miR-1抑制SDF-1表达和分泌调控HMSC-bm向肝癌细胞迁移

miR-1 regulates the expression and secretion of SDF-1 and inhibits human mesenchymal stem cell migration of bone marrow towards hepatoma cells

目的明确肿瘤抑制性micro RNA-1(miR-1)能否通过抑制SDF-1的表达和分泌,调控骨髓间充质干细胞(HMSC-bm)向肿瘤组织的转移。方法 (1)分别利用miR-1表达质粒pSuper-miR-1和miR-1反义寡核苷酸,在肝癌细胞系HepG2中过表达或下调miR-1,Western blot和酶联免疫吸附实验分别检测肝癌细胞蛋白裂解物和培养上清液中SDF-1的表达和分泌水平;(2)构建融合SDF-1 3'UTR的pGL3重组萤光素酶报告基因载体,将其与pSuper-miR-1共转染HEK293细胞,利用双萤光报告基因检测系统检测重组萤光素酶的表达情况。(3)利用pSuper-miR-1或miR-1反义寡核苷酸转染接种于Transwell下层小室的HepG2细胞过表达miR-1或下调miR-1水平,检测Transwell上层小室HMSC-bm向下层小室HepG2肝癌细胞的迁移情况。结果 (1)在肝癌细胞,HepG2过表达miR-1能够显著抑制SDF-1蛋白表达和分泌,而下调miR-1水平则能够诱导SDF-1的表达和分泌。(2)miR-1能够抑制携带SDF-1 3'UTR的pGL3重组萤光素酶报告基因活性。(3)在Transwell下层小室的HepG2细胞中过表达miR-1后,上层小室中HMSC-bm向下层小室迁移细胞数量显著减少,而下调下层小室HepG2细胞中miR-1表达水平,则明显增加HMSC-bm向HepG2肝癌细胞的迁移。结论 miR-1能够直接抑制肝癌细胞SDF-1的表达和分泌,并藉此降低肝癌细胞对HMSC-bm的趋化作用。

Objective To investigate whether miR-1 could regulate the migration of HMSC-bms towards hepatoma cells by targeting SDF-1.Methods （1） HepG2 cells were transfected with miR-1 expressing plasmid pSuper-miR-1 or antisense oligonucleotide of miR-1 to over-express or down-regulate miR-1 levels in HepG2 cells.Western blot and ELISA （Enzyme Linked Immunosorbent Assay） were performed to determine the expression and secretion of SDF-1 protein of HepG2 cells.（2） HEK293 cells were co-transfected with miR-1 expressing plasmid pSuper-miR-1 or control empty vector pSuper and recombined luciferase reporter plasmid pGL3-SDF-1-3＇UTR which contained the SDF-1-3＇UTR.Dual luciferase reporter detection system was used to determine the luciferase activities of HEK293 cells after the transfection.（3） HepG2 cells seeded in lower chambers of transwell plate were transfected with pSupre-miR-1 or antisense oligonucleotide of miR-1 to over-express or down-regulate miR-1 levels in HepG2 cells.Then the upper chambers seeded with HMSC-bms were put upon the lower chambers.12 hours later,HMSC-bms transferred through the mini-pores of upper chambers were stained and counted.Results （1） Ectopic expression of miR-1 inhibited the expression and secretion of SDF-1 protein in HepG2 cells,down-regulation of miR-1 by antisense oligonucleotide significantly increased the expression and secretion of SDF-1.（2）Ectopic expression of miR-1 decreased the luciferase activities of recombined luciferase reporter plasmid pGL3-SDF-1-3＇ UTR.（3） Ectopic expression of miR-1 in HepG2 cells seeded in lower chambers reduced the migration of HMSC-bms seeded in upper chambers towards the HepG2 cells.Oppositely,down-regulation of miR-1 in HepG2 promoted the migration of HMSC-bms towards the HepG2 cells.Conclusions miR-1 can inhibit the cell migration of HMSC-bm towards the hepatoma cells by targeting SDF-1.