MiR-548a-3p Promotes Keratinocyte Proliferation Targeting PPP3R1 After Being Induced By IL-22

Objective:Based on the previous studies on miRNA and mRNA microarrays,which identified the differentially expressed miRNAs and mRNAs in HaCaT cells after stimulated by IL-22,our research detected the biological function of miR-548a-3p on keratinocytes and its target gene.Furthermore,the molecular mechanisms of miRNA proliferating function on human keratinocytes under the inducement of IL-22 was investigated from the perspective of epigenetics.Methods:(1)Human immortalized keratinocytes(HaCaT cell line)were divided into the experimental group and the control group.The experimental group was disposed with IL-22 treatment for 24h with the dose of 100ng/ml and no treatment was given to the control group.Three independent experiments were conducted respectively.Total RNA was extracted by RNAiso plus reagent and its concentration and purity were examined to make sure that it could be used in following experiments.(2)One differentially expressed miRNA was selected from the miRNA microarray results in HaCaT cells,and RT-qPCR was conducted to testify if the microarray results were correct.U6 was set to be the internal reference and three independent experiments were conducted respectively.6 cases of patients with psoriasis vulgaris and 6 cases of normal controls were selected according to the inclusion criteria.RT-qPCR was performed respectively on their lesions and skin tissue samples to detect the expression of miRNAs.The results were also described with the 2-△△Ct method.(3)In order to verify the biological function of miRNAs on human keratinocytes,mimics,inhibitor,the negative control of mimics and inhibitor of the differently expressed miRNA transfected HaCaT cells respectively,and CCK-8 cell proliferation experiment was implemented.(4)To screen the target genes that could be controlled by the miRNA,first we compared the results of miRNA and mRNA microarrays,and selected genes that were negatively correlated.Then we looked up the literatures,and predicted the target genes using bioinformatics database.Results above were integrated in order to screen the downstream target genes of the miRNA.We performed dual luciferase reporter gene assay to verify the screened target genes.Dual luciferase reporter gene assay was performed to determine if there was complementary combination between the miRNA and the mRNA 3 ’UTR of its target gene by detecting ratio of the firefly luciferase and the renilla luciferase.(5)Western Blot was conducted to confirm the target gene again and detected the expression of the target gene after the differentially expressed miRNA was overexpressed.To detect the expression of the target gene in psoriasis vulgaris patients,we performed immunohistochemistry in paraffin sections of 6 lesion samples as well as in 6 normal control.These results could help determine whether the target gene protein was functional on keratinocytes.Results:(1)The miRNA and mRNA microarray results showed that a total of 20 miRNAs were differentially expressed more than 2 times.Among them,5 miRNAs were downregulated and 15 were upregulated.MiR-548a-3p was selected as the experimental object of the next stage,and 39 target genes were predicted for miR-548a-3p.(2)RT-qPCR results showed that stimulating HaCaT cells with IL-22 for 24h could significantly increase the expression level of miR-548a-3p,about 17.91-fold,which was obviously higher than the control group,and the difference was statistically significant(P<0.05).Stimulating HaCaT cells with IL-22 for 24h could also significantly increase the expression level of miR-548a-3p in lesions of 6 patients with psoriasis vulgaris,about 7.63-fold and the difference was statistically significant(P<0.05).(3)CCK-8 results proved that in the group transfected with miR-548a-3p mimics,the proliferating ability of HaCaT cells was significantly enhanced compared with the control group(P<0.05),whereas the proliferating ability of HaCaT was significantly reduced after HaCaT being transfected by miR-548a-3p inhibitors(P<0.05).(4)To accomplish the luciferase assay,report gene vectors carrying the wild or mutant type of 3’UTR region of PPP3R1 were constructed.Then we carried out co-transfection assay to HaCaT cells according to a grouping arrangement as:wild type + miR-548a-3p mimics,wild type + miR-548a-3p negative control,mutant + miR-548a-3p mimics,mutant + miR-548a-3p negative control.The results indicated that in the group transfected with wild type vector and miR-548a-3p mimics,the activity of luciferase was significantly lower than that in the control group,with statistically significance(P<0.05).As for the group transfected with mutant type vector and miR-548a-3p mimics,the activity of luciferase showed no significant change(P>0.05).(5)We referred to the results of bioinformatics analysis and related literatures,and concluded PPP3R1 as a downstream target gene of miR-548a-3p and planned to verify that.Immunohistochemistry results showed that the expression of Calcineurin B(the protein coded by PPP3R1)was downregulated in 6 lesion samples of patients with psoriasis vulgaris compared with normal control(P<0.05).(6)Western Blot was performed to verify the expression of PPP3R1 protein in the target gene.Results revealed that compared to the control group,in the miR-548a-3p mimics transfected group,the expression of Calcineurin B protein obviously declined,and the difference was statistically significant(P<0.05).The results above all implied that it was very likely that PPP3R1 was a direct target gene of miR-548a-3p.Conclusions:1.After the stimulation of IL-22,the expression level of miR-548a-3p in keratinocytes was increased,and its expression level was also increased in the skin lesions of patients with psoriasis vulgaris.2.MiR-548a-3p overexpression can significantly facilitate the proliferation of HaCaT cells.3.It was highly probable that PPP3R1 was the target gene of miR-548a-3p.MiR-548a-3p might promote keratinocyte proliferation through controlling Calcineurin B protein.