Role of the long non-coding RNA HOTAIR/miR-126 axis in an in vitro psoriasis model

Abstract

Psoriasis is a T-cell-mediated inflammatory skin disease that is characterized by excessive keratinocyte proliferation and persistent skin inflammation. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) are dysregulated in a number of inflammatory conditions. In the present study, an in vitro psoriasis cell model was established. Human HaCaT keratinocytes were activated using the inflammatory factor IL-22. Briefly, HaCaT cells were starved in serum-free DMEM for 24 h and then stimulated with 100 ng/ml IL-22 in serum-free DMEM for 24 h. Previous research indicated that HOX transcript antisense RNA (HOTAIR) may participate in the development of psoriasis. First, reverse transcription-quantitative PCR (RT-qPCR) analysis was performed to detect HOTAIR expression. The results indicated that HOTAIR expression was reduced in IL-22-stimulated HaCaT cells. Subsequently, a dual-luciferase reporter assay was performed to verify the binding site between HOTAIR and microRNA (miR)-126. The RT-qPCR results indicated that miR-126 expression was increased in IL-22-stimulated HaCaT cells. Moreover, the effects of HOTAIR and miR-126 on IL-22-stimulated HaCaT cell proliferation and apoptosis were assessed. HaCaT cells were transfected with control-plasmid, HOTAIR-plasmid, HOTAIR-plasmid + mimic control or HOTAIR-plasmid + miR-126 mimic for 24 h. At 24 h post-transfection, the cells were stimulated with 100 ng/ml IL-22 for 24 h and experiments were conducted. IL-22 induced cell proliferation and suppressed apoptosis. However, HOTAIR-plasmid inhibited cell viability and induced apoptosis in IL-22-stimulated HaCaT cells. In addition, the western blotting results indicated that HOTAIR-plasmid increased cleaved caspase-3 expression and the cleaved caspase-3/caspase-3 ratio, whereas the HOTAIR-plasmid-mediated effects were reversed by miR-126 mimic. Collectively, the results of the present study demonstrated that the lncRNA-HOTAIR/miR-126 axis may be implicated in the regulation of psoriasis progression and may serve as a potential therapeutic target for psoriasis.

Keywords: interleukin-22-induced HaCaT cells; long non-coding RNA-HOX transcript antisense RNA; microRNA-126; psoriasis.

Figure 1

HOTAIR expression in IL-22-stimulated HaCaT cells. Reverse transcription-quantitative PCR analysis was performed to detect the relative expression of HOTAIR in IL-22-treated HaCaT cells. Data are presented as the mean ± SD. ^**P<0.01 vs. control group. HOTAIR, HOX transcript antisense RNA; IL, interleukin.

Figure 2

Binding sites between HOTAIR and miR-126. (A) Potential binding sites between HOTAIR and miR-126. (B) The dual-luciferase reporter gene assay was performed to confirm the binding sites between HOTAIR and miR-126. (C) Reverse transcription-quantitative PCR analysis was performed to detect miR-126 expression in IL-22-treated HaCaT cells. Data are presented as the mean ± SD. ^**P<0.01 vs. mimic control group; ^##P<0.01 vs. control group. HOTAIR, HOX transcript antisense RNA; miR, microRNA; wt, wild-type; mut, mutant; IL, interleukin.

Figure 3

Transfection efficiency of HOTAIR and miR-126 in HaCaT cells. (A) RT-qPCR analysis was performed to detect HOTAIR expression in the control-plasmid and HOTAIR-plasmid groups. (B) RT-qPCR analysis was performed to detect miR-126 expression in the mimic control and miR-126 mimic groups. (C) RT-qPCR analysis was performed to detect miR-126 expression in the HOTAIR-plasmid + mimic control and HOTAIR-plasmid + miR-126 mimic groups. Data are presented as the mean ± SD. ^**P<0.01 vs. control-plasmid group; ^##P<0.01 vs. mimic control group; ^&&P<0.01 vs. HOTAIR-plasmid + mimic control group. HOTAIR, HOX transcript antisense RNA; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; IL, interleukin.

Figure 4

Effects of HOTAIR on IL-22-stimulated HaCaT cell proliferation and apoptosis. HaCaT cells were transfected with control-plasmid, HOTAIR-plasmid, HOTAIR-plasmid + mimic control or HOTAIR-plasmid + miR-126 mimic for 24 h. After 24 h, cells were stimulated with 100 ng/ml IL-22 for a further 24 h. (A) The MTT assay was conducted to detect cell proliferation at 24, 48 and 72 h. (B) Flow cytometry was performed to detect cell apoptosis. (C) Cell apoptosis rate. (D) Caspase-3 activity. (E) Western blotting was performed to measure cleaved caspase-3 and caspase-3 protein expression levels in different groups. (F) Cleaved caspase-3/caspase-3 ratio. Data are presented as the mean ± SD.^*P<0.05, ^**P<0.01 vs. control group; ^#P<0.05, ^##P<0.01 vs. IL-22 + control-plasmid group; ^&P<0.05, ^&&P<0.01 vs. IL-22 + HOTAIR-plasmid + mimic control group.HOTAIR, HOX transcript antisense RNA; miR, microRNA; IL, interleukin.

Figure 5

HOTAIR-plasmid reduces miR-126 expression in IL-22-induced HaCaT cells. HaCaT cells were transfected with control-plasmid, HOTAIR-plasmid, HOTAIR-plasmid + mimic control or HOTAIR-plasmid + miR-126 mimic for 24 h. After 24 h, cells were stimulated with 100 ng/ml IL-22 for a further 24 h. Reverse transcription-quantitative PCR analysis was performed to measure miR-126 expression. Data are presented as the mean ± SD. ^**P<0.01 vs. control group; ^##P<0.01 vs. IL-22 + control-plasmid group; ^&&P<0.01 vs. IL-22 + HOTAIR-plasmid + mimic control group. HOTAIR, HOX transcript antisense RNA; miR, microRNA; IL, interleukin.