MiR-646 Inhibited Cell Proliferation And EMT-induced Metastasis By Targeting FOXK1 In Gastric Cancer

BackgroundmiRNA is a kind of endogenous short sequences of single small non-coding RNA,its main biological function is involved in gene transcriptional regulation,and give priority to with negative regulation.In recent years,studys found that the expression of microRNAs was closely related to the occurrence of a wide variety of tumor development,including the gastric cancer.A lot of microRNAs mediated proliferation,invasion and metastasis in tumor by targeting negative regulation of the downstream key molecular transcription.Forkhead-box K1(FOXK1),a forkhead family transcription factor,is involved in development and metabolism in a wide variety of tumor.Our previous studies showed that FOXK1 induces the epithelial-mesenchymal transition(EMT)in tumor cells,maintains the invasive potential of GC and appears to play a crucial role in the metastatic progression of human carcinomas.We have developed an algorithm to predict the targeting of FOXK1 by miR-646.However,whether this miRNA participates in the regulation of FOXK1 expression in GC remains unknown.In this study,we aim to investigate the effects of the miR-646 on gastric cancer cell biological characteristics,and clear whether the miR-646 can be targeted to FOXK1 and inhibited the proliferation and metastasis of gastric cancer,in hope to find the theoretical basis for potential therapeutic targets in gastric cancer.MethodsFirst of all,qRT-PCR and in situ hybridization experiment testing the expression of miR-646 in gastric cancer cells and gastric cancer surgical specimens and analyze its correlation with clinical pathology data.By transfected miR-646 mimics or inhibitors to over-express or knock-down the expression of miR-646 in gastric cancer cells,and reuse EdU proliferation experiment,Soft agar assay,Wound healing experiment and Transwell invasion experiment to assess their impact on the biological characteristics of gastric cancer cells.Then,by observing the cell phenotype,immunofluorescence test and Western blot detection EMT related markers to observe the effects of miR-646 on gastric cancer cells EMT and its impact of TGF-beta inducing EMT.Dual luciferase assay confirmed the target genes of miR-646 and found its binding site.the in vitro cell experiment and build nude mice subcutaneously into tumor and metastatic tumor model were further validated the effect of miR-646/FOXK1.Finally,take the Western blot,IHC and ISH to clarify the molecular mechanism of miR-646/FOXK1 on gastric cancer.Results1.MiR-646 expression is markedly down-regulated in GC cell lines and tissues.Low miR-646 expression is associated with tumor size,invasion dEPth,lymph node metastasis and TNM stages,but no significant association was observed between miR-646 expression and age,gender or differentiation.2.Transient transfection of GC cells with miR-646 inhibited their growth and migration.Moreover,miR-646 influenced the expression of EMT-associated proteins.TGF-?1 treatment significantly suppressed the expression of miR-646,and overexpression of this miRNA counteracted the influence of the TGF-?1-inducedEMT phenotype.3.Overexpression of miR-646 inhibits FOXK1 3 'UTR dual luciferase activity,but mutation its binding sites,the fuction of inhibition disappear;4.MiR-646 inhibits gastric cancer cell FOXK1 expression,and the expression FOXK1 reversible miR-646 for inhibitory effect on the activity of invasion and metastasis of gastric cancer cells;5.In vivo,it inhibited the FOXK1-mediated proliferation and EMT-induced metastasis.Consistently,inverse correlations were also observed between the expression of miR-646 and FOXK1 in human GC tissue samples.Furthermore,miR-646 regulated AKT/mTOR signaling after FOXK1.ConclusionsMiR-646 expression is markedly down-regulated in GC cell lines and tissues,and inhibiting gastric cancer cell proliferation,invasion and metastasis.it play a role of tumor suppressor in gastric cancer;MiR-646 through targeted on FOXK1 and AKT/mTOR pathway to inhibit the EMT process of gastric cancer cell,and inhibit the invasion and metastasis of gastric cancer.