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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 tumor suppressor microRNA-1 (miR-1) regulates the metastasis of bone marrow-derived mesenchymal stem cells (HMSC-bm) to tumor tissue by inhibiting the expression and secretion of SDF-1. Methods (1) Overexpression or down-regulation of miR-1 in HepG2 cells was carried out by using miR-1 expression plasmid pSuper-miR-1 and miR-1 antisense oligonucleotide respectively. Western blot and enzyme-linked immunosorbent assay The expression and secretion of SDF-1 in protein lysates and culture supernatants of hepatocellular carcinoma cells were detected; (2) The pGL3 recombinant luciferase reporter gene vector was fused with 3’UTR of SDF-1, 1 was co-transfected into HEK293 cells and the double luciferase reporter assay was used to detect the expression of recombinant luciferase. (3) Transfection of pSp-miR-1 or miR-1 antisense oligonucleotide into HepG2 cells transplanted into Transwell’s lower chamber overexpression miR-1 or downregulate miR-1 level, Transwell upper chamber HMSC-bm to the lower Cell HepG2 hepatoma cell migration. Results (1) Overexpression of miR-1 in HepG2 significantly inhibited SDF-1 protein expression and secretion in HepG2 cells, while down-regulation of miR-1 induced SDF-1 expression and secretion in HepG2 cells. (2) miR-1 can inhibit the pGL3 recombinant luciferase reporter gene carrying SDF-1 3’UTR. (3) When miR-1 was overexpressed in HepG2 cells in Transwell lower chamber, the number of HMSC-bm migrating cells in the upper chamber was significantly reduced, whereas downregulation of miR-1 expression in HepG2 cells in the lower chamber significantly increased HMSC -bm to HepG2 hepatoma cells. Conclusion miR-1 can directly inhibit the expression and secretion of SDF-1 in hepatoma cells, and thus reduce the chemotactic effect of HMSC-bm on hepatoma cells.