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 cels 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 cels 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 cels 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 cels 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 cels to measure the luciferase.
RESULTS AND CONCLUSION: Compared with the negative control group, the proliferative and cloning capacities of cels in the transfection upregulation group were higher; the number of cels was significantly decreased at G1 stage and increased at S stage, and the apoptotic cels 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 cels 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 cels and reduce cel apoptosis, but downrelation of let-7f exhibits an inhibitory effect. Overexpression of IL-6 can suppress the proliferation of bone marrow mesenchymal stem cels, which is considered to be a target gene of let-7f, and let-7f may suppress the expression of IL-6 to promote the cel proliferation.