DISCUSSION
Our findings confirmed H19 upregulation in neutrophils in patients with acute ischemic stroke, which is positively associated with the severity of neurological deficits. Moreover, we observed miR-29b downregulation in neutrophils in patients with acute ischemic stroke, which was negatively associated with H19 levels. Additionally, we demonstrated that H19 regulates C1QTNF6 expression by sponging miR-29b, which facilitates IL-1β and TNF-α release by leukocytes and BBB disruption during cerebral ischemic injury. Our findings may provide new insights into the targeted treatment of ischemic stroke.
Emerging evidence regarding peripheral immune cells has provided insights into novel inflammatory mechanisms contributing to neuronal cell death in cerebral ischemic injury(Huang, Hussain, & Chang, 2021). Leukocytes are a sensitive indicator of inflammatory stimuli in patients with ischemic stroke(L. Wang et al., 2020). In patients with ischemic stroke, there is altered leukocyte expression of multiple ncRNAs, including H19, which is crucially involved in the inflammatory response of ischemic stroke(J. Wang et al., 2017). We previously reported that H19 increases the immune response by increasing plasma TNF-α and IL-1β levels, as well as decreasing plasma IL-10 levels, after ischemic stroke; moreover, it affects the subsequent pathological outcome(J. Wang et al., 2017). Wan et al. found that H19 upregulates MCAO-induced IL-1β and IL-18 expression by triggering caspase-1 signals; further, H19 excision exerted anti-inflammatory effects by inhibiting the production of these cytokines(Wan et al., 2020). Additionally, H19 functions as a competitive endogenous RNA for regulating neuronal apoptosis by sponging different miRNAs. Cheng et al. reported H19 upregulation in patients with diabetes mellitus, which directly suppresses miR-29b levels (Cheng et al., 2019). A recent study reported that H19 inhibition ameliorated OGD-induced hippocampal neuronal apoptosis and increased inflammatory cytokine levels by directly targeting miR‑29b(Xu, Wang, Meng, & Xu, 2021). We observed significant H19 upregulation in the leukocytes of patients with acute ischemic stroke and MCAO rats; moreover, H19 excision suppressed IL-1β and TNF-α secretion by directly targeting miR-29b. Moreover, we identified the binding site of miR-29b in H19 using the Starbase database. Our findings suggest that H19 and miR-29b interactions are crucially involved in inflammatory signalling during cerebral ischemic injury.
Multiple basic studies have demonstrated that miR-29b is a key mediator of the ischemic cascade in regulating inflammation and neuronal apoptosis (Jia, Hao, Wang, & Qu, 2015). There is decreased miR-29b expression in the ischemia region and in OGD-activated microglia, which contributes to injury in focally ischemic brains (H. Wang, Li, Gao, & Liao, 2019). Additionally, miR-29b can inhibit neuronal apoptosis by suppressing TNF-α and IL-1β release in OGD-activated microglia(H. Wang et al., 2019). There are significantly decreased blood miR-29b levels in patients with ischemic stroke, as well as decreased blood and brain levels of miR-29b in patients with cerebral ischemic injury(Y. Wang et al., 2015). This suggests that miR-29b in the brain and circulating blood could be involved in regulating the post-stroke immune response, which is consistent with our findings. We found that miR-29b excision promoted neuronal apoptosis and upregulated TNF-α and IL-1β expression by targeting C1QTNF6. Similarly, Wang et al. reported that miR-29b promoted IL-1β, IL-6, and IL-8 expression by inhibiting C1QTNF6 in human bronchial epithelial cells (J. Wang et al., 2020).
C1QTNF6 is an important member of the C1QTNF family. It was originally found to exert inflammatory-regulating effects in several diseases(Kirketerp-Moller, Bayarri-Olmos, Krogfelt, & Garred, 2020). C1QTNF6 is crucially involved in alleviating OGD-induced inflammatory molecules, including TNF-α, IL-1β, IL-6, and IL-10, in PC12 cells. (Y. Li, Sun, Gu, & Gao, 2020). CIQTNF6 overexpression protects against OGD-induced inflammation and cell apoptosis by activating PI3K/Akt signalling(Y. Li et al., 2020). CIQTNF6 induces IL-10 expression in macrophages, which may represent a novel target for pharmacological therapy in inflammatory diseases(Kim, Lee, Park, & Park, 2010). Consistent with previous findings, we found that CIQTNF6 is among the target genes of miR-29b and is significantly downregulated in leukocytes of patients with stroke and MCAO rats. However, C1QTNF6 overexpression with miR-29b inhibition promoted MCAO-induced expression of pro-inflammatory cytokines in leukocytes, including TNF-α and IL-1β. Peripheral inflammation is a common contributing factor to ischemic stroke deterioration(Meng et al., 2019). Ischemic stroke involves BBB damage; moreover, peripheral inflammatory factors, including IL-1β and TNF-α, are more likely to aggravate BBB damage and even worsen stroke outcomes(Yuan, Liu, & Qi, 2020). IL-1β and TNF-α could reduce tight junction expression or false adjacent blood vessels and further destroy BBB integrity and normal function (Rodrigues & Granger, 2015). Neutrophils produce various pro-inflammatory cytokines that affect BBB function, including IL-1β and TNF-α, which can further promote neutrophil recruitment to the brain parenchyma and exacerbate inflammation(Koh et al., 2015Koh et al., 2015). We observed increased IL-1β and TNF-α secretion after C1QTNF6 overexpression in the peripheral leukocytes of MCAO rats. Additionally, increased IL-1β and TNF-α levels in the brain promote leukocyte migration into the brain parenchyma and increase BBB permeability, which forms a vicious cycle. Consistent with our in vivo study, C1QTNF6 overexpression promoted IL-1β and TNF-α expression in HL-60 cells. IL-1β and TNF-α upregulation can directly cause apoptosis of endothelial cells, which is an important BBB component. Taken together, our findings support the hypothesis that C1QTNF6 induces BBB damage by promoting IL-1β and TNF-α secretion; moreover, C1QTNF6 suppression exerts neuroprotective effects.
In summary, this is the first study to demonstrate H19 and C1QTNF6 upregulation, as well as miR-29b downregulation, in the leukocytes of patients with ischemic stroke and MCAO rats. H19 promotes C1QTNF6 expression by directly targeting miR-29b in the leukocytes of MCAO rats. Furthermore, C1QTNF6 upregulation in leukocytes aggravates cerebral ischemic injury and promotes BBB damage through IL-1β and TNF-α upregulation. Our findings demonstrate the inflammation-modulatory effect of the H19/miR-29b/C1QTNF6 axis in the process of cerebral ischemia injury.