Effect of lentivirus-mediated small interfering RNA on mitogen- and stress-activated protein kinase 1 in spinal cord injury of rats_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHONG Zexiang ¹ , ZHOU Yinan ¹ , FENG Sisi ² , HUANG Yu ¹ ,  CHEN Xuanwei ¹

1. Department of Spine Surgery, the First Affiliated Hospital, Fujian Medical University, Fuzhou Fujian, 350005, P.R.China;
2. Department of Pathology and Pathophysiology, Fujian Medical University, Fuzhou Fujian, 350005, P.R.China;

Corresponding author：

CHEN Xuanwei, Email: fjcxw@126.com

Keywords：

Lentivirus; small interfering RNA; mitogen- and stress-activated protein kinase 1; proliferating cell nuclear antigen; spinal cord injury; rat

DOI：

10.7507/1002-1892.201801093

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ObjectiveTo investigate the expression changes and the repair effect of mitogen and stress- activated protein kinase 1 (MSK1) on spinal cord injury (SCI) in rats.MethodsOne 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 T₁₀ 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.ResultsWestern 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).ConclusionLentivirus-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.

Citation： ZHONG Zexiang, ZHOU Yinan, FENG Sisi, HUANG Yu, CHEN Xuanwei. Effect of lentivirus-mediated small interfering RNA on mitogen- and stress-activated protein kinase 1 in spinal cord injury of rats. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(7): 941-950. doi: 10.7507/1002-1892.201801093 Copy

1.	Colak A, Kaya M, Karaoğlan A, et al. Calpain inhibitor AK 295 inhibits calpain-induced apoptosis and improves neurologic function after traumatic spinal cord injury in rats. Neurocirugia (Astur), 2009, 20(3): 245-254.
2.	Dray C, Rougon G, Debarbieux F. Quantitative analysis by in vivo imaging of the dynamics of vascular and axonal networks in injured mouse spinal cord. Proc Nat Acad Sci U S A, 2009, 106(23): 9459-6494.
3.	Ueno M, Yamashita T. Strategies for regenerating injured axons after spinal cord injury-insights from brain development. Biologics, 2008, 2(2): 253-264.
4.	Deak M, Clifton AD, Lucocq JM, et al. Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB. EMBO J, 1998, 17(15): 4426-4441.
5.	Kaiser M, Wiggin GR, Lightfoot K, et al. MSK regulate TCR-induced CREB phosphorylation but not immediate early gene transcription. Eur J Immunol, 2007, 37(9): 2583-2595.
6.	Vermeulen L, De Wilde G, Van Damme P, et al. Transcriptional activation of the NF-κB p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1). EMBO J, 2003, 22(6): 1313-1324.
7.	Kim HG, Lee KW, Cho YY, et al. Mitogen- and stress-activated kinase 1-mediated histone H3 phosphorylation is crucial for cell transformation. Cancer Res, 2008, 68(7): 2538-2547.
8.	Wiggin GR, Soloaga A, Foster JM, et al. MSK1 and MSK2 are required for the mitogen- and stress-induced phosphorylation of CREB and ATF1 in fibroblasts. Mol Cell Biol, 2002, 22(8): 2871-2881.
9.	Tan BT, Jiang L, Liu L, et al. Local injection of Lenti-Olig2 at lesion site promotes functional recovery of spinal cord injury in rats. CNS Neurosci Ther, 2017, 23(6): 475-487.
10.	Hu J, Lang Y, Zhang T, et al. Lentivirus-mediated PGC-1α overexpression protects against traumatic spinal cord injury in rats. Neuroscience, 2016, 328: 40-49.
11.	Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma, 1995, 12(1): 1-21.
12.	Graeber MB, Li W, Rodriguez ML. Role of microglia in CNS inflammation. Febs Letters, 2011, 585(23): 3798-3805.
13.	Myer DJ, Gurkoff GG, Lee SM, et al. Essential protective roles of reactive astrocytes in traumatic brain injury. Brain, 2006, 129(Pt 10): 2761-2772.
14.	Stirling DP, Cummins K, Mishra M, et al. Toll-like receptor 2-mediated alternative activation of microglia is protective after spinal cord injury. Brain, 2014, 137(Pt 3): 707-723.
15.	Bao Y, Qin L, Kim E, et al. CD36 is involved in astrocyte activation and astroglial scar formation. J Cereb Blood Flow Metab, 2012, 32(8): 1567-1577.
16.	Cheng P, Alberts I, Li X. The role of ERK1/2 in the regulation of proliferation and differentiation of astrocytes in developing brain. Int J Dev Neurosci, 2013, 31(8): 783-789.
17.	Zhu P, Zhan L, Zhu T, et al. The Roles of p38 MAPK/MSK1 Signaling Pathway in the Neuroprotection of Hypoxic Postconditioning Against Transient Global Cerebral Ischemia in Adult Rats. Mol Neurobiol, 2014, 49(3): 1338-1349.
18.	Ananieva O, Darragh J, Johansen C, et al. The kinases MSK1 and MSK2 act as negative regulators of Toll-like receptor signaling. Nat Immunol, 2008, 9(9): 1028-1036.
19.	Gong P, Xu X, Shi J, et al. Phosphorylation of mitogen- and stress-activated protein kinase-1 in astrocytic inflammation: a possible role in inhibiting production of inflammatory cytokines. PLoS One, 2013, 8(12): e81747.
20.	Ananieva O, Darragh J, Johansen C, et al. The kinases MSK1 and MSK2 act as negative regulators of Toll-like receptor signaling. Nat Immunol, 2008, 9(9): 1028-1036.
21.	Hughes JP, Staton PC, Wilkinson MG, et al. Mitogen and stress response kinase-1 (MSK1) mediates excitotoxic induced death of hippocampal neurones. J Neurochem, 2003, 86(1): 25-32.
22.	Choi YS, Karelina K, Alzate-Correa D, et al. Mitogen- and stress-activated kinases regulate progenitor cell proliferation and neuron development in the adult dentate gyrus. J Neurochem, 2012, 123(5): 676-688.
23.	Ning B, Li Z, Zhu N, et al. Traumatic brain injury induces a downregulation of MSK1 in rat brain cortex. J Mol Neurosci, 2013, 49(2): 380-386.
24.	Lau PJ, Kolodner RD. Transfer of the MSH2.MSH6 complex from proliferating cell nuclear antigen to mispaired bases in DNA. J Biol Chem, 2003, 278(1): 14-17.

1. Colak A, Kaya M, Karaoğlan A, et al. Calpain inhibitor AK 295 inhibits calpain-induced apoptosis and improves neurologic function after traumatic spinal cord injury in rats. Neurocirugia (Astur), 2009, 20(3): 245-254.
2. Dray C, Rougon G, Debarbieux F. Quantitative analysis by in vivo imaging of the dynamics of vascular and axonal networks in injured mouse spinal cord. Proc Nat Acad Sci U S A, 2009, 106(23): 9459-6494.
3. Ueno M, Yamashita T. Strategies for regenerating injured axons after spinal cord injury-insights from brain development. Biologics, 2008, 2(2): 253-264.
4. Deak M, Clifton AD, Lucocq JM, et al. Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB. EMBO J, 1998, 17(15): 4426-4441.
5. Kaiser M, Wiggin GR, Lightfoot K, et al. MSK regulate TCR-induced CREB phosphorylation but not immediate early gene transcription. Eur J Immunol, 2007, 37(9): 2583-2595.
6. Vermeulen L, De Wilde G, Van Damme P, et al. Transcriptional activation of the NF-κB p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1). EMBO J, 2003, 22(6): 1313-1324.
7. Kim HG, Lee KW, Cho YY, et al. Mitogen- and stress-activated kinase 1-mediated histone H3 phosphorylation is crucial for cell transformation. Cancer Res, 2008, 68(7): 2538-2547.
8. Wiggin GR, Soloaga A, Foster JM, et al. MSK1 and MSK2 are required for the mitogen- and stress-induced phosphorylation of CREB and ATF1 in fibroblasts. Mol Cell Biol, 2002, 22(8): 2871-2881.
9. Tan BT, Jiang L, Liu L, et al. Local injection of Lenti-Olig2 at lesion site promotes functional recovery of spinal cord injury in rats. CNS Neurosci Ther, 2017, 23(6): 475-487.
10. Hu J, Lang Y, Zhang T, et al. Lentivirus-mediated PGC-1α overexpression protects against traumatic spinal cord injury in rats. Neuroscience, 2016, 328: 40-49.
11. Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma, 1995, 12(1): 1-21.
12. Graeber MB, Li W, Rodriguez ML. Role of microglia in CNS inflammation. Febs Letters, 2011, 585(23): 3798-3805.
13. Myer DJ, Gurkoff GG, Lee SM, et al. Essential protective roles of reactive astrocytes in traumatic brain injury. Brain, 2006, 129(Pt 10): 2761-2772.
14. Stirling DP, Cummins K, Mishra M, et al. Toll-like receptor 2-mediated alternative activation of microglia is protective after spinal cord injury. Brain, 2014, 137(Pt 3): 707-723.
15. Bao Y, Qin L, Kim E, et al. CD36 is involved in astrocyte activation and astroglial scar formation. J Cereb Blood Flow Metab, 2012, 32(8): 1567-1577.
16. Cheng P, Alberts I, Li X. The role of ERK1/2 in the regulation of proliferation and differentiation of astrocytes in developing brain. Int J Dev Neurosci, 2013, 31(8): 783-789.
17. Zhu P, Zhan L, Zhu T, et al. The Roles of p38 MAPK/MSK1 Signaling Pathway in the Neuroprotection of Hypoxic Postconditioning Against Transient Global Cerebral Ischemia in Adult Rats. Mol Neurobiol, 2014, 49(3): 1338-1349.
18. Ananieva O, Darragh J, Johansen C, et al. The kinases MSK1 and MSK2 act as negative regulators of Toll-like receptor signaling. Nat Immunol, 2008, 9(9): 1028-1036.
19. Gong P, Xu X, Shi J, et al. Phosphorylation of mitogen- and stress-activated protein kinase-1 in astrocytic inflammation: a possible role in inhibiting production of inflammatory cytokines. PLoS One, 2013, 8(12): e81747.
20. Ananieva O, Darragh J, Johansen C, et al. The kinases MSK1 and MSK2 act as negative regulators of Toll-like receptor signaling. Nat Immunol, 2008, 9(9): 1028-1036.
21. Hughes JP, Staton PC, Wilkinson MG, et al. Mitogen and stress response kinase-1 (MSK1) mediates excitotoxic induced death of hippocampal neurones. J Neurochem, 2003, 86(1): 25-32.
22. Choi YS, Karelina K, Alzate-Correa D, et al. Mitogen- and stress-activated kinases regulate progenitor cell proliferation and neuron development in the adult dentate gyrus. J Neurochem, 2012, 123(5): 676-688.
23. Ning B, Li Z, Zhu N, et al. Traumatic brain injury induces a downregulation of MSK1 in rat brain cortex. J Mol Neurosci, 2013, 49(2): 380-386.
24. Lau PJ, Kolodner RD. Transfer of the MSH2.MSH6 complex from proliferating cell nuclear antigen to mispaired bases in DNA. J Biol Chem, 2003, 278(1): 14-17.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of lentivirus-mediated small interfering RNA on mitogen- and stress-activated protein kinase 1 in spinal cord injury of rats

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