| Objective:By using miRNA and mRNA microarrays, we detected the differentially expressed miRNAs and mRNAs in IL-22 stimulated HaCaT cells to explore the molecular mechanisms of proliferation induced by IL-22 from the perspective of epigenetics.Methods:(1) The experiment was performed on human immortalized keratinocytes (HaCaT cell line). The experimental group received IL-22 (100 ng/ml) stimulation for 24h and the control group received no stimulation. It was conducted by two independent experiments. Total RNA was extracted by Trizol agent and the differentially expressed miRNAs and mRNAs were screened out using Invitrogen’s microRNA microarray 4.0 and mRNA expression microarray 2.0.(2) RT-qPCR was used to verify the miRNA microarray results in HaCaT cells. Then the verified differentially expressed miRNA was testified in 6 cases of patients with psoriasis vulgaris and in normal control tissues. U6 was selected as internal reference and data was processed by 2-△△Ct.(3) By integrating differentially expressed miRNAs and mRNAs data mentioned above, we got the potential target genes whose expression trends were opposite to their miRNAs. Then we applied the bioinformatics methods and literatures to further analyze the target gene. The predicted target gene was tested in HaCaT cells stimulated by IL-22 for 24h using RT-qPCR and β-actin was selected as internal reference. Immunohistochemistry in paraffin sections was used to detect the expression of target gene in 6 cases of patients with psoriasis vulgaris and in normal control.(4) Proliferation assay was performed to testify the biological function of differentially expressed miRNA in HaCaT cells by transfecting the specific mimics, inhibitors and control respectively.(5) Luciferase assay and Western Blot were used to confirm the target gene.Results:(1) It showed that a total of 15 miRNAs were upregulated and 5 were downregulated more than 2 times. There were 7 candidate taget genes for miR-122-5p.(2) RT-qPCR results showed that compared with the control, miR-122-5p expression was upregulated both in HaCaT cells (about 3.50-fold, P<0.05) stimulated by IL-22 for 24h and in 6 cases of patients with psoriasis vulgaris (about 2.26-fold, P＜0.05) significantly.(3) By integrating bioinformatics methods, we determined Sprouty2 (Spry2) as a downstream target gene to be verified. RT-qPCR results showed that Spry2 expression was downregulated (about 1.72-fold, P<0.05) in HaCaT cells stimulated by IL-22 for 24h compared with the control. Immunohistochemistry results showed that the expression of Spry2 was lower in 6 cases of patients with psoriasis vulgaris than in control (P＜0.05).(4) CCK-8 results showed that compared with control, proliferation was significantly increased in transfecting with miR-122-5p mimetic group, whereas was significantly reduced in transfecting with miR-122-5p inhibitors group (P<0.05).(5) Luciferase assay showed that constructed 3’UTR of Spry2 reporter vectors in wild/mutant types were co-transfected with miR-122-5p into HaCaT cells respectively. Compared with the control, fluorescence luciferase activity in wild-type group was significantly reduced (P<0.05) and in mutant-type group it was no significant difference (P>0.05). Western Blot results showed that compared with control, Spry2 protein expression was significantly decreased in transfecting miR-122-5p mimics group, while was significantly increased in transfecting miR-122-5p inhibitors group (P<0.05). This suggested that Spry2 was a direct target gene of miR-122-5p.Conclusions:The expression of miR-122-5p was upregulated both in IL-22-stimulated keratinocytes and patients with psoriasis vulgaris. Overexpression of miR-122-5p promoted proliferation in HaCaT cells and low-expression of miR-122-5p inhibited prolifetation in HaCaT cells. Spry2 was the target gene of miR-122-5p. |
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