MiR-103 alleviates autophagy and apoptosis by regulating SOX2 in LPS-injured PC12 cells and SCI rats

Document Type: Original Article

Authors

1 Department of Spine Surgery, The Fifth Affiliated Hospital of Sun Yat-Sen University, Zhuhai, Guangdong 519000, China

2 Department of Anaesthesiology, The Fifth Affiliated Hospital of Sun Yat-Sen University, Zhuhai, Guangdong 519000, China

3 Department of Gastroenterological Surgery, The Fifth Affiliated Hospital of Sun Yat-Sen University, Zhuhai, Guangdong 519000, China

Abstract

Objective(s): Recent studies revealed that microRNAs (miRNAs) may play crucial roles in the responses and pathologic processes of spinal cord injury (SCI). This study aimed to investigate the effect and the molecular basis of miR-103 on LPS-induced injuries in PC12 cells in vitro and SCI rats in vivo.
Materials and Methods: PC12 cells were exposed to LPS to induce cell injuries to mimic the in vitro model of SCI. The expression of miR-103 and SOX2 in PC12 cells were altered by transient transfections. Cell viability and apoptotic cell rate were measured by CCK-8 assay and flow cytometry assay. Furthermore, Western blot analysis was performed to detect the expression levels of apoptosis- and autophagy- related proteins, MAPK/ERK pathway- and JAK/STAT pathway-related proteins. In addition, we also assessed the effect of miR-103 agomir on SCI rats.
Results: LPS exposure induced cell injuries in PC12 cells. miR-103 overexpression significantly increased cell viability, reduced cell apoptosis and autophagy, and opposite results were observed in miR-103 inhibition. miR-103 attenuated LPS-induced injuries by indirect upregulation of SOX2. SOX2 overexpression protected PC12 cells against LPS-induced injuries, while SOX2 inhibition expedited LPS-induced cell injuries. Furthermore, miR-103 overexpression inhibited MAPK/ERK pathway and JAK/STAT pathway through upregulation of SOX2. We also found that miR-103 agomir inhibited cell apoptosis and autophagy in SCI rats.
Conclusion: This study demonstrates that miR-103 may represent a protective effect against cell apoptosis and autophagy in LPS-injured PC12 cells and SCI rats by upregulation of SOX2 expression.

Keywords

Main Subjects


1. Lee BB, Cripps RA, Fitzharris M, Wing PC. The global map for traumatic spinal cord injury epidemiology: update 2011, global incidence rate. Spinal Cord 2014; 52:110-116.

2. Yunta M, Nieto-Díaz M, Esteban FJ, Caballero-López M, Navarro-Ruíz R, Reigada D, et al. MicroRNA Dysregulation in the Spinal Cord following Traumatic Injury. Plos One 2012; 7:e34534.

3. Nieto-Diaz M, Esteban FJ, Reigada D, Muñoz-Galdeano T, Yunta M, Caballero-López M, et al. MicroRNA dysregulation in spinal cord injury: causes, consequences and therapeutics. Front Cell Neurosci 2014; 8:53.

4. Ning B, Gao L, Liu RH, Liu Y, Zhang NS, Chen ZY, et al. microRNAs in Spinal Cord Injury: Potential Roles and Therapeutic Implications. Int J Biol Sci 2014; 10:997-1006.

5. Bareyre FM, Schwab ME. Inflammation, degeneration and regeneration in the injured spinal cord: insights from DNA microarrays. Trends Neurosci 2003; 26:555.

6. Nesic O, Svrakic NM, Xu GY, Mcadoo D, Westlund KN, Hulsebosch CE, et al. DNA microarray analysis of the contused spinal cord: Effect of NMDA receptor inhibition. J Neurosci Res 2002; 68:406.

7. Krichevsky AM. MicroRNA profiling: from dark matter to white matter, or identifying new players in neurobiology. Scientificworldjournal 2007; 7:155.

8. Li Z, Yu X, Shen J, Law PTY, Chan MTV, Wu WKK. MicroRNA expression and its implications for diagnosis and therapy of gallbladder cancer. Oncotarget 2015; 6:13914-13921.

9. Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 2008; 19:92-105.

10. Hu JZ, Huang JH, Zeng L, Wang G, Cao M, Lu HB. Anti-apoptotic effect of microRNA-21 after contusion spinal cord injury in rats. J Neurotrauma 2013; 30:1349.

11. Mourelatos Z, Dostie J, Paushkin S, Sharma A, Charroux B, Abel L, et al. miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev 2002; 16:720.

12. Li M, Liu Z, Zhang Z, Liu G, Sun S, Sun C. miR-103 promotes 3T3-L1 cell adipogenesis through AKT/mTOR signal pathway with its target being MEF2D. Biol Chem 2015; 396:235-244.

13. Martello G, Rosato A, Ferrari F, Manfrin A, Cordenonsi M, Dupont S, et al. A MicroRNA targeting dicer for metastasis control. Cell 2010; 141:1195-1207.

14. Trajkovski M, Hausser J, Soutschek J, Bhat B, Akin A, Zavolan M, et al. MicroRNAs 103 and 107 regulate insulin sensitivity. Nature 2011; 474:649-653.

15. Parra P, Serra F, Palou A. Expression of adipose microRNAs is sensitive to dietary conjugated linoleic acid treatment in mice. Plos One 2010; 5:e13005.

16. Li G, Li Y, Li X, Ning X, Li M, Yang G. MicroRNA identity and abundance in developing swine adipose tissue as determined by Solexa sequencing. J Cell Biochem 2011; 112:1318–1328.

17. Xie HM, Bing L, Lodish HF. MicroRNAs induced during adipogenesis that accelerate fat cell development are downregulated in obesity. Diabetes 2009; 58:1050-1057.

18. Moncini S, Salvi A, Zuccotti P, Viero G, Quattrone A, Barlati S, et al. The role of miR-103 and miR-107 in regulation of CDK5R1 expression and in cellular migration. Plos One 2011; 6:e20038.

19. Zhang H, Wu F, Kong X, Yang J, Chen H, Deng L, et al. Nerve growth factor improves functional recovery by inhibiting endoplasmic reticulum stress-induced neuronal apoptosis in rats with spinal cord injury. J Transl Med 2014; 12:130.

20. Guo X, Chen Y, Liu Q, Wu J, Wang L, Tang X, et al. Ac-cel, a novel antioxidant, protects against hydrogen peroxide-induced injury in PC12 cells via attenuation of mitochondrial dysfunction. J Mol Neurosci 2013; 50:453-461.

21. Li R, Yin F, Guo YY, Zhao KC, Ruan Q, Qi YM. Knockdown of ANRIL aggravates H2O2-induced injury in PC-12 cells by targeting microRNA-125a. Biomed Pharmacother 2017; 92:952.

22. Zhou Y, Cui Z, Xia X, Liu C, Zhu X, Cao J, et al. Matrix metalloproteinase-1 (MMP-1) expression in rat spinal cord injury model. Cell Mol Neurobiol 2014; 34:1151-1163.

23. Liebscher T, Schnell L, Schnell D, Scholl J, Schneider R, Gullo M, et al. Nogo‐A antibody improves regeneration and locomotion of spinal cord–injured rats. Ann Neurol 2005; 58:706.

24. Hutchison ER, Okun E, Mattson MP. The Therapeutic Potential of microRNAs in Nervous System Damage, Degeneration, and Repair. Neuromolecular Med 2009; 11:153.

25. Nakanishi K, Nakasa T, Tanaka N, Ishikawa M, Yamada K, Yamasaki K, et al. Responses of microRNAs 124a and 223 following spinal cord injury in mice. Spinal Cord 2010; 48:192-196.

26. Liu NK, Wang XF, Lu QB, Xu XM. Altered microRNA expression following traumatic spinal cord injury. Exp Neurol 2009; 219:424-429.

27. Bhalala OG, Srikanth M, Kessler JA. The emerging roles of microRNAs in CNS injuries. Nat Rev Neurol 2013; 9:328-339.

28. Hu JZ, Huang JH, Zeng L, Wang G, Cao M, Lu HB. Anti-apoptotic effect of microRNA-21 after contusion spinal cord injury in rats. J Neurotrauma 2013; 30:1349-1360.

29. Jee MK, Jung JS, Im YB, Jung SJ, Kang SK. Silencing of miR20a is crucial for Ngn1-mediated neuroprotection in injured spinal cord. Hum Gene Ther 2012; 23:508-520.

30. Hong Z, Feng Z, Sai Z, Tao S. PER3, a novel target of miR-103, plays a suppressive role in colorectal cancer in vitro. BMB Rep 2014; 47:500-505.

31. Chen HY, Lin YM, Chung HC, Lang YD, Lin CJ, Huang J, et al. miR-103/107 promote metastasis of colorectal cancer by targeting the metastasis suppressors DAPK and KLF4. Cancer Res 2012; 72:3631.

32. Park JK, Peng H, Katsnelson J, Yang W, Kaplan N, Dong Y, et al. MicroRNAs-103/107 coordinately regulate macropinocytosis and autophagy. J Cell Biol 2016; 215:667-685.

33. Peng H, Park J, Katsnelson J, Yang W, He C, Lavker RM. miRs-103/107 maintain autophagy: a process critical to stem cell maintenance. Invest Ophthalmol Vis Sci 2015; 56:2069-2069.

34. Gubbay J, Collignon J, Koopman P, Capel B, Economou A, Münsterberg A, et al. A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature 1990; 346:245-250.

35. Pevny LH, Nicolis SK. Sox2 roles in neural stem cells. Int J Biochem Cell Biol 2010; 42:421-424.

36. Episkopou V. SOX2 functions in adult neural stem cells. Trends Neurosci 2005; 28:219-221.

37. Miyagi S, Masui S, Niwa H, Saito T, Shimazaki T, Okano H, et al. Consequence of the loss of Sox2 in the developing brain of the mouse. FEBS Lett 2008; 582:2811-2815.

38. Jiang J, Li Z, Yu C, Chen M, Tian S, Sun C. MiR-1181 inhibits stem cell-like phenotypes and suppresses SOX2 and STAT3 in human pancreatic cancer. Cancer Lett 2015; 356:962-970.

39. Kim NH, Lee MY, Park SJ, Choi JS, Oh MK, Kim IS. Auranofin blocks interleukin‐6 signalling by inhibiting phosphorylation of JAK1 and STAT3. Immunology 2007; 122:607.

40. Tsuda M, Kohro Y, Yano T, Tsujikawa T, Kitano J, Tozaki-Saitoh H, et al. JAK-STAT3 pathway regulates spinal astrocyte proliferation and neuropathic pain maintenance in rats. Brain 2011; 134:1127-1139.

41. Yamauchi K, Osuka K, Takayasu M, Usuda N, Nakazawa A, Nakahara N, et al. Activation of JAK/STAT signalling in neurons following spinal cord injury in mice. J Neurochem 2006; 96:1060-1070.

42. Herrmann JE, Imura T, Song B, Qi J, Ao Y, Nguyen TK, et al. STAT3 is a Critical Regulator of Astrogliosis and Scar Formation after Spinal Cord Injury. J Neurosci 2008; 28:7231-7243.

43. Thoennissen NH, Iwanski GB, Doan NB, Okamoto R, Lin P, Abbassi S,et al. Cucurbitacin B Induces Apoptosis byInhibition of the JAK/STAT Pathway and Potentiates Antiproliferative Effects of Gemcitabine on Pancreatic Cancer Cells. Cancer Res 2009; 69:5876-5884.

44. Xu Z, Wang BR, Wang X, Kuang F, Duan XL, Jiao XY, et al. ERK1/2 and p38 mitogen-activated protein kinase mediate iNOS-induced spinal neuron degeneration after acute traumatic spinal cord injury. Life Sci 2006; 79:1895-1905.

45. Genovese T, Esposito E, Mazzon E, Muià C, Di PR, Meli R, et al. Evidence for the role of mitogen-activated protein kinase signaling pathways in the development of spinal cord injury. J Pharmacol Exp Ther 2008; 325:100-114.

46. Ding D, Xu H, Liang Q, Xu L, Zhao Y, Wang Y. Over-expression of Sox2 in C3H10T1/2 cells inhibits osteoblast differentiation through Wnt and MAPK signalling pathways. Int Orthop 2012; 36:1087-1094.

47. Chen S, Li X, Lu D, Xu Y, Mou W, Wang L, et al. SOX2 regulates apoptosis through MAP4K4-survivin signaling pathway in human lung cancer cells. Carcinogenesis 2014; 35:613.

48. Gao X, Chen G, Gao C, Zhang DH, Kuan SF, Stabile LP, et al. MAP4K4 is a novelMAPK/ERKpathway regulator required for lung adenocarcinoma maintenance. Mol Oncol 2017; 11:628-639.

49. Collins CS, Hong J, Sapinoso L, Zhou Y, Liu Z, Micklash K, et al. A small interfering RNA screen for modulators of tumor cell motility identifies MAP4K4 as a promigratory kinase. Proc Natl Acad Sci U S A 2006; 103:3775-3780.