The LncRNA H19/miR-1-3p/CCL2 axis modulates lipopolysaccharide (LPS) stimulation-induced normal human astrocyte proliferation and activation
Introduction
Astrocytes are regarded as the most abundant glial cell type within the central nervous system (CNS), and play an essential role in a variety of structural and physiological functions [1], [2]. Reactive astrocyte proliferation post spinal cord injury (SCI) leads to the formation of glial scars, thus impeding axon regeneration [3], [4]. Further investigations of the factors that might modulate astrocyte proliferation and activation, as well as the underlying mechanisms, can provide novel strategies for SCI repair.
After injury, astrocytes exhibit changes in phenotype and morphology and enhance the expression of intermediate filaments, including glial fibrillary acidic proteins (GFAP), S100β, nestin, and Vimentin [1], [5], [6]. Moreover, reactive astrocytes help release proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukins IL-1 and IL-6, CXCL10, and CCL2, which regulate the mechanisms of inflammation and secondary damage [7], [8]. CCL2, a typically highly modulated inflammatory chemokine, is capable of driving myeloid recruitment to CNS injury sites [9], [10]. During CNS injury and diseases, CCL2 is mainly expressed by reactive astrocytes [11], [12]. For example, astrocyte-derived CCL2 and the CCL2/CCR2 pathway are linked to M1 polarization and increased migratory capacity, suggesting that this pathway might emerge as an effective target for improving CNS inflammation [13]. More importantly, CCL2 expression increases after nerve injury [14], [15], [16]. Based on these previous findings, in the present study, we aimed to develop an in-depth understanding of reactive astrocyte-derived CCL2.
In our previous study, we demonstrated that the miR-140/BDNF axis inhibits the proliferation of normal human astrocytes and LPS-mediated secretion of IL-6 and TNF-α [17]. MicroRNAs (miRNAs), an evolutionarily conserved large class of short noncoding RNAs, are capable of regulating post-transcriptionally gene expression [18]. In recent decades, increasing attention has been paid to miRNAs and lncRNAs (long noncoding RNAs), another family of noncoding RNAs, because of their complicated effects on normal biological processes and pathogenesis. miRNAs can suppress translation or lead to the degradation of downstream target mRNAs by binding to their 3′UTR [19], [20]. By competitively binding to miRNAs, lncRNAs counteract miRNA-mediated inhibition of downstream target mRNAs by acting as ceRNAs (competitive endogenous RNAs) [21], [22]. Previous studies have reported that several lncRNA-miRNA-mRNA axes play essential roles in SCI repair [23], [24], [25]. Thus, we speculated that lncRNA-miRNA-mRNA axes could also affect reactive astrocyte-derived CCL2 and astrocyte dysfunction.
In the present study, we downloaded and analyzed online microarray expression profiles reporting differentially expressed lncRNAs in activated and inactivated astrocytes in response to multiple stimuli to identify lncRNAs related to CCL2 and astrocyte proliferation. Among the differentially expressed lncRNAs, H19 attracted our attention because of its oncogenic role in glioma through the promotion of glioma cell proliferation. LncRNA H19 has long been reported to act as an oncogenic lncRNA by promoting glioma cell proliferation [26], [27], [28]. Hyperproliferation is one of the key features of activated glial cells. More importantly, H19 overexpression has been reported to directly induce the activation of astrocytes and microglia and the release of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in the hippocampus, whereas H19 knockdown inhibited status epilepticus-induced glial cell activation [29]. Thus, H19 was chosen for further experiments.
Since increasing evidence indicates that a lncRNA acts as a ceRNA for miRNA by competing for miRNA binding with downstream target mRNA, therefore counteracting miRNA-mediated inhibition of downstream target mRNA expression, we speculated that a miRNA might link H19 and CCL2 to mediate the role of H19 in astrocyte activation. Next, miRNAs that might simultaneously target H19 and CCL2 were analyzed using five online tools (TargetScan, starBase, mirDIP, miRDB, and miRTarBase) and miR-1-3p was selected. The specific effects of H19 and miR-1-3p on normal human astrocytes (NHAs) were examined, respectively. The predicted miR-1-3p binding to H19 and the CCL2 3′UTR, and the dynamic effects of H19 and miR-1-3p on CCL2 expression and NHA activation and proliferation were investigated. In summary, we attempted to identify a lncRNA-miRNA-mRNA network that exerts a critical effect on NHA activation and proliferation.
Section snippets
Cell line, cell transfection, and cell treatment
Normal human astrocytes (NHAs, isolated from the spinal cord) were obtained from ScienCell (Cat. #1820, Carlsbad, CA, USA) and cultured in astrocyte medium (AM, Cat. #1801, ScienCell) supplemented with FBS (ScienCell).
H19 knockdown in NHAs was achieved by transfection of si-H19#1/#2/#3 (GenePharma, Shanghai, China). miR-1-3p overexpression or inhibition in NHAs was established by transfection of miR-1-3p mimics or miR-1-3p inhibitor (GenePharma). All cell transfections were performed using
Expression of H19 in normal human astrocytes (NHAs) based on online data and experimental results
H19 has been reported to promote human glioma cell proliferation [26], [27] and hippocampal glial cell activation [29]. Here, we downloaded and analyzed online microarray expression profiles reporting differentially expressed lncRNAs in activated and inactivated astrocytes in response to multiple stimuli, including LPS treatment, EGF treatment, and MCAO surgery. According to GSE35338 and GSE5282, H19 expression in NHAs was significantly upregulated by any of LPS treatment (GSE35338, Fig .1A),
Discussion
Herein, we found that lncRNA H19 acted as an upregulated lncRNA in NHAs in response to LPS stimulation, and H19 was positively correlated with CCL2. H19 silencing in NHAs significantly attenuated the promoting effects of LPS stimulation on NHA proliferation and activation as manifested by inhibited cell viability and DNA synthesis capacity, reduced NHA activation markers, and reduced inflammatory factor concentrations (CCL2, IL-6, and TNF-α). miR-1-3p directly bound to H19 and the CCL2 3′UTR.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
Project supported by the Hunan Province Science Foundation of China (Grant No. 2018JJ3752).
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