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.