The Long Non-coding RNA H19 Regulates The Expression Of Aquaporin 3 Through The Competitive Adsorption Of MicroRNA-874 In The Mucosal Barrier Of The Small Intestine

Background:The intestinal barrier,which consists of mechanical,biological,immunological and chemical barriers,plays a central role in maintaining internal homeostasis.Our previous study showed that AQP3 plays an important role in maintaining normal function of the intestinal barrier.Furthermore,the effect and underlying mechanism of miRNA-874 on regulation of the intestinal barrier through targeting the 3'UTR of AQP3 was also confirmed.Long noncoding RNA(IncRNA),defined as noncoding RNA more than 200 nucleotide in length,were once considered to be simply transcriptional 'noise' or cloning artefacts in past decades.However,recent studies demonstrated lncRNA may function as competing endogenous RNA(ceRNA)that'sponge' miRNA,thereby modulating the derepression of miRNA targetsand imposing an additional level of post-transcriptional regulation.Inspired by the ceRNA regulatory network and our previous studies,we hypothesized that AQP3 protein and the intestinal barrier function may also be regulated by an lncRNA that serves as a ceRNA by sponging miRNA-874.Methods:In the first part of the study,to investigate whether the expression of miRNA-874 is regulated by lncRNA,bioinformatics analysis was used in combination to predict lncRNA which might target miRNA-874.Bioinformatics analysis revealed a putative lncRNA,IncRNA H19,containing a putative 7-mer-binding motif binding site within miRNA-874.RIP and luciferase assays were used to validate the direct binding of the miRNA-874 response elements on the predicted lncRNA transcript.In the second part of the study,we firstly used immunohistochemistry,Western blot and qRT PCR technology to detecte the IncRNA H19,miRNA-874 and AQP3 expression levels and their correlation in hunman diseased small-bowel mucous tissue,mice intestinal ischemia-reperfusion injury model and cell model in vitro.Then,we developed an in vitro cultured epithelial monolayer system,utilizing the Caco-2 cell line with different expressions of H19 and miRNA-874 following H19 and miRNA-874 knockdown and/or overexpressionin cells.We detect the LY permeability,TEER and E.coli translocation which could reflect the function of intestinal barrier.Lastly,tight junction proteins,were detected by western blot in Caco-2 cells with both H19 overexpression and knockdown.Results:1.IncRNA H19 containing a putative 7-mer-binding motif binding site within miRNA-874.2.H19 knockdown in Caco-2 cells resulted in the up-regulation of the miRNA-874 and down-regulation of the AQP3 protein compared with the negative control.Conversely,the level of miRNA-874 was significantly reduced and the protein level of AQP3 was significantly increased in Caco-2 cells following overexpression of H19.The results of rescue experiment indicated that the positive relationship between H19 and AQP3 could be reverted by overexpression and knockdown of miRNA-874 in Caco-2 cells.3.miRNA-874 was significantly up-regulated and H19,AQP3 was significantly down-regulated in proximal compared with the distal tissues of diseased small-bowel mucous.4.The results of qRT-PCR showed a time-dependent increase in miRNA-874 expression and a time-dependent decrease in H19 expression.The results of IHC and western blot showed that ischaemia reperfusion induced a time-dependent decrease of AQP3 protein expression after 15 min of SMA occlusion.5.The results of qRT-PCR and western blot showed that the level of AQP3 protein and H19 expression were significantly reduced in a time-dependent manner in hypoxic and serum-free conditions.In contrast,the level of miRNA-874 was significantly increased with incubation time.6.The results showed that knockdown of H19 in Caco-2 cells caused an time-dependent increase in paracellular permeability compared with control groups,as measured by decreased TEER,increased LY permeability and E.coli translocation.In contrast,the paracellular permeability of the Caco-2 monolayer with overexpressed H19 showed a tendency to decrease.The results of rescue experiment showed that the negative regulation of Caco-2 cell monolayer paracellular permeability by H19 could be reverted by overexpression or knockdown of miRNA-874.7.The results of western blot showed that the expression of occludin and claudin-1 was significantly positively changed with H19 in Caco-2 cells.Expression of the other tight junction proteins did not change significantly.Conclusion:In summary,this study demonstrated that H19 down-regulation is a characteristic molecular change in intestinal barrier dysfunction,and weinvestigated the biological roles of H19,which may function as a ceRNA to regulate the expression of AQP3 through competition with miRNA-874.Further studies appears that the knockdown of H19 promoted the increase of paracellular permeability.In contrast,the level of paracellular permeability tends to be lower in the Caco-2 cell line with H19 overexpression compared with the control group.Moreover,our results showed two tight junction proteins,occludin and claudin-1,were significantly decreased in Caco-2 cells treated with siH19 compared with the negative control.All these findings confirmed that H19 plays an important role in intestinal barrier dysfunction through targeting miRNA-874.