early diagnosis,organ system toxicity caused by chemotherapy and drug resistance.Therefore,it is particularly important to find new therapeutic targets for gastric cancer drugs.MicroRNA has been reported to suppress proliferation and migration by regulating the expression of its target genes.Objective:The aim of this study was to investigate whether miR-4316 inhibits proliferation and migration by downregulating VEGF-A in vitro and its clinical significance in gastric cancer.Methods:In this study,we used four cell lines which are GES-1 as normal gastric cell line,and gastric cancer cell lines MGC-803,BGC-823,and SGC-7901.First,we investigated miR-4316 expression in human tissues and all cell lines.The miR-4316 and VEGF-A gene expression was detected by qRT-PCR,BGC-823 and SGC-7901 cell lines have selected in this study.Secondly,VEGF-A and c-Met protein level were determined by western blot after the cells SGC-7901 and BGC-823 in each transfected group(miR-4316-mimic,miR-4316-inhibitor or N.C)or co-transfected group(si-VEGF-A,miR-4316-mimic+si-VEGF-A,miR-4316-inhibitor+si-VEGF-A,or N.C)were cracked after transfected 48 h.The function of miR-4316 was analyzed through cell proliferation,migration and colony formation experiments.The effect of miR-4316 on the proliferation of gastric cancer cells was studied through the CCK-8 experiment.Through each group(miR-4316 mimic,miR-4316 inhibitor and NC)or(si-VEGF-A,miR-4316 mimic + si-VEGF-A,miR-4316 inhibitor + si-VEGF-A And N.C)scratch test to analyze the effect of miR-4316 regulating VEGF-A on migration ability of gastric cancer cell lines.Immunofluorescence stain assay was used to detect the expression of VEGF-A after transfection of miR-4316 or co-transfection of miR-4316 with VEGF-A in each group(miR-4316-mimic,miR-4316-inhibitor or N.C)or co-transfected with(si-VEGF-A,miR-4316-mimic+si-VEGF-A,miR-4316-inhibitor+si-VEGF-A,or N.C).The bioinformatics analaysis and luciferase reporter gene assay were applied to confirm the relationship between mi RNA and VEGF-A on VEGF-A 3-untranslated region(3-UTR).We also carried out in vivo xenograft nude mice by inoculation the gastric cancer cell line BGC-823 transfected with miR-4316-mimic then measured the volume and the weight each mice.Results:The results of qRT-PCR indicated that miR-4316 expression level was significantly downregulated in human tissues of GC compared with their adjacent tissues as well as in GC cell lines MGC-803,SGC-7901 and BGC-823 compared with their control GES-1 cell lines.Then SGC-7901 and BGC-823 cell lines were selected because miR-4316 expression was down-regulated in both of cells SGC-7901 and BGC-823.MiR-4316 was transfected using miR-4316-mimic and miR-4316-inhibitor into SGC-7901 and BGC-823 cell lines and the results have been shown that miR-4316 was up-regulated by miR-4316-mimic and downregulated by miR-4316-inhibitor detected by qRT-PCR.After transfected miR-4316-mimic and miR-4316-inhibitor into SGC-7901 and BGC-823 cell lines,the results were showed that the miR-4316-mimic inhibited cell proliferation,cell migration and cell colony formation,in contrast,miR-4316-inhibitor were promoted cell proliferation,cell migration and cell colony formation,the results have been demonstrated by CCK-8,wound healing,transwell,and colony formation assay.The target gene of miR-4316 was validated by luciferase reporter gene assay.First,the regulation of miR-4316 for VEGF-A transcription was showed in SGC-7901 and BGC-823 cells.Then,we verified the regulatory effect of miR-4316 on VEGF-A gene,this result suggested that VEGF-A gene is a target of miR-4316.VEGF-A expression was significantly high in gastric cancer tissues compared with their control tissues and it also was high in cancer cell lines MGC-803,SGC-7901 and BGC-823 compared with GES-1 and analyzed by qRT-PCR.After transfected SGC-7901 and BGC-823 cells by miR-4316-mimic and miR-4316-inhibitor the results showed that VEGF-A was significantly low expressed by miR-4316-mimic and significantly high expressed by miR-4316-inhibitor,the results were detected by qRT-PCR.The western blot's results showed that VEGF-A and c-Met expression were low expressed in miR-4316-mimic transfected cells than N.C while VEGF-A expression was high expressed in miR-4316-inhibitor transfected cells than N.C in both of SGC-7901 and BGC-823.The immunofluorescence assay results indicated that level of VEGF-A was lower than N.C while VEGF-A level was higher than N.C in both of SGC-7901 and BGC-823 cell lines.The results showed that VEGF-A expression was significantly decreased than N.C after transfected with si-VEGF-A in both of cell lines,this detected by qRT-PCR assay.After transfected and co-transfected the GC cell lines with si-VEGF-A,miR-4316-mimic+si-VEGF-A and miR-4316-inhibitor plus si-VEGF-A into both of cell lines.VEGF-A and c-Met level were significantly decreased in si-VEGF-A groups,while VEGF-A and c-Met were more decreased in miR-4316 plus si-VEGF-A than N.C,while no significance with miR-4316-inhibitor plus si-VEGF-A compared with N.C,this was observed by Western blot assay.Additionally,the CCK-8,colony formation assay,wound healing assay,and transwell assays results have showed that si-VEGF-A and miR-4316-mimic plus si-VEGF-A were significantly inhibited proliferation,cell growth,wound closure and cell migration compared with N.C,but the results were no changed in miR-4316-inhibitor plus si-VEGF-A compared with N.C.The immunofluorescence assay result showed that VEGF-A level was reduced in si-VEGF-A,and miR-4316-mimic plus si-VEGF-A and no significance with miR-4316-inhibitor plus si-VEGF-A when it compared with N.C.The schematic diagram showed the pathway of miR-4316.In vivo experment the tumor growth was significantly reduced in transfected group with miR-4316-mimic-BGC-823 compared with N.C in nude mice.Conclusion:The present study demonstrated that miR-4316 can significantly suppress gastric cancer cells proliferation and migration by directly targeting down-regulating VEGF-A.Our results indicated that miR-4316 could act as a tumor suppressor gene in gastric cancer and this indicated that Mi R-4316 might be a potential therapeutic target for gastric cancer. |
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