RESUMEN
Objective: To investigate the expression changes and the repair effect of mitogen and stress- activated protein kinase 1 (MSK1) on spinal cord injury (SCI) in rats. Methods: One hundred and twenty male Sprague Dawley (SD) rats (weighing 220-250 g) were used for the study, 70 of them were randomly divided into sham-operation group and SCI group ( n=35), the rats in SCI group were given SCI according to Allen's method, and the sham-operation group only opened the lamina without injuring the spinal cord; spinal cord tissue was collected at 8 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, and 7 days after invasive treatment, each group of 5 rats was used to detect the expression of MSK1 and proliferating cell nuclear antigen (PCNA) by Western blot assay. Another 20 SD rats were grouped by the same method as above ( n=10). In these rats, a negative control lentiviral LV3NC dilution was injected at a depth of approximately 0.8 mm at the spinal cord T 10 level. The results of transfection at 1, 3, 5, 7, and 14 days after injection were observed under an inverted fluorescence microscope to determine the optimal transfection time of the virus. The other 30 SD rats were randomly divided into group A with only SCI, group B with a negative control lentiviral LV3NC injected after SCI, and group C with MSK1 small interfering RNA (siRNA) lentivirus injected after SCI, with 10 rats each group. The Basso, Beatlie, Bresnahan (BBB) score of hind limbs was measured at 1, 3, 5, 7, and 14 days after treatment; spinal cord tissue collected at the optimal time point for lentivirus transfection was detected the expression changes of MSK1 and PCNA by Western blot and the localization by immunofluorescence staining of MSK1 and PCNA proteins. Results: Western blot assay showed that there was no significant changes in the expression of MSK1 and PCNA at each time points in the sham-operation group. In the SCI group, the expression of MSK1 protein was gradually decreased from 8 hours after injury to the lowest level at 3 days after injury, and then gradually increased; the expression change of PCNA protein was opposite to MSK1. The expression of MSK1 in SCI group was significantly lower than that in the sham-operation group at 1, 2, 3, and 5 days after injury ( P<0.05), and the expression of PCNA protein of SCI group was significantly higher than that of the sham-operation group at 8 hours and 1, 2, 3, 5, and 7 days after injury ( P<0.05). The fluorescence expression of both the SCI group and the sham-operation group has be found and peaked at 7 days. There was a positive correlation between fluorescence intensity and time in 7 days after transfection. With the prolongation of postoperative time, the BBB scores of groups A, B, and C showed a gradually increasing trend. The BBB score of group C was significantly lower than those of groups A and B at 5, 7, and 14 days after treatment ( P<0.05). After transfection for 7 days, Western blot results showed that the relative expression of MSK1 protein in group C was significantly lower than that in groups A and B ( P<0.05); and the relative expression of PCNA protein was significantly higher than that in groups A and B ( P<0.05). Immunofluorescence staining showed that MSK1 was expressed in the nuclei of the spinal cord and colocalized with green fluorescent protein, neuronal nuclei, and glial fibrillary acidic protein (GFAP). The relative expression area of MSK1 positive cells in group C was significantly higher than that in group B ( P<0.05), and the relative expression areas of PCNA and GFAP positive cells were significantly lower than those in group B ( P<0.05). Conclusion: Lentivirus-mediated MSK1 siRNA can effectively silence the expression of MSK1 in rat spinal cord tissue. MSK1 may play a critical role in the repair of SCI in rats by regulating the proliferation of glial cells.
RESUMEN
Objective Abnormal activation of mitogen-and stress-activated kinase (MSK1) plays an important role in the development of various cancers.This study was to explore the effect of small interfering RNA (siRNA)-mediated MSK1-silencing on the proliferation of human nasopharyngeal carcinoma (NPC) cells and its underlying mechanism.Methods The siRNA vector targeting MSK1 was constructed and transfected into CNE2 cells, and the NPC cell line stably expressing MSK1 was established.Then the cells were divided into a blank control (without transfection of the plasmid), a negative control (with stable transfection of the negative control plasmid), and an experimental group (with stable transfection of the positive recombinant plasmid).The expressions of MSK1 mRNA and protein were detected by real-time quantitative PCR and Western blot, respectively, the proliferation of the cells determined by CCK-8 and colony formation assays, the cell cycles analyzed by flow cytometry, the level of histone H3 phosphorylation at Ser10 examined by Western blot, and The transcriptional activity and expression of the c-jun protein measured by dual-luciferase reporter gene assay and Western blot.Results Compared with the blank control, the inhibition rates of cell proliferation at 48, 72 and 96 hours were significantly reduced in the experimental group (P<0.05), and so were the colony formation ability of the cells (P<0.01) and the expression and transcriptional activity of the c-jun protein (P<0.05).In comparison with the negative control, the experimental group showed significant decreases in the rate of cell growth after 24 hours, the inhibition rates of cell proliferation at 48, 72 and 96 hours (P<0.05), the number of formed colonies ([221.00±20.08] vs [99.67±15.57] / 300 cells, P<0.01), the proportion of S-phase cells (P<0.01), and the expression of the c-jun protein in the CNE2 cells ([100.00±0.00] vs [48.77±10.71] %, P<0.05), but a remarkable increase in the percentage of G0/G1-phase cells (P<0.01).Furthermore, histone H3 phosphorylation at Ser10 was markedly reduced (P<0.01) but no significant change was observed in the expression of the total c-jun protein in the experimental group.Conclusion Knockdown of MSK1 using siRNA can significantly inhibit the growth and proliferation of CNE2 cells, which may be closely related to the decreased phosphorylation of histone H3 and subsequently down-regulated transcriptional activity of c-jun.