let-7f对骨髓间充质干细胞增殖的影响

背景：microRNA let-7f和炎性因子白细胞介素6对骨髓间充质干细胞的具体作用及两者之间是否存在联系尚不可知。 目的：分析let-7f及白细胞介素6表达水平对骨髓间充质干细胞增殖的影响及两者的关系。 方法：①构建let-7f过表达及抑制慢病毒载体，分别转染SD大鼠骨髓间充质干细胞，实验分为4个组：转染上调组(转染LV-rno-let-7f-up)、转染下调组(转染LV-rno-let-7f-down)、阴性转染组(转染空病毒)、未转染组(不进行病毒转染)。 qRT-PCR检测各组细胞let-7f表达水平。MTT法、流式细胞术和ELISA等方法检测不同let-7f表达水平下细胞增殖情况及白细胞介素6表达水平，qRT-PCR及Western blot检测各组Cyclin D1 mRNA和蛋白表达水平。②预测let-7f潜在的靶基因，构建野生型/突变型白细胞介素6 3’UTR报告基因载体，分别与let-7f/let-7f inhibitor共转染293T细胞系，测定荧光素酶活性。 结果与结论：let-7f转染上调组较未转染组及阴性转染组细胞增殖能力及克隆能力明显增强，G1期细胞数明显减少，S期明显增多，凋亡减少(P < 0.05)；转染下调组结果与其相反。let-7f转染上调组细胞培养液中的白细胞介素6表达水平低于未转染组及阴性转染组(P < 0.05)；转染下调组细胞培养液中的白细胞介素6则明显升高(P < 0.05)；Western blot、定量PCR显示let-7f转染上调组Cyclin D1蛋白及mRNA表达上调(P < 0.05)；转染下调组表达均下调(P < 0.05)；未转染组与阴性转染组所有结果无明显差异(P > 0.05)。野生型白细胞介素6 3’UTR报告基因载体与let-7f共转染细胞的荧光素酶活性显著减低(P < 0.05)。结果表明上调let-7f表达水平可显著促进骨髓间充质干细胞增殖活性及克隆形成能力，减少凋亡，而下调let-7f表达水平则出现抑制作用。白细胞介素6过表达可抑制骨髓间充质干细胞增殖，考虑白细胞介素6是let-7f的靶基因，let-7f可能通过抑制白细胞介素6表达而促进细胞增殖。 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 

let-7f effects on the proliferation of bone marrow mesenchymal stem cells

BACKGROUND: There is no clear understanding about the effect of let-7f and interleukin-6 (IL-6) on the proliferation of bone marrow mesenchymal stem cells and their relationship. OBJECTIVE: To explore the effects of expression levels of let-7f and IL-6 on the proliferation of bone  marrow mesenchymal stem cells and their relationship. METHODS: (1) LV-rno-let-7f-up and LV-rno-let-7f-down were constructed and transfected into bone marrow mesenchymal stem cells of Sprague-Dawley rats, respectively. Then, there were four groups in the study: transfection upregulation group transfected with LV-rno-let-7f-up), transfection inhibition group (transfected with LV-rno-let-7f-down), negative control group (transfected with FU-RNAi-NC-LV), and untransfected group. The expression level of let-7f in each group was detected by qRT-PCR. The proliferation ability of cells and expression levels of IL-6 when let-7f expression was at different levels were detected by MTT, flow cytometry and ELISA. The expression of Cyclin D1 at mRNA and protein levels was detected by qRT-PCR and western blot, respectively. (2) To predict the potential target gene of let-7f, the wild-type/mutant IL-6 3’UTR reporter gene vectors were constructed, and cotransfected with let-7f/let-7f inhibitor respectively into the 293T cells to measure the luciferase. RESULTS AND CONCLUSION: Compared with the negative control group, the proliferative and cloning capacities of cells in the transfection upregulation group were higher; the number of cells was significantly decreased at G1 stage and increased at S stage, and the apoptotic cells were reduced in number (P < 0.05). However, the transfection inhibition group had opposite results. The expression level of IL-6 in the transfection upregulation group was lower than that in the untransfected group and negative control group (P < 0.05); while in the transfection inhibition group, the expression level of IL-6 was significantly increased (P < 0.05). The expression of Cyclin D1 at mRNA and protein levels was up-regulated in transfection upregulation group (P < 0.05) and down-regulated in the transfection inhibition group (P < 0.05), but there was no significant difference between the negative control group and untransfected group (P > 0.05). Luciferase activity of cells transfected with wide-type IL-6 3’UTR and let-7f was significantly reduced (P < 0.05). These findings indicate that up-regulation of let-7f can promote the proliferative and cloning capacities of bone marrow mesenchymal stem cells and reduce cell apoptosis, but downrelation of let-7f exhibits an inhibitory effect. Overexpression of IL-6 can suppress the proliferation of bone marrow mesenchymal stem cells, which is considered to be a target gene of let-7f, and let-7f may suppress the expression of IL-6 to promote the cell proliferation.