Clin Exp Otorhinolaryngol.  2024 May;17(2):122-136. 10.21053/ceo.2023.00025.

MicroRNA-145-5p Regulates the Epithelial-Mesenchymal Transition in Nasal Polyps by Targeting Smad3

• Zhang M
• Peng X
• Liang X
• Wang W
• Yang Y
• Xu F
• Lu X
• Geng D
• Li M

• KMID: 2556003
• DOI: http://doi.org/10.21053/ceo.2023.00025

Abstract

Objectives
. The annual prevalence of chronic rhinosinusitis (CRS) is increasing, and the lack of effective treatments imposes a substantial burden on both patients and society. The formation of nasal polyps in patients with CRS is closely related to tissue remodeling, which is largely driven by the epithelial-mesenchymal transition (EMT). MicroRNA (miRNA) plays a pivotal role in the pathogenesis of numerous diseases through the miRNA-mRNA regulatory network; however, the specific mechanism of the miRNAs involved in the formation of nasal polyps remains unclear.
Methods
. The expression of EMT markers and Smad3 were detected using western blots, quantitative real-time polymerase chain reaction, and immunohistochemical and immunofluorescence staining. Differentially expressed genes in nasal polyps and normal tissues were screened through the Gene Expression Omnibus database. To predict the target genes of miR-145-5p, three different miRNA target prediction databases were used. The migratory ability of cells was evaluated using cell migration assay and wound healing assays.
Results
. miR-145-5p was associated with the EMT process and was significantly downregulated in nasal polyp tissues. In vitro experiments revealed that the downregulation of miR-145-5p promoted EMT. Conversely, increasing miR-145-5p levels reversed the EMT induced by transforming growth factor-β1. Bioinformatics analysis suggested that miR-145-5p targets Smad3. Subsequent experiments confirmed that miR-145-5p inhibits Smad3 expression.
Conclusion
. Overall, miR-145-5p is a promising target to inhibit nasal polyp formation, and the findings of this study provide a theoretical basis for nanoparticle-mediated miR-145-5p delivery for the treatment of nasal polyps.

Keyword

Rhinosinusitis; Nasal Polyps; Epithelial-Mesenchymal Transition; MicroRNAs

Figure

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  Fig. 1. Expression of epithelial-mesenchymal transition markers in nasal polyp tissue. (A) Nasal structure and scheme of the study design. (B-E) Western blot analysis of protein expression in normal tissues (N1, N2, N3) and nasal polyps (P1, P2, P3). ImageJ was used to calculate the relative expression rate. (F-H) Quantitative real-time polymerase chain reaction (qRT-PCR) for mRNA expression in normal tissues and nasal polyp tissues. (I) Representative images of immunohistochemical staining in normal tissues and nasal polyp tissues. Scale bar=100 μm (left) and 20 μm (right). All data are shown as mean±standard deviation. n=3 per group. SMA, smooth muscle actin. *P<0.05, **P<0.01, as compared to the control group (normal tissues).

• 

  Fig. 2. miR-145-5p was significantly low-expressed in nasal polyp tissues. (A) Heatmap of the differential expression of genes between nasal polyp tissues and normal tissues. The red hue signifies elevated gene expression, whereas the blue shade corresponds to diminished gene expression. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of miRNAs. Darker hues indicate a more robust correlation. (C) KEGG enrichment analysis of miR-145-5p. (D-J) The expression levels of miRNAs in normal and nasal polyp tissues were verified through quantitative real-time polymerase chain reaction. The levels of miRNAs were normalized utilizing U6. All data are shown as mean±standard deviation. n = 3 per group. *P<0.05, **P<0.01.

• 

  Fig. 3. Transforming growth factor (TGF)-β1 induced the epithelial-mesenchymal transition (EMT) and inhibited the expression of miR-145-5p in human nasal epithelial cells (HNEpCs). (A) Experimental protocol for observing morphological changes. (B-E) Western blot analysis of the expression levels of EMT markers in HNEpCs treated with various concentrations of TGF-β1. (F-I) Western blot analysis was conducted to evaluate fluctuations in the expression levels of EMT markers in HNEpCs after varying time intervals. (J) After 48 hours with 10 ng/mL TGF-β1, changes in the cell morphology of HNEpCs. Scale bar=100 μm (left) and 20 μm (right). (K-N) The mRNA expression levels of miR-145-5p and EMT markers in HNEpCs, treated with 10 ng/mL TGF-β1 for 48 hours. All data are shown as mean±standard deviation. n = 3 per group. NS, not significant; SMA, smooth muscle actin. *P<0.05, **P<0.01.

• 

  Fig. 4. The downregulation of miR-145-5p promoted the epithelial-mesenchymal transition and cell migration. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) for verification of miR-145-5p inhibitor transfection efficiency. (B-D) qRT-PCR for quantification of α-smooth muscle actin (SMA), N-cadherin, and vimentin mRNA expression levels. (E-H) Western blot assay for evaluation of E-cadherin, N-cadherin, and vimentin protein expression. (I, J) Immunofluorescence staining analysis to assess the expression levels of E-cadherin and vimentin. Scale bar=50 μm. (K) The scratch assay was utilized to assess the migratory capacity of cells. Scale bar=100 μm. (L) Cell migration assay was employed to evaluate of cell migratory capability. Scale bar=100 μm. (M) Observation of cell morphology under a light microscope. Scale bar=100 μm. All data are shown as mean±standard deviation. n=3 per group. NS, not significant; DAPI, 4´,6-diamidino-2-phenylindole. *P<0.05, **P<0.01.

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  Fig. 5. The upregulation of miR-145-5p inhibited the epithelial-mesenchymal transition and cell migration. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) for verification of miR-145-5p mimic transfection efficiency. (B-E) Western blotting for detecting the protein expression levels of E-cadherin, N-cadherin, and vimentin. (F) Observation of cellular morphological changes under the light microscope. Scale bar=100 μm. (G-I) qRT-PCR was employed to detect the mRNA expression levels of vimentin, α-smooth muscle actin (SMA), and N-cadherin. (J) Schematic diagram of the cell migration assay experiment. (K, L) Cell migration assay and scratch assays were utilized to assess the migratory capacity of cells. Scale bar=100 μm. (M, N) Immunofluorescence staining detection of E-cadherin and vimentin expression. Scale bar=50 μm. All data are shown as mean±standard deviation. n=3 per group. TGF, transforming growth factor. *P<0.05, **P<0.01.

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  Fig. 6. miR-145-5p inhibited Smad3 expression by targeting the 3’-untranslated region (UTR) sequence. (A) Candidate genes targeted by miR-145-5p were screened using a Venn diagram. (B) A luciferase reporter assay verified the binding between miR-145-5p and Smad3 mRNA 3’-UTR. (C) Kyoto Encyclopedia of Genes and Genomes enrichment analysis was conducted on the six candidate target genes. (D) miR-145-5p was identified to have a complementary relationship with Smad3 through the TargetScan database. (E-H) Western blot analysis examining changes in Smad3 expression levels. (I, J) Quantitative real-time polymerase chain reaction was utilized to detect the mRNA expression levels of Smad3. (K) Immunofluorescence staining was used to detect alterations in the expression of Smad3. Scale bar=50 μm. All data are shown as mean±standard deviation. n=3 per group. NC, negative control; WT, wild-type; NS, not significant; TGF, transforming growth factor. *P<0.05, **P<0.01.

• 

  Fig. 7. miR-145-5p exerted its inhibitory effect on the epithelial-mesenchymal transition by directly downregulating Smad3. (A, B) Western blotting was used to detect Smad3 and p-Smad3 expression in nasal polyp tissues and normal tissues. (C, D) Quantitative real-time polymerase chain reaction (qRT-PCR) and representative images of immunohistochemical staining were used to detect Smad3 expression in nasal polyp tissues and normal tissues. Scale bar=100 μm (left) and 20 μm (right). (E) Assessment of Smad3 transfection efficiency through qRT-PCR. (F, G) Assessment of Smad3 transfection efficiency through Western blotting. (H-J) qRT-PCR was employed to assess the mRNA expression of α-smooth muscle actin (SMA), N-cadherin, and vimentin. (K-N) Western blotting was employed to assess the expression of E-cadherin, N-cadherin, and vimentin. (O) Diagram of the mechanism of nasal polyp formation. All data are shown as mean±standard deviation. n = 3 per group. LV, lentivirus vector; TGF, transforming growth factor. *P<0.05, **P<0.01.

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