MiR-155 Promotes Gastric Cancer Growth And Invasion By Negatively Regulating Transforming Growth Factor Beta Receptor 2

Background: Gastric cancer(GC)is one of the most common malignancies worldwide,because of occult and poor prognosis,it has high mortality rates.China is a big country of GC,and the new cases of GC account for about 40% of the world.With the intensive studies of the molecular signal transduction pathway of the GC,new therapeutic targets for GC receive extensive attention.Transforming growth factor-β receptor 2(TGFβR2)causes a variety of biological reactions including cell proliferation,differentiation,migration,apoptosis and immune response by transmitting the extracellular signal causes a variety of biological reactions by transferring the extracellular signal TGF-β to the cell.The inactivation of TGFβR2 can cause the conduction disturbance of the TGF-β signaling pathway,leading to the growth,invasion and metastasis of cancer cells.MicroRNAs(miRNAs)are highly conservative,non coding single strand small molecule RNA.They repress translation by binding with the complementary sequences in 3’-untranslated regions(3’-UTR)of target mRNAs,which regulate gene expression efficiently.miRNAs are closely involved in the proliferation,differentiation,apoptosis and other pathological processes of cancer cells.Our previous studies have discovered that the copy number of miR-155 in serum of gastric cancer patients was significantly higher than control group.And we also find that the TGFbR2 protein levels,but not mRNA levels,are downregulated in GC tissues,and the levels of miR-155 are significantly increased in GC tissues.Objective:This study aimed to explore the underlying mechanism of abnormal expression of TGFβR2 in GC,select upstream miRNA which can directly target TGFβR2.And further explore the effects of miRNA-TGFβR2 pathway in the biological behavior including proliferation,invasion and apoptosis of GC,which defines new molecular targets and provides new basis and ideas for the target therapy of GC.Methods:Clinical tissue specimen experiments: Analysis of the expression of TGFβR2 and the level of miR-155 in GC tissue.We collected GC tissues and the paired non-tumor tissues from patients who underwent surgical resection at the Tianjin Medical University Cancer Institute and Hospital.Then we detected the TGFβR2 protein and mRNA expression level respectively by using Western blot and qRT-PCR.Then,we tested the expression levels of miR-155 in the above-mentioned tissues.Target prediction: The potential upstream miRNA of TGFβR2 was selected by using bioinformatics tools including miRanda(http://www.microrna.org/),TargetScan(http: //www.targetscan.org/),and PicTar(http://pictar.mdc-berlin.de/).In vitro experiments: The effects of miR-155-TGFβR2 signaling pathway on the biological behavior of GC cell line SGC7901 and MGC803 were analyzed.We further verified whether miR-155 can directly regulate the expression of TGFβR2 by luciferase report experiments.MiR-155 mimics and miR-155 inhibitors were transfected into SGC7901 and MGC803 cells,respectively.After 48 hours,protein and RNA were extracted,and the expression levels of miR-155,TGFβR2 mRNA and TGFβR2 protein were detected.Then the effect of miR-155 on the regulation of TGFβR2 was clarified.In order to up-regulate and down-regulate the expression level of miR-155 in GC cell line,the miR-155 mimics and miR-155 inhibitors were transfected into SGC7901.Then the effects of miR-155 on biological behavior of transfected cells were verified by cell function test(cell scratch test,Transwell and cell proliferation assay).The over-expression or knockdown of TGFβR2 was achieved by transfected GC cells with over-expressed TGFβR2 plasmid or TGFβR2 siRNA.Then the functional roles of TGFβR2 in biological behavior of GC cells were tested.Furthermore,we simultaneously over-expressed miR-155 and TGFβR2,the effects of miR-155-TGFβR2 signaling pathway on the proliferation,invasion and metastasis of gastric cancer cells were verified.In vivo experiments: To verify the function of miR-155 and TGFβR2 in the growth of human GC cell xenografts in nude mice,the SGC7901 cells were transfected with miR-155 over-expressing lentivirus or TGFβR2 over-expressing plasmid respectively.Then above cells and SGC7901 cells as control group were harvested and injected subcutaneously in the armpit of mice.After 4 weeks,we sacrificed the mice and separated tumors.Then we observed and recorded the tumor sizes and weights clearly.HE staining experiments and Immunohistochemistry revealed the expression of TGFβR2.Results:Clinical tissue specimen experiments: The TGFβR2 protein levels,but not mRNA levels,were down-regulated in GC tissues,and the levels of miR-155 were significantly increased in GC tissues.Target prediction: We predicted that miR-155 can directly target the 3’-UTR of TGFβR2 mRNA by employing bioinformatics tools.In vitro experiments: MiR-155 promotes the proliferation,invasion and inhibition of apoptosis of GC cells by negative regulation of TGFβR2.The luciferase assay showed that miR-155 can directly bind to the 3’UTR of TGFβR2 mRNA and regulate the expression of TGFβR2.After transfection of miR-155 mimics and miR-155 inhibitor in GC cell lines,the expression level of miR-155 in the cells was up and down respectively.Meanwhile,the level of TGFβR2 protein decreased and increased respectively.However,there was no significant alter in the level of TGFβR2 mRNA by qRT-PCR.Cell function experiments showed that transfection of miR-155 mimics and miR-155 inhibitors promoted and inhibited the proliferation and migration of GC cells respectively.Over-expression of TGFβR2 partly repressed GC cell growth.Furthermore,TGFβR2 siRNA leads to a sharp decrease in protein expression,thus significantly promoting cell proliferation and suppressing GC cell apoptosis.In vivo experiments: Over expression of miR-155 group increased significantly compared with control group,the expression level of miR-155 increased,and the level of TGFβR2 protein expression decreased.Meanwhile,the level of TGFβR2 mRNA did not alter significantly.TGFβR2 over-expression group had opposite results.Conclusion:MiR-155 promotes cell growth and invasion by negatively regulating TGFβ R2.