Formin Like-2 Regulate The Expression Of The Matrix Metal Enzyme-2 Through Rho GTPases Signaling Pathways In Hepatoma Carcinoma Cell

Primary hepatocellular carcinoma(HCC)is one of the most common digestive system malignant tumor,high incidence,recurrence,metastasis rate and mortality rate is high,the prognosis is not ideal.Therefore,liver cancer early detection,early diagnosis and prevention of recurrence,have very important significance for clinical treatment and prognosis.Formin homology protein2(FMNL-2)and the matrix metal enzyme2(MMP-2)are involved in the invasion and metastasis of primary hepatocellular carcinoma.FMNL-2 can regulate the structure change of actin cytoskeleton and pseudopodia movement.MMP-2 is closely related to tumor cells to break through the basement membrane barrier by enzymolysis extracellular matrix and basement membrane protein collagen.Studies have shown that FMNL-2 lead to tumor cell migration was achieved by Rho related signaling pathways.FMNL-2 participate in platy pseudopod extend of Rho related proteins Cdc42 in cell migration process.Other results suggest that Rock2 regulation MMP-2 in the primary liver cancer cells on the invasive migration.Earlier experiments have proved that FMNL-2 regulation MMP-2 expression,in this study intends to down-regulate FMNL-2 by proposed by gene silencing technology,as well as Y27632 blocking Rho GTPases,Detection Rho A,Racl,Cdc42,MMP-2 protein and m RNA change,thus determine whether FMNL-regulating the MMP-2 by Rho GTPases signal pathway.This study provide theoretical basis for the research and development of the Rho signal pathway as the target of anti-tumor drugs.Objective:Confirm FMNL-2 and the relationship between the MMP-2.Clarify whether FMNL-2 regulate MMP-2 though Rho GTPases signal pathway.Methods:1.Application of gene silencing technology build FMNL-2 lower expression of liver cancer cell lines.MHCC 97 H liver cancer cell lines as normal control group.Detection of two kinds of cell lines FMNL-2 and MMP-2 gene and protein expression,to determine the control relationship.2.Detect Rho A,Rac1,Cdc42 three kinds of gene expression of the situation in low expression group and 97 h two kinds of liver cancer cell lines,to verify that the Rho GTPases is downstream regulatory by FMNL-2.In addition,select specific downstream genes,through the differences gene expression of Rho A,Rac1 and Cdc42.3.Inhibit Rho GTPases by Y27632,MHCC 97 H as control group,detection the expression of FMNL-2 and MMP-2,to verify the relationship between Rho GTPases and MMP-2.Thereby explain FMNL regulate the expression of MMP-2 by Rho GTPases pathway.Results:1.si RNA down regulated FMNL-2 expression in MHCC 97 H cell line,MMP-2 expression decreased accordingly.Western blot and rt-PCR results show that after down-regulated FMNL-2 the expression of MMP-2 descend,and the difference has statistical significance(P < 0.05),suggesting FMNL-2 can regulate the expression of MMP-2.In addition,after FMNL-2 down-regulated,the expression of Rho A invariant,Cdc42 slightly decreased,but the difference has not statistically significance,and Rac1 m RNA expression decreased significantly,the difference has statistically significant(P < 0.05).2.Y27632 selective inhibition of Rac1 activate in MHCC 97 H.With 30μmol/L Y27632 treatment of MHCC 97 H cells,activation of Rac1 protein expression significant reduced relative to normal control group,the difference has statistical significance(P < 0.05).3.Rac1 expression decreased via Y27632,MMP-2 expression decreased accordingly.Western blot results show that Rac1 activity declined,the protein expression of MMP-2 reduced significantly relative to normal control group,the difference has statistically significant(P < 0.05).Conclusion:1.Rac1 is downstream target genes of FMNL-2.2.FMNL-2 regulate the expression of MMP-2 in MHCC 97 H cell line via Rac1.