MiRNA-330-3p Promotes Brain Metastasis Of Non-small Cell Lung Cancer By Activating MAPK/ERK Signaling Through GRIA3

Part one: miR-330-3p affects the biological behavior of NSCLC and HUVECs in vitro and in vivoObjective: To investigate the biological role of miR-330-3p in NSCLC cells in vitro and in vivo,and further to explore the effect of miR-330-3p on migration,invasion and angiogenesis in HUVECs.Methods: In this study,the expression of miR-330-3p in The normal human bronchial epithelial cell line BEAS-2B and NSCLC cells(A549,H460,HCC827,H1975 and PC-9)was quantified by quantitive real-time PCR(q RT-PCR).And we also used q RT-PCR quantified the expression of miR-330-3p in lung tissue samples of NSCLC patients with or without brain metastasis(BM).Stable over-expression and knock-down of miR-330-3p in NSCLC cells was constructed with lentivirus.The effects of miR-330-3p on NSCLC cells(A549 and HCC827 cells)were investigated using assays of cell viability,migration,invasion,cell cycle,apoptosis,western blotting,immunohistochemical and immunofluorescence staining.A xenograft nude mouse model and in situ brain metastasis model were used to observe tumor growth and brain metastasis.HUVECs were co-cultured with conditioned medium(CM),obtained from NSCLC cells over-expression or knock-down of miR-330-3p or empty-vector transfection.Wound healing,transwell and tube formation assayes were employed to examine the effect of miR-330-3p on HUVECs.Results: The expression of miR-330-3p was up-regulated in NSCLC cells compared with BEAS-2B,and in patients with BM compared with BM-(P<0.05).MTT assay showed that overexpressing miR-330-3p promoted proliferation of NSCLC cells at 24 and 48h(P<0.05).PI staining revealed over-expressing miR-330-3p increased the percentage of cells in the S phase,and decreased the percentage of cells in the G1 phase in both A549 and HCC827 cells,indicating that over-expressing miR-330-3p could lead to S arrest.Western blot showed that cylin D1,cell cycle associated protein,was up-regulated in A549 and HCC827 cells over-expression of miR-330-3p.Flow cytometry showed that apoptosis was inhibited in over-expressing miR-330-3p in both A549 cells(3.06% vs.5.64% in lentivirus transfection alone)and HCC827 cells(3.42% vs.7.41%).In contrast,cell apoptosis was increased in miR-330-3p-knocked down A549 cells(16.66% vs.5.64%)and HCC827 cells(13.41% vs.7.41%)(P<0.05).Detection of the expression of apoptosis-associated proteins(Bax,Bcl-2,and caspase3)exhibited that that over-expressed miR-330-3p increased Bcl-2 and reduced Bax expression,and miR-330-3p knockdown increased the expression of cleaved caspase3.The wound healing assay demonstrated that the migratory ability of A549 and HCC827 cells over-expressing miR-330-3p was significantly higher than that of cells transfected with empty lentivirus(P<0.05).Similarly,the transwell migration assay showed that miR-330-3p over-expression significantly increased the migratory ability of NSCLC cells(P<0.001).Moreover,the transwell invasion assay revealed that the invasiveness of NSCLC cells over-expressing miR-330-3p was significantly higher than that of the cells transfected with the empty lentivirus(P<0.05).These results indicated that miR-330-3p over-expression significantly promoted the migration,invasion and angiogenesis of NSCLC cells in vitro.Culture of HUVEC cells in conditioned medium extracted from culture supernatant of A549 and HCC827 cells showed that migration and invasion of HUVEC cells were enhanced by miR-330-3p over-expression in A549 and HCC827 cells and decreased by miR-330-3p knockdown(P<0.05 for both).Tube formation assay showed that tube was compact and complete in A549 and HCC827 cells over-expressing miR-330-3p,and the tube was incomplete and flutty in cells knocking down miR-330-3p.Additionally,miR-330-3p over-expression elevated the level of VEGFA expression.A xenograft tumor model by subcutaneously injecting into the front flank of nude mice the A549 and HCC827 cells stably over-expressing miR-330-3p,cells with miR-330-3p knockdown or cells transfected with empty vector.The results showed that tumors injected with miR-330-3p-knock down A549 and HCC827 cells grew more slowly and were of small size as compared with the tumors injected with cells with empty vector(P<0.05),The survival time was shorter in mice injected with H460 and H1975 cells over-expressing miR-330-3p(P<0.05 vs.tumors injected with empty vector cells).Furthermore,immunohistochemical analysis revealed that miR-330-3p over-expression increased the expression of PCNA and cyclin D1 and decreased the expression of caspase3 and BAX in tumors when compared with the NC group.Knocking down miR-330-3p decreased PCNA and cyclin D1 expression,and increased caspase3 and Bax expression in tumors.Immunofluorescence staining revealed that CD34 expression was down-regulated and stained vessels were less in tumors injected with miR-330-3p-knockdown cells.The brain metastasis model showed that more metastatic foci in mice receiving NSCLC cells over-expressing miR-330-3p than in those receiving cells transfected with empty vector or non-transfected cells,Most mice receiving A549 and HCC827 cells with miR-330-3p-knockn down did not have tumor foci.These finding suggested that miR-330-3p could promote the growth of metastatic tumors.Conclusion: miR-330-3p could promote the ability of proliferation,migration,invasion,angiogenesis and tumorigenesis,inhibit apoptosis of NSCLC cells.Conversely,knocking down miR-330-3p could inhibited the ability of proliferation,migration,invasion,angiogenesis and tumorigenesis,promoted apoptosis of NSCLC cells.Part two: miR-330-3p exerted biological effect on NSCLC cells by activating MAPK/ERK signaling pathway through GRIA3Objective: Identifying the target of miR-330-3p,and investigating the underlying machanism of miR-330-3p in regulation of migration and invasion in NSCLC cellsMethods: Bioinformatics analysis(Target Scan,PicTar,HOCTar,Tarbase,miRanda and micro Cosmwere)was used to forcast the gene targets of miR-330-3p.Candidate targets were analyzed by whole genome analysis of A549 cells over-expressing miR-330-3p vs.cells transfected with empty lentivirus.RT-PCR and Western blotting were used to detect the effect of miR-330-3p on the target at m RNA and protein levels.Dual luciferase reporter assay was used to confirm the target of miR-330-3p.Plasmid over-expressing miR-330-3p was transfected in A549 and HCC827 cells,the transfective efficiency was detected by western blotting.MTT and transwell assay were used to examine the ability of migration and invasion of A549 and HCC827 cell over-expressing GRIA3.Furthermore,pathway enrichment analysis of A549 cells over-expressing miR-330-3p was made to identify the pathway associated with migration and invasion in NSCLC cells.Western blotting was used to detect the pathway.Additionally,the inhibitors of pathway were used to confirm the pathway.Results: Bioinformatics analysis using Target Scan,PicTar,HOCTar,Tarbase,miRanda and micro Cosm identified 4 genes: BMI-1,GRIA3,SOSTDC1 and AGTR2.Candidate targets were analyzed by whole genome analysis of A549 cells over-expressing miR-330-3p vs.Cells transfected with empty lentivirus.Then we selected GRIA3 as our research target.In a luciferase reporter vector containing the wild-type(Wt)or mutant(Mut)sequences of the GRIA3 3’-UTR targeted by miR-330-3p,co-transfection with a miR-330-3p mimic inhibited the luciferase activity of the Wt reporter gene(P=0.048).Activity of the Mut reporter gene was not affected.The expression of GRIA3 in A549 or HCC827 cells was increased by over-expressing miR-330-3p(P=0.01 and P =0.008 vs.cells transfected with empty lentivirus,respectively),and decreased by knocking down miR-330-3p(P=0.009 and P=0.006,respectively)at m RNA level.Western blot yielded highly similar results.GRIA3 was negatively correlated with the expression of miR-330-3p.GRIA3 expression was significantly lower in the 62 NSCLC patients with BM vs.in those without BM both in serum and tissues(P<0.05.These results demonstrated that miR-330-3p could directly suppress the expression of GRIA3 in NSCLC cells by directly targeting the GRIA3 3’UTR.Over-expression of GRIA3 decreased cell viability and migration in both cell lines.Pathway enrichment analysis of A549 cells over-expressing miR-330-3p identified a number of genes that could be categorized into apoptosis,cell cycle,mitogen-activated protein kinase(MAPK),and NF-κB.Over-expressing miR-330-3p decreased total AKT and ERK in A549 cells,and increased p-AKT and p-ERK in HCC827 cells.Treatment with the MEK1/2 inhibitor U0126 increased the levels of GRIA3 in A549 and HCC827 cells that overexpressed miR-330-3p as well as in cells expressing anti-miR-330-3p.The PI3K/AKT inhibitor LY294002 treatment did not affect GRIA3 expression.The migration and invasion of A549 and HCC827 cells over-expressing miR-330-3p was inhibited by U0126 but not LY294002.Conclusions: miR-330-3p may promote migration and invasion of NSCLC cells by activating MAPK/ERK signaling pathway through targeting GRIA3.