LINC01272 activates epithelial-mesenchymal transition through miR-153-5p in Crohn's disease

Abstract

Objectives: Long noncoding RNAs (lncRNAs) have different functions in different diseases. There is little research on the functions of lncRNAs in Crohn's disease (CD). By using RNA-seq technology, we identified a lncRNA associated with Crohn's disease. However, the mechanism of lncRNA regulation remains unknown. This study aimed to determine the association of LINC01272 with epithelial cell-mesenchymal transition and the underlying mechanism in CD.

Methods: RNA was detected by qRT-PCR. The interaction of protein and RNA was determined by RNA binding protein immunoprecipitation. Luciferase reporter assays were used to detect the targeted miRNA of LINC01272. Tissue fibrosis was observed by Masson and H&E staining. Protein expression was determined by western blotting and immunofluorescence.

Results: LINC01272 was highly expressed in patients with CD. Knockdown of LINC01272 inhibited TGF-β1-induced epithelial-mesenchymal transition (EMT). Additionally, LINC01272 regulated TGF-β1-induced EMT through the miR-153-5p axis, and knockdown of LINC01272 inhibited EMT in CD mice in vivo.

Conclusion: LINC01272 activated the epithelial-mesenchymal transition through miR-153-5p in CD.

Keywords: Crohn’s disease; EMT; LINC01272; miR-153-5p.

Figure 1

LINC01272 is highly expressed in CD. A. The relative expression of LINC01272 in the control group and CD group. B. Correlation analysis of the expression of LINC01272 in CD tissues and plasma samples. ***P<0.001.

Figure 2

Knockdown of LINC01272 inhibits TGF-β1-induced EMT in IEC-6 cells. A. Relative expression of LINC01272 in TGF-β1-treated IEC-6 cells detected by qRT-PCR. B. The transfection efficiency of sh-LINC01272 detected by qRT-PCR. C. Relative expression of E-cadherin, CK, collagen I, collagen III, α-SMA and N-cadherin mRNA detected by qRT-PCR. D. Expression of E-cadherin, CK, collagen I, collagen III, α-SMA and N-cadherin protein detected by western blot. E. Expression of collagen I and collagen III detected by immunofluorescence. F. Cell migration ability detected by wound-healing assay. G. Cell invasion ability detected by transwell assay. *P<0.05, ***P<0.001.

Figure 3

MiR-153-5p targets the 3’UTR of LINC01272. A. Subcellular localization experiment. B. Targeting relationship between LINC01272 and miR-153-3p predicted by bioinformatics. C. The transfection efficiency detected by qRT-PCR. D. A luciferase reporter assay verified the relationship between LINC01272 and miR-153-3p. E. RNA binding protein immunoprecipitation experiments verified the relationship between LINC01272 and miR-153-3p. F. Relative expression of miR-153-3p in TGF-β1-treated IEC-6 cells. G. Relative expression of miR-153-3p in the control group and CD group. H. Relative expression of miR-153-3p before and after silencing LINC01272. I. Pearson’s correlation analysis of the relative expression of LINC01272. *P<0.05, ***P<0.001.

Figure 4

LINC01272 regulates TGF-β1-induced EMT through the miR-153-5p axis. A. Relative expression of E-cadherin, CK, collagen I, collagen III, α-SMA and N-cadherin mRNA detected by qRT-PCR. B. Expression of E-cadherin, CK, collagen I, collagen III, α-SMA and N-cadherin protein detected by western blot. C. Expression of collagen I and collagen III detected by immunofluorescence. D. Cell migration ability detected by wound-healing assay. E. Cell invasion ability detected by transwell assay. *P<0.05; **P<0.01; ***P<0.001.

Figure 5

Knockdown of LINC01272 inhibits EMT in CD mice in vivo. A. DAI scores of the control group, TNBS group and TNBS+sh-LINC01272 group. B. Pathological manifestations of colon tissue of mice in each group. C, D. Protein levels of EMT-related proteins and fibrosis proteins in each group of mice. E. The mRNA levels of EMT-related proteins and fibrosis proteins in each group of mice. **P<0.01; ***P<0.001.