LncRNA NBR2 Affects The Proliferation,imgration And Invasion Of Gastric Cancer Cells Through MiR-3619-5p

Objective: lncRNA NBR2 is dysregulated in Multiple cancers.Previous studies in our group indicate that lncRNA NBR2 is related to the malignant progression of GC cells.This study aims to explore the mechanism of lncRNA NBR2 in promoting the proliferation,migration and invasion of GC cells.methods1.The expression of lncRNA NBR2 in gastric cancer and normal tissues was analyzed by UALCAN databases(http://ualcan.path.uab.edu),and It is verified at the cell level by real-time fluorescent quantitative PCR(q RT-PCR).2.miRNAs binding to lncRNA NBR2 were predicted through the starbase databases(http://starbase.sysu.edu.cn/index.php).The wild-type vector lncRNA NBR2-wt and mutant vector lncRNA NBR2-mu were constructed,and the binding between miR-3619-5p and lncRNA NBR2 was detected by dual luciferase reporter system.3.The expression of miR-3619-5p in gastric cancer cells SGC7901,MGC803,BGC823 and immortalized gastric mucosal epithelial cell GES-1was detected by q RT-PCR.4.The lncRNA NBR2 interfering vector(sh-NBR2),miR-3619-5p mimic,miR-3619-5p inhibitor and corresponding negative control were transiently transfected into BGC823 cell line by Lipofectamine 8000.sh-NBR2 and miR-3619-5p inhibitor were also co-transfected into BGC823 cell line.Transfection efficiency was detected by fluorescence microscopy and q RTPCR.5.The expression of miR-3619-5p in BGC823 cells after lncRNA NBR2 knockdown was analyzed by q RT-PCR.6.The effects of lncRNA NBR2 and miR-3619-5p on the biological behavior of BGC823 cells were investigated by CCK8 proliferation test,wound-healing test,Transwell migration and invasion test,and then the functional relationship was analyzed between lncRNA NBR2 and miR-3619-5p.Results1.UALCAN database showed that lncRNA NBR2 was highly expressed in gastric cancer tissues compared with normal tissues(P < 0.0001),and lncRNA NBR2 was generally overexpressed in different gastric cancer stages,lymph node metastasis and histological types of gastric cancer.The q RT-PCR confirmed that compared with gastric mucosal epithelial cell GES-1,the expression of lncRNA NBR2 was increased in three gastric cancer cell lines(P<0.05),especially in BGC823 cells.2.Starbase database predicted that there were binding sites between miR-3619-5p and lncRNA NBR2.Dual luciferase reporter gene assay showed that miR-3619-5p significantly decreased the luciferase activity of lncRNA NBR2-wt(P < 0.0001),while miR-3619-5p failed to down-regulate the luciferase activity of lncRNA NBR2-mu(P=0.9971),indicating the binding between miR-3619-5p and lncRNA NBR2.3.q RT-PCR verified that the expression level of miR-3619-5p in gastric cancer SGC7901,MGC803 and BGC823 cells was markedly lower than that in GES-1 cells(P<0.05),and BGC823 was down-regulated most evidently.After interfering with lncRNA NBR2 in BGC823 cells,the expression of miR-3619-5p increased significantly,indicating that lncRNA NBR2 has a negative effect on miR-3619-5p.4.The results of CCK8 proliferation experiment revealed that OD value of BGC823 cells at 450 nm in sh-NBR2 group was significantly lower than that in NC group(P < 0.0001),which suggests that the interference lncRNA NBR2 decreased the proliferation of gastric cancer BGC823 cells.The OD value of BGC823 cells at 450 nm in miR-3619-5p mimic group decreased vs m-NC group(P=0.0078)and control group(P=0.0010),while OD value in miR-3619-5p inhibitor group increased significantly vs i-NC group(P<0.0012)and control group(P<0.0061),suggesting that miR-3619-5p can inhibit cell proliferation.Campared with sh-NBR2 group,the OD value of BGC823 cells co-transfected with sh-NBR2 and miR-3619-5p inhibitor increased significantly(P=0.0102),which indicated that miR-3619-5p could partially reverse the stimulative effect of lncRNA NBR2.5.The wound-healing assay displayed that the cell mobility ratio of shNBR2 group was evidently reduced compared with that of control group(P=0.0014)and NC group(P=0.0061).Compared with m-NC group,miR-3619-5p mimic group delayed the migration of cells at the scratch site(P=0.0083),while miR-3619-5p inhibitor promoted cell scratch healing compared with iNC group(P<0.0001).And down-regulation of miR-3619-5p partially reversed the migration inhibitory effect caused by lncRNA NBR2 interference(P=0.0076).6.Transwell migration assay showed that the number of migrating cells in sh-NBR2 group was significantly lower than that in NC group and control group(P < 0.0001),suggesting that interference lncRNA NBR2 weakened the migration ability of gastric cancer cells.Mi R-3619-5p mimic transfection cut down the number of cell migration clearly compared to control group and mNC group(P < 0.0001),while miR-3619-5p inhibitor transfection have the opposite effect vs control group and i-NC group(P < 0.0001),which demonstrated that miR-3619-5p inhibited cell migration.The number of migratory cells in miR-3619-5p inhibitor and sh-NBR2 co-transfected group was significantly higher than that in sh-NBR2 group(P<0.0001),indicating that down-regulated miR-3619-5p partially reversed the anti-migration effect caused by lncRNA NBR2 interference.7.Transwell invasion assay showed that the number of invasive cells in the sh-NBR2 group was significantly less than that in control group and NC group(P<0.0001),indicating that interference with lncRNA NBR2 inhibited cell invasion.Compared with control group and m-NC group,the number of invasive cells in miR-3619-5p mimic group decreased(P<0.0001),however invasive cells in miR-3619-5p inhibitor group were significantly higher than those in control group and i-NC group(P<0.0001),which implied that miR-3619-5p can suppress the migration of BGC823 cells.Compared with shNBR2 group,the number of invasive cells increased significantly after cotransfection with miR-3619-5p inhibitor(P < 0.0001),indicating that the down-regulation of miR-3619-5p partially reverse anti-invasion effect of shNBR2.Conclusions1.Down-regulation of lncRNA NBR2 inhibits the proliferation,migration and invasion of gastric cancer cells.2.miR-3619-5p has an inhibitory effect on the proliferation,migration and invasion of gastric cancer cells.3.miR-3619-5p partially reversed the promotion effects of lncRNA NBR2 on the proliferation,migration and invasion of gastric cancer cells.