MiR-10b Promotes Cell Invasion Through RhoC-AKT Signaling Pathway By Targetting HOXD10in Gastric Cancer

Objective: To explore the role of miR-10b in gastric cancer invasionanddiscloseits the possible molecular mechanism.Methods:qRT-PCR and in situ hybridizationexamine the expression of miR-10bin primary gastric carcinoma samples. Western blotting examines the expression ofmiR-10b in different gastric cancer cell lines. MKN45cells are transfected with orwithout miR-10b inhibitor, the transwell analysis detects the effect of miR-10b on cellmigration and invasion. Bioinformatic method, qRT-PCR andluciferase reporterassayare used to identify the target of miR-10b. Overexpressed miR-10b or HOXD10in MNK45cells, transwell analysis explores whether HOXD10is involved in the wayof miR-10b promotes the gastric cancer invasion. siRNA or kinase inhibitor block thedownstream target protein or signaling pathway, transwell analysis to further examinethe related mechanism underlying the role of miR-10b during gastric cancer invasion.Finally, immunohistochemistryand western blotexamine whether HOXD10isdownregulated in metastasis gastric cancer and poor differentiated gastric cancer celllines.Results:miR-10b expression levels weredramatically elevated in lymphoma nodemetastasis-positive tumor tissues compared with lymphoma node metastasis-freetumor tissues and the adjacent non-neoplastic gastric samples.These data revealedmiR-10b would be involved in the process of gastric cancer metastasis.miR-10b couldnot be detected in normal gastric cells nor in well-differentiatedgastric cancercells.The weak expression of miR-10b was observed in cells with moderatelydifferentiation.And miR-10b was highly expressed in poorly differentiated gastriccancer cell lines. These results indicated that miR-10b expression level was positivelyrelated to metastatic ability of gastric cancer cells. Inhibition of miR-10b activity bymiR-10b inhibitor had no obviously effects on migration of MNK45cells, whileseverely blocked cell invasion ability. And overexpression of miR-10b in GES-1cellslargely enhanced cells invasiveness. These results suggestted miR-10b selectivelyregulated invasion under the context of gastric cancer cells.HOXD10was identified as the target molecule of miR-10b by usingcomputational methods. In GES-1cells,miR-10b overexpression induced the decrease of HOXD10protein, while could notalter mRNA level of HOXD10. The result of luciferase reporter gene analysis showedthat miR-10b overexpression onlyremarkably reduced luciferase activity of reportergene with wild-type, but not mutant HOXD103’UTR. Overexpression HOXD10lacking3’UTR in GES-1cells overexpressing miR-10b completely interfered withmiR-10b-stimulated enhancement of cell invasiveness,indicating its important role asthe target of miR-10bin this process.In GES-1cells, overexpressed miR-10b resultedin decrease of HOXD10protein, while led to upregulation of RhoC. In addition,knockdown of RhoC by siRNA partially abrogated the invasiveness induced bymiR-10b overexpression. miR-10b overexpression led to robust phosphorylation ofAKT inGES-1cells, which was completely blocked by knockdown of RhoC. InGES-1cells overexpressing miR-10b, inhibition of AKT activity by AKT inhibitor IIIpartially interfered with cell invasion. Hence, RhoC seemed to promote gastric cancercell invasion induced by miR-10b through AKTsignaling pathway.HOXD10proteinwere evidently decreased in poorly differentiated gastric cancer cells compared withthe other cell lines with low metastatic capability. In tumor tissues, protein levels ofHOXD10were dramatically downregulated in metastasis-positive tissues. Thus,changes of HOXD10expression in cell lines and species of gastric cancer suggestedthe suppressive role of HOXD10in gastric cancer invasion.Conclusion:high levels of miR-10b are associated with the metastatic degree ingastric cancer patients and gastric cancer cell lines. In addition, miR-10boverexpression promoted invasiveness of gastric cells by targeting HOXD10, whichpartially passed through RhoC-AKT signaling. These results showed novelmechanism underlying invasion of gastric cancer cells and supply novel therapeutictargets in inhibiting metastasis in gastric cancer.