LIN28 expression in rat spinal cord after injury

Abstract

LIN28, an RNA-binding protein, is known to be involved in the regulation of many cellular processes, such as embryonic stem cell proliferation, cell fate succession, developmental timing, and oncogenesis. However, its expression and function in central nervous system still unclear. In this study, we performed an acute spinal cord contusion injury (SCI) model in adult rats and investigated the dynamic changes of LIN28 expression in spinal cord. Western blot and immunohistochemistry analysis revealed that LIN28 was present in normal spinal cord. It gradually increased, reached a peak at 3 day, and then nearly declined to the basal level at 14 days after SCI. Double immunofluorescence staining showed that LIN28 immunoreactivity was found in neurons, astrocytes and a handful of microglia. Interestingly, LIN28 expression was increased predominantly in astrocytes but not in neurons. Moreover, the colocalization of LIN28 and proliferating cell nuclear antigen was detected after injury. Western blot showed that LIN28 participated in lipopolysaccharide (LPS) induced astrocytes inflammatory responses by NF-κB signaling pathway. These results suggested that LIN28 may be involved in the pathologic process of SCI, and further research is needed to have a good understanding of its function and mechanism.

Fig. 1

Behavior analysis and histology of the spinal cord of experimental animals. a Time course and degree of functional recovery in rats after SCI. Rats (n = 3) were killed at each time point (at 6 h and 12 h and at 1, 3, 5, 7, and 14 days post-injury). Data are reported as mean ± values of open-field locomotion BBB scores. b Histopathologic analysis of spinal cord from sham and injured rats at day 3 was used by H&E staining with paraffin histopathology sections. No lesion was seen in the sham group (a, c). Edema, vacuoles, irregularly shaped spaces, axon degradation, and disorders of the organization were evident in white matter at day 3 after injury (arrows in d). The majority of typical characteristics of neuronal apoptosis including nuclear fragmentation, nuclear disappearance, and nuclear pyknosis were found in gray matter (arrows in b). Scale bars, 20 μm (a–d)

Fig. 2

Time-dependent expression of LIN28 protein in rat spinal cord after SCI. Spinal cord tissues from rats at various survival times after SCI were homogenized and subjected to immunoblot analysis. Sample immunoblots probed with LIN28 and GAPDH are shown above (a). The bar chart below demonstrates the ratio of LIN28 to GAPDH for each time point (b). Data are mean ± SEM (n = 3; *p < 0.05, significantly different from the sham groups)

Fig. 3

Dose-response effects’ between the SCI and LIN28 expression. Spinal cord tissues from rats at various degrees of contusion induced by different heights (0, 6.25, 12.5, 25, 50, 75, 100, and 125 mm) at day 1 after SCI were homogenized and subjected to immunoblot analysis. Sample immunoblots probed with LIN28 and GAPDH are shown above (a). The bar chart below demonstrates the ratio of LIN28 to GAPDH for each height point (b). Data are mean ± SEM (n = 3; *p < 0.05, significantly different from the sham group)

Fig. 4

Immunohistochemical expression of LIN28 in adult rat spinal cord. Low-power views of cross-sections immunostained with antibody specific for LIN28 in sham spinal cord (a) and 3 day after injury (b). Higher-power views in the ventral horn (c, d) and white matter (e, f) of the spinal cord. Quantitative analysis of LIN28-positive cells/mm² between sham and at day 3 after SCI (g). *p < 0.05 compared with sham spinal cord. Error bars represent SEM. Scale bars, 200 μm (a, b) and 20 μm (c–g)

Fig. 5

Double immunofluorescence staining for LIN28 and different phenotype-specific markers in the spinal cord. In the adult rat spinal cord 2 mm to epicenter at day 3 after SCI, horizontal sections were labeled with LIN28 (red) and different phenotype-specific markers (green), such as NeuN, GFAP, S100B and Iba1. The colocalization of LIN28 with different phenotype specific markers are shown in c, f, i, l, o, r, u, x and negative control in y. Quantitative analysis of different phenotype-specific markers positive cells expressing LIN28 (/mm²) in sham spinal cord and at 3 day after SCI (*p < 0.05, the changes of LIN28 expression after SCI were clear in astrocytes, but not in neurons) compared with the sham group (z). Error bars represent SEM. Scale bars, 20 μm (a–m) (Color figure online)

Fig. 6

Association of LIN28 with the cell proliferation after SCI. Westernblot analysis of PCNA in spinal cord after SCI. The expression of PCNA was increased after SCI and peaked at day 3 (A). Double immunofluorescence staining is for PCNA, GFAP, and LIN28 in spinal cord after SCI (B). In adult spinal cord at 3 days after injury, sections labeled with PCNA (e) and GFAP (d) and the colocalization of PCNA with GFAP (yellow) were shown in the spinal cord (f). The majority of reactive astrocytes were PCNA-positive at 3 days after SCI (f). Moreover, there was colocalization between LIN28 and PCNA (l). However, we observed hardly any expression of PCNA in sham groups (b, h). Scale bars, 20 μm (b)

Fig. 7

The expression and correlation of LIN28 and NF-κB signaling pathway during LPS induced astrocytes activation. Astrocytes were treated with LPS (1 μg/ml) for indicated times. INOS, p65, p50, IκBα, p-IκBα, p-p65, LIN28, and GAPDH expression was determined by Western blot analysis (a). Quantification of INOS, p65, p50, IκBα, p-IκBα, p-p65, and LIN28 protein levels. (n = 3, *p < 0.05) (b). The p-p65,LIN28 expression was analyzed by Western blot. Astrocytes in the presence or absence of LPS (1 μg/ml) or PDTC (100 uM) for 6 h were analyzed (c). Quantification of p-p65,LIN28 protein levels. (n = 3, *p < 0.05) (d)