Long noncoding RNA H19 act as a competing endogenous RNA of Let-7g to facilitate IEC-6 cell migration and proliferation via regulating EGF.

Intestinal mucosal injury is one of the most significant complications of burns. In our previous study, it was found that autophagy could alleviate burn-induced intestinal injury, but the underlying mechanisms are still unclear. Irregular expression of long noncoding RNAs (lncRNAs) is present in many diseases, including burns. However, the relationship between lncRNAs and intestinal mucosal injury requires further elucidation. In this study, we established a burn mice model and detected the expression level of autophagy-related proteins. Then, H19 content after autophagy intervention was tested in vitro and in vivo. The interaction of H19 with Let-7g and that of Let-7g with epidermal growth factor (EGF) were verified by dual-luciferase reporter assays. We found that the expression of the autophagy-associated proteins LC3-II and Beclin-1 was raised in the intestinal tract of the burn mice model. Similarly, the transfection of H19 raised autophagy levels. H19 was elevated after autophagy intervention in vitro and in vivo. H19 overexpression was able to promote IEC-6 cell migration and proliferation. Let-7g was suppressed by the overexpression of H19 and the combination of Let-7g mimic was able to abolish the physiological effect of H19. Moreover, the suppression of Let-7g increased the expression of EGF protein, which heightened IEC-6 cell migration and proliferation. Besides this, dual-luciferase assays revealed that Let-7g was a direct target of H19 as well as the EGF gene. Taken together, autophagy-mediated H19 increases in mouse intestinal tract after severe burn and functions as a sponge to Let-7g to regulate EGF, which suggests that H19 serves as a potential therapeutic target and biomarker for intestinal mucosal injury after burns.

Epidermal growth factor regulation by autophagy-mediated lncRNA H19 in murine intestinal tract after severe burn.

To investigate the regulation of epidermal growth factor (EGF) by autophagy-mediated long non-coding RNA (lncRNA) H19 in the intestinal tracts of severely burned mice. C57BL/6J mice received third-degree burns to 30% of the total body surface area. Rapamycin and 3-methyladenine (3-MA) were used to activate and inhibit autophagy, and the changes in LC3 and Beclin1 levels were assessed by Western blotting. The effect of autophagy on lncRNA H19 was detected by qRT-PCR. Adenovirus-mediated overexpression of lncRNA H19 in IEC-6 cells was used to assess the effects of lncRNA H19 on EGF and let-7g via bioinformatics analysis, Western blotting and qRT-PCR. let-7g mimic/inhibitor was used to overexpress/inhibit let-7g, and qRT-PCR and Western blotting were used to detect the effects of let-7g on EGF. The expression levels of LC3-II, Beclin1 and lncRNA H19 were increased in intestinal tissues and IEC-6 cells after rapamycin treatment but were reversed after 3-MA treatment. LC3-II, Beclin1 and lncRNA H19 levels increased in intestinal tissues after the burn, and these increases were more significant after rapamycin treatment but decreased after 3-MA treatment. The lncRNA H19 overexpression in IEC-6 cells resulted in increased and decreased expression levels of EGF and let-7g, respectively. Furthermore, overexpression and inhibition of let-7g resulted in decreased and increased expression of EGF, respectively. Taken together, intestinal autophagy is activated after a serious burn, which can increase the transcription level of lncRNA H19. lncRNA H19 may regulate the repair of EGF via let-7g following intestinal mucosa injury after a burn.

Identification of Differentially Expressed Long Noncoding RNAs in Mice Intestines After Severe Burns and a Preliminary Study into the Key Gene H19.

The intestine is considered the key organ in stress response to severe burns and injury to the intestine after severe burns can be fatal. However, the injury and subsequent repair of intestinal tissues after severe burns at the genetic level are poorly understood. Long noncoding RNAs (lncRNAs) have important functions in regulating many biological processes, including gene transcription and translation. Autophagy is a process of intracellular degradation and reutilization of cytoplasmic proteins and organelles. We herein analyzed the genome-wide expression profile of lncRNAs and mRNAs after severe burns in the intestines of mice by lncRNA microarray. Quantitative reverse transcription-polymerase chain reaction was performed to verify the reliability of microarray analysis results, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used for bioinformatics analysis of differentially expressed mRNAs. The common regulatory network between the top 10 differentially expressed lncRNAs and trans-related mRNAs were visualized by Cytoscape (v3.7.2). Next, we hypothesized that H19 is the key gene for intestinal mucosal repair. After H19 was overexpressed, the changes in downstream autophagy protein expression levels were observed. GO and KEGG analysis indicated that the differentially expressed mRNAs were mainly enriched in a cell cycle- and mitosis-related genes. Overexpression of lncRNA-H19 showed that the autophagy-related gene Trim21 was upregulated, while HIF1α was downregulated. LncRNA-H19 played a key role in repairing the intestinal mucosa, and overexpression of lncRNA-H19 activated autophagy and migration of intestinal epithelial cells (IEC-6).