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PVT1促进在胶质瘤C6细胞微环境中的BMSCs增殖和迁移

PVT1 Promoted the Proliferation and Migration Ability of BMSCs in Glioma C6 Microenvironment

    摘要
  • 摘要:
      目的  探讨与胶质瘤C6细胞间接共培养后骨髓间充质干细胞（BMSCs）增殖和迁移能力的变化以及长链非编码RNA（lncRNA）浆细胞瘤变异异位基因1（plasmacytoma variant translocation 1 gene，PVT1）在这种变化中的作用。
      方法  分离、培养、鉴定BMSCs后，采用Transwell小室将生长状态良好的BMSCs与胶质瘤C6细胞间接共培养（共培养组），单独培养的正常BMSCs为对照组。CCK8及软琼脂克隆形成实验检测2组细胞的增殖能力，流式细胞术检测细胞周期，划痕实验及Transwell法检测细胞的迁移能力，实时定量PCR（qRT-PCR）检测PVT1基因的表达。在共培养组BMSCs中转染si-PVT1（si-PVT1组）和si-NC（si-NC组），转染后重复上述检测，并增加qRT-PCR检测周期蛋白基因CyclinD1及迁移相关基因基质金属蛋白酶2、9（ MMP2、 MMP9）的表达。
      结果  所用BMSCs具有成骨、成脂分化能力。与对照组相比，共培养组BMSCs体积变小，排列紊乱，细胞间接触抑制消失，增殖及迁移能力增强，S期及G2期细胞比例增加，PVT1表达增加（P<0.05）。与si-NC组比较，si-PVT1组抑制共培养BMSCs的增殖及迁移能力，G1期细胞比例增加，S期细胞比例减少，PVT1、CyclinD1及 MMP2、MMP9 mRNA表达也降低（P<0.05）。
      结论  胶质瘤C6细胞微环境中BMSCs增殖及迁移能力增强可能与PVT1高表达有关。

     

    Abstract:
      Objective  To investigate the changes in the proliferation and migration ability of bone marrow mesenchymal stem cells (BMSCs) after indirect co-culturing with glioma C6 cells, and to examine the role of plasmacytoma variant translocation 1 gene (PVT1), a long non-coding RNA (lncRNA), in these changes.
      Methods  After separation, cultivation and identification of BMSCs, BMSCs of good growth condition were picked out and indirectly co-cultured with glioma C6 cells in Transwell chambers. These cells are henceforth referred to as the co-culture group. Normal BMSCs cultured separately were the control group. CCK-8 and soft agar colony formation assay were used to examine the proliferation ability of the two groups of cells. Flow cytometry was used to examine the cell cycle. Wound healing assay and Transwell assay were used to explore the migration ability of the cells. Quantitative real-time PCR (qRT-PCR) was used to examine the genetic expression level of PVT1 in the two groups. The above-mentioned tests were repeated after the co-cultured BMSCs were transfected with si-PVT1 (si-PVT1 group) and si-NC (si-NC group). In addition, qRT-PCR was done to evaluate the expression of CyclinD1, a cell cycle protein gene, and matrix metalloproteinases 2 and 9 (MMP2 and MMP9), the migration-related genes in the si-PVT1 and si-NC transfected co-cultured BMSCs.
      Results  The BMSCs used in the present study possess the capability of osteogeneic and adipogenic differentiation. Compared with the control group, the co-cultured BMSCs had smaller size, disorderly arrangement and the lack of intercellular contact inhibition. The proliferation and migration ability was significantly enhanced, the proportions of S and G2 phase cells greatly increased and the expression level of PVT1 was significantly up-regulated (P<0.05) in the co-cultured group in comparison with those of the control group. When compared with the si-NC group, the si-PVT1 group showed inhibited proliferation and migration ability of the co-cultured BMSCs; the percentage of G1 phase cells increased, while that of S phase decreased; the expression of PVT1, CyclinD1, MMP2 and MMP9 mRNA also decreased (P<0.05) in the si-PVT1 group.
      Conclusion  The enhanced proliferation and migration ability of BMSCs in the glioma C6 microenvironment may be associated with the up-regulated expression of PVT1 .

     

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