MiR-7 Suppresses Tumor Progression Via Directly Targeting MAP3K9 In Pancreatic Cancer

Background:Pancreatic cancer(PC)lacks an effective treatment due to that it is difficult to be diagnosed at the early stage,leading to its high mortality rate.In recent years,lots of studies have shown that the cancer progression is involved in many signaling pathways including MAPK signaling pathway.It has been known that MAP3K9 is the upstream kinase in MAPK signaling pathway.The imbalance of MAP3K9 expression is closely correlated to carcinogenesis.Our previous experimental data showed that down-regulation of MAP3K9 suppressed human PC cell proliferation and induced apoptosis.We also found that MAP3K9 overexpression promoted human PC cell proliferation and inhibited apoptosis.Some miRNAs such as miR-34 a and miR-148 b are involved in the regulation of MAPK signaling through targeting the expression of MAP3K9.However,there are few reports about whether miR-7 could target the MAP3K9 and then participate in the MAPK signaling pathway.Purposes:We aim to investigate whether MAP3K9 is the direct target of miR-7 and whether miR-7 suppresses tumor progression via directly targeting MAP3K9 in pancreatic cancer.Our study will provide the theoretical and experimental evidences for clinical targeted therapies of pancreatic cancer.Methods:The PC cell lines including BxPC-3,PANC-1,and Patu8988 were studied for the detection of miR-7 expression by qRT-PCR.miR-7 inhibitors were transfected in PC cells which express high levels of miR-7.miR-7 mimics were transfected into PC cells with low expression of miR-7.MTT assay,Transwell assay,wound healing assay,flow cytometry,Western Blot,and in vivo experiments were used to observe the changes in the malignant biological behavior of cells and the effect on MAP3K9 expression after transfection.The PC xenograft model was established to observe theeffect of miR-7 on tumor growth in vivo.Luciferase activity analyzes the binding of miR-7 to MAP3K9 3’UTR.Combined use of miR-7 mimics and MAP3K9 overexpression co-treatment or co-treatment of miR-7 inhibitor and MAP3K9 siRNA in PC cells and observe changes in the malignant biological behavior of PC cells.Thereby,our results could determine whether miR-7 coukd directly regulate MAP3K9 and then affect the progress of PC.Results:The expression of miR-7 in BxPC-3 cells was significantly higher than that of PANC-1 and Patu-8988 cells.After miR-7 inhibitor was transfected into BxPC-3 cells,the proliferation,migration and invasion abilities of cells were obviously enhanced,and the cell apoptosis was impaired.After miR-7 mimics were transfected with PANC-1 and Patu-8988 cells,cell proliferation,migration and invasion abilities were significantly inhibited,and apoptosis was increased.In vivo experiments showed that the over-expression of miR-7 significantly inhibited tumor growth in mice.Luciferase activity analysis showed that miR-7 bound to the wild-type MAP3K9 3’UTR sequence,but did bind to the mutant MAP3K9 3’UTR sequence.Down-regulation of miR-7 expression in BxPC-3 cells increased the expression of MAP3K9,whereas PANC-1 and Patu-8988 cells exhibited lower expression of MAP3K9 after miR-7 up-regulation.Consistent with this result,the expression of MAP3K9 in the miR-7 overexpression group was significantly lower in animal experimental tumor tissues than in the control group.Knockdown of MAP3K9 impaired miR-7 down-regulation-mediated cell proliferation promotion and apoptosis inhibition.Overexpression of MAP3K9 reduced the inhibitory effect of miR-7mimics on cell proliferation and invasion in PANC-1 and Patu-8988 cells.Conclusions:miR-7 overexpression not only retarded the proliferation,but also inhibited the abilities of migration and invasion of human PC cells,and also induced cell apoptosis.Moreover,MAP3K9 is a direct target gene of miR-7.Furthermore,miR-7 n down-regulated the expression of MAP3K9 in human PC cells,thereby inhibiting cell proliferation and induction of apoptosis.All together,miR-7 exerts its anti-tumor activity via targeting MAP3K9 in PC cells.