MicroRNA-338Inhibits Growth, Invasion, Metastasis And Epithelial-mesenchymal Transition Of Gastric Cancer By Targeting NRP1Expression

Objective:NRP1as multifunctional non-tyrosine-kinase receptors play critical roles intumor progression. MicroRNAs (miRNAs) are an important class ofpervasive genes that are involved in a variety of biological functions,particularly cancer. It remains unclear whether miRNAs can regulate theexpression of NRP1. The goal of this study was to identify miRNAs thatcould inhibit the growth, invasion and metastasis of gastric cancer bytargeting NRP1expression.Methods:1. The upstream miRNA of NRP1was predict by miRNA profiles andprediction tools.2. The expression levels of mature miRNA in gastric cancer (n=41) andadjacent normal mucosa tissues (n=24) were determined usingquantitative PCR, and the expression of miRNA in gastric cancer cell lines(SGC7901, HGC27, AGS, MKN45and N87) and normal gastric mucosa cell line (GES1) were also detected by quantitative PCR.3. The wild-type and mutant NRP1PsicheckTM-2vector was constructed.Lipofectamine2000was used to cotransfect MKN45cells with thehsa-miRNA and PsicheckTM-2Dual-Luciferase miRNA target expressionvectors containing wild-type or mutant target sequences. The fireflyluciferase activity was measured using the Dual Luciferase Assay18h aftertransfection.4. We infected the gastric cancer cell lines AGS and MKN45withLV-hsa-miRNA, and then gastric cancer cell migration,invasion,proliferation and apoptosis was determined by cell viability andclonability assays, transwell assays and the fluorescence-activated cellsorting (FACS) analysis. Rescue experiments were also performed. Weconstruct a plasmid containing the NRP1coding sequence but lacking the3’UTR of the NRP1mRNA. To determine gastric cancer cell migration,invasion,proliferation and apoptosis in AGS cells infected withLV-hsa-miRNA or cont-miR, with or without NRP1restoration,4groupswere set up:①C ontrolgroup②o verexpressed miR-338group③overexpressed NRP1group④o verexpressed miR-338and NRP1group.5. Primary tumor growth was examined after the orthotopic injection of1×106AGS cells with the forced expression of miR-338or cont-miR, withor without NRP1restoration. 6. The phosphorylation and total expression levels of ERK1/2, Akt andP38MAPK was detected by western blot in AGS cells infected withLV-hsa-mir-338or cont-miR. Rescue experiments were also performed asabove.7. SiNRP1plasmid was constructed, and the expression of N-cadherin,vimentin, fibronectin, E-cadherin and SNAIL was detected by western blotin AGS cells transfected with siNRP1or siC. The expression ofmesenchymal and epithelial markers was also examined by western blot inAGS cells infected with LV-hsa-mir-338or cont-miR, with or withoutNRP1restoration.Results:1. We predicted that the upstream miRNA of NRP1was miR-338by bymiRNA profiles and prediction tools.2. The expression level of mature miR-338in the gastric cancer tissues wassignificantly lower than in the adjacent normal mucosa tissues (p<0.05).miR-338was decreased in the gastric cancer cell lines (SGC7901, HGC27,AGS, MKN45and N87) compared with the normal gastric mucosa cell line(GES1)(p<0.05).3. The co-transfection of miR-338and the wild-type NRP13’UTR caused asignificant decrease in luciferase units compared with the controls (p<0.05).However, the co-transfection of miR-338and the mutant NRP13’UTR didnot cause a decrease in luciferase units compared with the controls (p>0.05). These results suggest that miR-338targets NRP1directly.4. Gastric cancer cell proliferation, migration and invasion capacities weresignificantly reduced after LV-hsa-mir-338infection compared withcont-miR infection (p<0.05). Gastric cancer cell apoptosis was significantlyincreased after LV-hsa-mir-338infection compared with cont-miR infection(p<0.05). Gastric cancer cell migration, invasion,proliferation andapoptosis are restored after NRP1restoration. Those results show thatmiR-338inhibits gastric cancer cell migration, invasion and proliferationand promotes apoptosis by targeting NRP1.5. Primary tumor growth in AGS cells with the forced expression ofmiR-338was significantly decreased compared with the control group(p<0.05), but the overexpression of NRP1could restore tumor growth.These results indicated that miR-338reduces tumor growth by targetingNRP1in vivo.6. The phosphorylation levels of ERK1/2, Akt and P38MAPK in AGS cellsinfected with LV-hsa-mir-338were significantly decreased compared withthe control group (p<0.05). The phosphorylation levels of Erk1/2, Akt andP38MAPK were not significantly altered in the AGS cells with forcedmiR-338expression and NRP1restoration. So we conclude that miR-338regulates the phosphorylation of ERK1/2, P38MAPK and Akt via NRP1.7. The expression levels of fibronectin, vimentin, N-cadherin and SNAILwere significantly decreased and the expression of E-cadherin was significantly increased in the NRP1-depleted tumor cells compared with thecontrol group (p<0.05). The expression levels of fibronectin, vimentin,N-cadherin and SNAIL were decreased and the expression of E-cadherinwas increased in the AGS cells with the forced expression of miR-338compared with the control group (p<0.05). The expression of the abovemesenchymal markers in the miR-338-expressing cells was restored to thenormal level by the restoration of NRP1expression. These resultsdemonstrated that miR-338could inhibit EMT via NRP1in gastric cancercells.Conclusions:miR-338as a novel tumor suppressor gene was decreased in gastric cancer.Meanwhile, miR-338can decrease migratory, invasive, proliferative andapoptotic behaviors, as well as gastric cancer EMT, by attenuating theexpression of NRP1.