LncRNA MIR181A2HG inhibits keratinocytes proliferation through miR-223-3p/SOX6 axis

Abstract

Background: Psoriasis is a complex and recurrent chronic inflammatory skin disease, and the abnormal proliferation of keratinocytes plays a crucial role in the pathogenesis of psoriasis. Long non-coding RNAs (lncRNAs) play an indispensable role in regulating cellular functions. This research aims to explore the potential impact of lncRNA MIR181A2HG on the regulation of keratinocyte proliferation.

Methods: The expression level of MIR181A2HG and the mRNA level of KRT6, KRT16, and SOX6 were assessed using qRT-PCR. The viability and proliferation of keratinocytes were evaluated using CCK-8 and EdU assays. Cell cycle analysis was performed using flow cytometry. Dual-luciferase reporter assays were applied to test the interaction among MIR181A2HG/miR-223-3p/SOX6. Protein level was detected by Western blotting analysis.

Results: The findings indicated that psoriasis lesions tissue exhibited lower levels of MIR181A2HG expression compared to normal tissue. The overexpression of MIR181A2HG resulted in the inhibition of HaCaT keratinocytes proliferation. The knockdown of MIR181A2HG promoted cell proliferation. The dual-luciferase reporter assay and rescue experiments provided evidence of the interaction among MIR181A2HG, SOX6, and miR-223-3p.

Conclusions: The lncRNA MIR181A2HG functions as a miR-223-3p sponge targeting SOX6 to regulate the proliferation of keratinocytes, which suggested that MIR181A2HG/miR-223-3p/SOX6 might be potential diagnostic and therapeutic targets for psoriasis.

Keywords: MIR181A2HG; SOX6; keratinocytes; miR-223-3p; psoriasis.

Figure 1

MIR181A2HG was down-regulated in psoriatic skin lesions. (A) The datasets GSE13355, GSE14905 and GSE50790 were downloaded to analyze the expression of MIR181A2HG in skin tissues. (B) qRT-PCR was performed to detect the expression of MIR181A2HG in skin tissues from psoriasis patients and normal individuals. **P<0.01, ***P<0.001.

Figure 2

MIR181A2HG negatively regulated keratinocytes proliferation. (A) The efficiency of interference and overexpression of MIR181A2HG in HaCaT keratinocytes was detected. (B) The effects of interference or overexpression of MIR181A2HG on cell viability were assessed using CCK-8 kit. (C) HaCaT keratinocytes transfected for 48 hours were harvested and qRT-PCR was used to evaluate the effects of interference or overexpression of MIR181A2HG on KRT6 and KRT16 mRNA level. (D) Western blotting was applied to detect the effects on Cyclin A2, CDK4, Cyclin D1, KRT6 and KRT16 protein level. GAPDH served as an internal reference. (E) Flow cytometry was performed to analyze the effects on cell cycle distribution. *P<0.05, **P<0.01, ***P<0.001.

Figure 3

MIR181A2HG sponged miR-223-3p. (A) Venn diagram illustrates the intersection of upregulated miRNAs from GEO datasets (GSE145054 and GSE142582) and miRDB predicted targets of MIR181A2HG. (B) The binding site between MIR181A2HG and miR-223-3p. HaCaT keratinocytes were co-transfected with miR-223-3p/NC mimic and MIR181A2HG wide type (MIR181A2HG-WT)/MIR181A2HG mutant (MIR181A2HG-MUT) plasmid for 48 hours. Dual-luciferase reporter assay was performed to verify the interaction between MIR181A2HG and miR-223-3p. (C) Expression correlation of MIR181A2HG and miR-223-3p in dataset GSE142582. (D, E) qRT-PCR was used to detect the expression of MIR181A2HG, KRT6 and KRT16 in HaCaT keratinocytes with the transfection of miR-223-3p mimic/inhibitor. GAPDH served as an internal reference. (F) Western blotting was used to detect the protein level of Cyclin A2, CDK4, Cyclin D1, KRT6 and KRT16 in HaCaT keratinocytes with the transfection of miR-223-3p mimic/inhibitor. GAPDH served as an internal reference. (G) The rate of cell proliferation was assessed using EdU incorporation assays. Scale bar: 100 μm. (H) Flow cytometry was performed to analyze the effects on cell cycle distribution. *P<0.05, **P<0.01, ***P<0.001.

Figure 4

miR-223-3p targeted SOX6. (A) Venn diagram illustrates the intersection of downregulated genes from GEO datasets (GSE13355, GSE14905 and GSE50790) and TargetScan predicted targets of miR-223-3p. (B) qRT-PCR was performed to evaluate the effects of miR-223-3p mimic transfection on SOX6, F3 and ENPP5 mRNA level. GAPDH served as an internal reference. (C) Western blotting was used to detect the protein level of F3 and SOX6 in HaCaT keratinocytes with the transfection of miR-223-3p mimic/inhibitor. GAPDH served as an internal reference. (D) The binding site between miR-223-3p and SOX6. (E) HaCaT keratinocytes were co-transfected with miR-223-3p/NC mimic and SOX6 wide type (SOX6-WT)/SOX6 mutant (SOX6-MUT) plasmid for 48 hours. Dual-luciferase reporter assay was performed to verify the interaction between miR-223-3p and SOX6. (F) Expression correlation of miR-223-3p and SOX6 in dataset GSE142582. (G) qRT-PCR was used to detect the expression of SOX6 in HaCaT keratinocytes with the transfection of NC inhibitor/ miR-223-3p inhibitor. GAPDH served as an internal reference. (H) The efficiency of interference of si-SOX6 in HaCaT keratinocytes was detected. (I) The effects of SOX6 interference on cell viability were assessed using CCK-8 kit. (J) qRT-PCR was used to detect the expression of KRT6, KRT16 in HaCaT keratinocytes with the transfection of si-NC/si-SOX6. GAPDH served as an internal reference. (K) Western blotting was used to detect the protein level of Cyclin A2, CDK4, Cyclin D1 and KRT6 in HaCaT keratinocytes with the transfection of si-NC/si-SOX6. GAPDH served as an internal reference. (L) The rate of cell proliferation was assessed using EdU incorporation assays. Scale bar: 100 μm. (M) Flow cytometry was performed to analyze the effects on cell cycle distribution. *P<0.05, **P<0.01, ***P<0.001.

Figure 5

The modulation of MIR181A2HG could affect SOX6 expression. (A) Expression correlation of MIR181A2HG and SOX6 in datasets (GSE13355, GSE14905 and GSE50790). (B, C) HaCaT keratinocytes transfected for 48 hours were harvested and qRT-PCR/Western blotting was used to detect the expression of SOX6 in HaCaT keratinocytes with the transfection of si-MIR181A2HG/pcDNA3.1-MIR181A2HG. (D) CCK-8 kit was applied to detect the cell viability of HaCaT keratinocytes co-transfected with pcDNA3.1/pcDNA3.1-MIR181A2HG and si-NC/si-SOX6. (E, F) qRT-PCR and Western blotting were performed to detect the KRT6/KRT16 level in HaCaT keratinocytes co-transfected with pcDNA3.1/pcDNA3.1-MIR181A2HG and si-NC/si-SOX6. GAPDH served as an internal reference. *P<0.05, **P<0.01, ***P<0.001.

Figure 6

The MIR181A2HG/miR-223-3p/SOX6 axis regulated the proliferation of HaCaT keratinocytes. (A) CCK-8 kit was applied to detect the cell viability of HaCaT keratinocytes co-transfected with NC inhibitor/miR-223-3p inhibitor and si-NC/si-SOX6. (B, C) qRT-PCR and Western blotting were performed to detect the KRT6/KRT16 level in HaCaT keratinocytes co-transfected with NC inhibitor/miR-223-3p inhibitor and si-NC/si-SOX6. (D) CCK-8 kit was applied to detect the cell viability of HaCaT keratinocytes co-transfected with pcDNA3.1/pcDNA3.1-MIR181A2HG and NC mimic/miR-223-3p mimic. (E, F) qRT-PCR and Western blotting were performed to detect the KRT6/KRT16 level in HaCaT keratinocytes co-transfected with pcDNA3.1/pcDNA3.1-MIR181A2HG and NC mimic/miR-223-3p mimic. GAPDH served as an internal reference. *P<0.05, **P<0.01, ***P<0.001.