| Hepatocellular carcinoma(HCC)is one of the most morbidity and mortality malignant tumors in our country,and the recurrence and metastasis of HCC are the key factors influencing the poor prognosis.The molecular mechanisms of HCC development and progression are gradually revealed by advanced science and technology.Up to date,the target therapy and personalized therapy for HCC have attracted more attention.It is reported that miRNAs regulated the development and progression of tumor by silencing the m RNA 3?-UTR region of target genes in the post-transcription level.In our previous results,the content of miR-27 a has higher level in Hep G2 and Huh7 HCC cell lines than that of hepatic normal cell(L02),and the le vel of miR-27 a in HCC tumor tissue is higner than that of tumor adjancant tissues.Exogenous miR-27 a will promote the growth of tumor cells in co-culture system,suggesting that miR-27 a is carcinogenic miRNA.To investigate that miR-27 a may be a potential therapeutic target of HCC metastasis,we firstly confirm the relevance between miR-27 a and metastasis of HCC,and screen out the possible downstream target genes by bioinformatics method.Target Scan,Pic Tar and miRBase database were chosen for clustering analysis with combining the correlation with tumor metastasis in literature.We anchored SFRP1(secreted frizzled related protein)as the downstream target of miR-27 a,and found that miR-27 a negatively regulated SFRP1.SFRP1 gene promoter can silence the expression of SFRP1 by frequent DNA methylation,and then activate Wnt/?-catenin signaling pathway in HCC,in contrast to its non-methylation status in hepatic normal cell.Wnt/?-catenin signaling pathway will be suppressed when restoring the expression of SFRP1.Canonical Wnt/?-catenin signaling pathway play crucial role of regulating metastasis in different cancers through up-regulating ?-catenin expression.Metal matrix protein 7(MMP7)is a downstream target gene of ?-catenin.Therefore,we hypothesized that miR-27 a might inhibit downstream target SFRP1,sequently activate the Wnt/?-catenin signaling pathway to achieve the effect of promoting tumor metastasis.The present study set miR-27 a as an anti-metastasis target in HCC,using oligomeric nucleotide anti-miR-27 a complementarily combined with miR-27 a,and suppress the function of the endogenous miR-27 a of cancer cells or tissues.Giving that anti-miR-27 a is vulnerable to nucleic acid enzyme degradation and suffers from poor targeting to the HCC cells,our experiment took advantage of hyaluronic acid(HA),which could specifically bind to the overexpressed CD44 receptor in HCC cell membrane,and mediate endocytosis into target cancer cells,to improve the targeting to the HCC cells.The anti-miR-27 a is encapsulated in the center of the nanoparticles and HA is exposed on the surface of the composite nanoparticles.Herein,this study focused on the impact of HA-PEI/anti-miR-27 a composite nanoparticles on Hep G2 cell migration and relative mechanisms.Objective:This study aims to investigate the effects of HA-PEI/anti-miR-27 a nanoparticles on the Hep G2 cell migration,find out the direct downstream target of miR-27 a and its metastatic signaling pathways,and discuss the molecular mechanism of HA-PEI/antimiR-27 a nanoparticles on inhibiting the migration of Hep G2 cell.Our work will provide a novel target for the treatment of HCC metastasis,as well as shed light on the genetherapy with of HA-PEI/anti-miR-27 a nanoparticles.Methods:RT-PCR was used to detect the p H-responsive release of HA-PEI/anti-miR-27 a composite nanoparticles under acid condition in vitro;MTT method to determine the proper concentrations of HA-PEI/anti-miR-27 a composite nanoparticles on Hep G2 cell migration;Trans well cabin was used to establish Hep G2 cell migration model.Four groups was set up,including control group,HA-PEI group,HA-PEI/anti-miR-27 a group,HA-PEI/anti-miR-27 a N.C group;Scratch experiment further verified the role of HA-PEI/anti-miR-27 a on inhibiting the migration of Hep G2 cell;Adhesion experiment tested the effects of HA-PEI/anti-miR-27 a on adhesion capacity to extracellular matrix or cells;Western blot method to detect the expression of protein SFRP1,?-catenin and MMP7,which are related to HCC cell migration in the downstream of miR-27 a.Results:HA-PEI/anti-miR-27 a nanoparticles can enter the cell through targeting CD44 receptors on the cell membrane surface,then release the anti-miR-27 a and interact with miR-27 a in cellular acidic condition.HA-PEI/anti-miR-27 a composite nanoparticles has inhibitory effect on Hep G2 cell migration at the concentration of 50 n M;HA-PEI/anti-miR-27 a can inhibit the adhesion between Hep G2 cells,and Hep G2 cells and extracellular matrix;HA-PEI/anti-miR-27 a composite nanoparticles can increase the expression of SFRP1 protein in Hep G2 cells(P<0.05),and reduce the expression of ?-catenin and MMP7 protein in the Wnt/?-catenin signaling pathway(P<0.05).Conclusion:HA-PEI/anti-miR-27 a composite nanoparticles have inhibitory effect on Hep G2 cell migration,mainly through the release of anti-miR-27 a in tumor acid environment and the interaction with endogenous miR-27 a in Hep G2 cells to block miR-27 a function.The mechanism of HA-PEI/anti-miR-27 a composite nanoparticles inhibits Hep G2 cell migration might through increasing the expression of SFRP1 protein and reducing the expression of ?-catenin and MMP-7 protein in Wnt/?-catenin signaling pathway. |
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