Role of hippocampal circKcnk9 in visceral hypersensitivity and anxiety comorbidity of irritable bowel syndrome

Abstract

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by recurrent visceral pain and altered bowel habits (diarrhea or constipation). However, the molecular and pathological mechanisms are poorly understood. This study found neonatal colorectal distension to induce visceral hypersensitivity and anxiety. The expression of hippocampal circKcnk9, a novel circRNA, was significantly increased in IBS-like rats. Interestingly, CA1 shcircKcnk9 treatment inhibited long-term potentiation (LTP) and alleviated visceral hypersensitivity and anxiety in IBS-like rats, whereas overexpression of CA1 circKcnk9 induced LTP, visceral hypersensitivity, and anxiety in controls. Several experiments indicated that increased CA1 circKcnk9 acted as a miR-124-3p sponge, which resulted in the inhibitory effect of miR-124-3p on gene silencing. There was a negative correlation between circKcnk9 and miR-124-3p expression. As expected, CA1 administration of agomiR-124-3p decreased CA1 LTP, visceral hypersensitivity, and anxiety in the IBS-like rats. In contrast, CA1 treatment with antagomiR-124-3p induced LTP, visceral hypersensitivity, and anxiety in the controls. Furthermore, bioinformatic analysis and experimental data showed that EZH2 is a circKcnk9/miR-124-3p target gene, and increased EZH2 expression was involved in visceral hypersensitivity and anxiety in IBS-like rats by enhancing hippocampal synaptic plasticity. In conclusion, early life stress induces increased expression of circKcnk9 in the CA1 of IBS-like rats. Increased circKcnk9 expression regulates synaptic transmission and enhances LTP, leading to visceral hypersensitivity and anxiety in IBS-like rats. The underlying circKcnk9 signaling pathway is miR124-3p/EZH2. Increased circKcnk9 reinforces its sponging of miR124-3p and strongly suppresses miR124-3p activity, resulting in increased expression of the target gene EZH2. This study provides a new epigenetic mechanism for visceral hypersensitivity and anxiety in IBS-like rats.

Methyltransferase-like 3 modulates visceral hypersensitivity through regulating the nuclear export of circKcnk9 in YTHDC1-dependent manner

“…More recent studies suggested that m 6 A modification could mark long non-coding RNA (lncRNA) for processing. 23,24 Considering circKcnk9 was significantly associated with IBS in our previous study, 17 we speculated that circKcnk9 might contribute to the development of chronic visceral pain through m 6 A modification. fluorescent in situ hybridization results showed that circKcnk9 in IBS-like rats significantly co-localized with more c-fos in hippocampus as compared with the controls, indicating that circKcnk9 in hippocampus could activate neurons (Figure 4(a)).…”

“…40 It has been shown that m 6 A can also modulate the expression of circRNAs like other RNA molecules through regulating their generation, stability, or degradation. 41 Considering that our previous studies 17 have demonstrated that circKcnk9, a novel circRNA, was found to adsorb MiR-124-3p in the cytoplasm to induce visceral hypersensitivity in IBS-like rats, it is important to trace the changes of m 6 A modification associated with circKcnk9 in IBS-like rats. Recently, YTHDC1 and its homologous complex have attracted increasing attention.…”

“…Lentivirus and AAV (shcircKcnk9 and aavcicKcnk9) vector of production and verification came from BrainVTA (Wuhan, China) were performed as described. 17 AAV-METTL3 came from GeneChem (Shanghai, China), siYTHDC1 were designed and commercially synthesized (CRN2901, Cohesion Bioscience, London, Britain) (Figure S1). siMETTL3 was constructed by GenePharma (Shanghai, China).…”

“…fluorescent in situ hybridization results showed that circKcnk9 in IBS-like rats significantly co-localized with more c-fos in hippocampus as compared with the controls, indicating that circKcnk9 in hippocampus could activate neurons (Figure 4(a)). Next, the secondary structure of cicKcnk9 was predicted based on the base sequence of circKcnk9 17,25 (Figure 4(b)). The RNA fold prediction website revealed that circKcnk9 was highly m 6 A modified (Figure S4), and Figure 4(c) showed the specific structure (GGACA) of the circKcnk9 modification.…”

“…15,16 In our previous study, the expression of hippocampal circKcnk9, a novel circRNA, was identified that being significantly increased in IBS-like rats. 17 Up to now, the potential mechanisms of hippocampal METTL3 and circKcnk9 in the progression of IBS are still unknown.…”