MiR-20a-3p Regulates TGF-β1/Survivin Pathway To Affect Keratinocytes Proliferation And Apoptosis By Targeting SFMBT1 In Vitro

BackgroundPsoriasis is a common immune-mediated chronic inflammatory skin disease characterized by a clearly shapes and sizes of erythema,covered with thickened silvery scales.The disease affects the physical and mental health of patients seriously because of its long course,repeated attack,and difficult to cure.In addition,the recurrence and treatment of psoriasis imposes a heavier financial burden on patients.Therefore,it has become a focus and an urgent problem to be solved in the world to study the pathogenesis and new therapeutic targets of psoriasis.The exact etiology and pathogenesis of psoriasis are still unclear.It is widely accepted that the etiology of psoriasis involves genetic susceptibility and environmental that make innate and adaptive immune dysfunction,which leads to abnormal keratinocyte proliferation,differentiation and apoptosis.Recently,Th17 cells and the cytokines(interleukin-17A(IL-17A),IL-17F and IL-22)secreted by it play an important role in the pathogenesis of psoriasis.Increased levels of IL-22 have been detected in lesional skin and blood of patients with psoriasis,and there is a positive relevance in IL-22 and severity of psoriasis.IL-22 receptor is exclusively expressed on epithelial cells rather than immune cells and therefore is the uniquely communication between the immune system and keratinocytes.IL-22 could block the terminal differentiation of KC but also induce KC proliferation and migration in vitro.All evidence suggests that IL-22 plays a pivotal role in the pathogenesis of psoriasis.miRNAs(microRNAs)are small single-stranded noncoding RNAs(18-25nt).They can negatively regulate gene expression through translational repression by binding to the 3’ untranslated region(UTR)of the target mRNA,which play a key role in cellular process including proliferation,differentiation,apoptosis,invasion and metabolism.Recently,an increasing miRNAs abnormal expression was reported in lesional skin and blood of psoriasis patients.It is now clear that miRNAs can regulate proliferation,differentiation and cytokine responses of keratinocytes and so on.Therefore,understanding the role of miRNAs may provide potential insight in the pathogenesis and treatment of psoriasis.Using previous miRNA microarrays,we found that miR-20a-3p was down-regulated in psoriatic lesions and in HaCaT cells(human keratinocyte cell line)treated by IL-22 stimulation.Therefore,we selected miR-20a-3p to further study and investigated the possible functional role of miR-20a-3p in psoriasis and IL-22 induced keratinocyte proliferation.Moreover,From the direction of epigenetics,we further explored the mechanism of IL-22 in the pathogenesis of psoriasis from the perspective of epigenetics,and thus providing a new target for the treatment of psoriasis.Part 1 Effect of miR-20a-3p on keratinocytes proliferation,cell cycle and apoptosisUsing miRNA microarrays,we identified 116 differentially expressed miRNAs in the psoriasis skin tissues and healthy skin,of which we found that miR-20a-3p was downregulated in psoriatic lesions.In our previous work,we performed a microarray analysis to identify changes in miRNA and mRNA profiles in human keratinocytes exposed to IL-22,which plays a pivotal role in the pathogenesis of psoriasis.Of interest,we found that miR-20a-3p was also decreased in human keratinocytes treated with IL-22 compared with the control group.Therefore,we selected miR-20a-3p to further study and investigated the possible functional role of miR-20a-3p in psoriasis and provided a novel molecular basis for IL-22 induced keratinocyte proliferation from the perspective of epigenetics.ObjectiveTo study the expression level of miR-20a-3p in psoriasis tissues compared with healthy control skin;To detect the expression level of miR-20a-3p in HaCaT cells treated byIL-22 stimulation.To study the effect of miR-20a-3p on cell proliferation,cycle and apoptosis of keratinocytes in vitro.Methods(1)The skin tissues of 6 psoriasis patients and 6 healthy controls were collected.Total RNA was extracted by TRIzol reagent and the expression of miRNA-20a-3p was detected by RT-q PCR.(2)The experiment was performed on human immortalized keratinocytes(HaCaT cell line).The experimental group was stimulated with IL-22(100 ng/ml)for 24 h,and the control group was not stimulated.Total RNA was extracted by Trizol agent and the differentially expressed of miRNA-20a-3p was detected by RT-q PCR.(3)HaCaT cells were transfected with miR-20a-3p mimics and its negative control at a final concentration of 100nM or miR-20a-3p inhibitor and its negative control at a final concentration of 200nM.Cell proliferation was measured by a Cell Counting Kit-8(CCK-8)and 5-Ethynyl-2’-deoxyuridine(EdU)incorporation assay;Cell cycle and apoptosis was measured by flow cytometry and Annexin V-FITC/PI staining.(4)Western blot was used to detect the changes of the expression of cell cycle proteins related to G1/S transition and apoptosis-related protein after transfection with miR-20a-3p mimics,inhibitor and their respective controls into HaCaT cells.Results(1)The results of RT-qPCR showed that the expression level of miR-20a-3p in the skin lesions of psoriasis patients was down-regulated compared with the skin tissues of healthy controls.(2)The results of RT-qPCR showed that miR-20a-3p was downregulated in HaCaT cells treated by IL-22 stimulation.(3)The results of CCK8 assay showed that upregulation of miR-20a-3p suppressed proliferation while knockdown of miR-20a-3p had the opposite effect in HaCaT cells.For EdU assay,the EdU-positive rate(percentage of cells that underwent cell division)was significantly reduced by miR-20a-3p mimics(P<0.05).Conversely,inhibition of endogenous miR-20a-3p increased the EdU-positive rate compared with the control group(P<0.05).Flow-cy-tometric analysis showed that the proportion of cells at G1 phase increased(P<0.01)while that of cells at S phase decreased(P<0.05)after upregulation of miR-20a-3p in HaCaT cells.In contrast,knockdown of miR-20a-3p resulted in a reduced G1 phase and increased S phase,the differences were statistically significant(P<0.05).For cell apoptosis analysis,the fl ow cytometric results demonstrated that the percentage of early and late apoptotic cells increased after overexpression of miR-20a-3p compared with the control group(early apoptotic rate:1.79 ± 0.23 vs 1.20 ± 0.10,p<0.05;late apoptotic rate:22.9 ± 2.07 vs 14.17 ± 2.97,p<0.05).Conversely,in the miR-20a-3p knockdown group,early and late apoptotic decreased compared with the control group(early apoptotic rate:0.67 ± 0.05 vs 0.94 ± 0.11,p<0.05;late apoptotic rate:6.64 ± 0.76 vs 10.81 ± 0.48,p<0.01)(4)Western blot showed that HaCaT cells transfected with miR-20a-3p mimics decreased the expression of cell cycle control protein CDK2,CDK4,Cyclin D and phospho S811-Rb,and increased P21clipl protein expression.while knockdown of miR-20a-3p had the opposite effect in HaCaT cells.In addition,we found that upregulation of miR-20a-3p decreased the expression of apoptosis-related protein survivin,whereas knockdown of miR-20a-3p increased the expression of survivin in HaCaT cells.ConclusionsmiR-20a-3p was down-regulated in psoriatic lesions and in HaCaT cells treated with IL-22 stimulation.Functional experiments showed that miR-20a-3p in HaCaT cells could suppress proliferation,induce apoptosis,and result in cell cycle arrest(cells arrested in G1 phase)and suppress G1/S transition.Downregulated of miR-20a-3p in psoriasis may contribute to hyperproliferation and aberrant apoptosis of keratinocytes,which may lead to the occurrence of psoriasis Part 2 Mechanism of miR-20a-3p affecting proliferation,cell cycle and apoptosis of human keratinocytesThe above studies revealed that miR-20a-3p inhibits the proliferation of keratinocytes,promotes apoptosis,and arrests the cell cycle in the G1 phase.Next,the second part of this paper is to study the mechanism of miR-20a-3p on human keratinocyte proliferation,cell cycle and apoptosis.miRNA regulate diverse cellular processes by binding to the 3’UTR of target mRNAs.Therefore,searching for the target gene of miR-20a-3p is the key to study its mechanism.ObjectiveTo predict and identify the target protein of miRNA-20a-3p;To study the regulation of miRNA-20a-3p on TGF-beta 1/Survivin signaling pathway,and explore the mechanism of miRNA-20a-3p in the pathogenesis of psoriasis.Methods(1)We used Targetscans/Mirbase/picTar and other target gene prediction software to predict the possible target genes of miRNA-20a-3p,and the target gene SFMBT1 protein was verified by dual-luciferase reporter assay and western blot experiment.(2)Immunohistochemical staining was used to detect the expression of target protein SFMBT1 in 6 cases of vulgaris psoriasis lesions and 6 healthy controls.The expression of SFMBT1 protein was detected in IL-22 stimulated keratinocytes using western blot.(3)HaCaT cells were transfected with the SFMBT1-specific siRNA and its negative control.Cell proliferation was measured by a Cell Counting Kit-8(CCK-8)and 5-Ethynyl-2’-deoxyuridine(EdU)incorporation assay;Cell cycle and apoptosis was measured by flow cytometry and Annexin V-FITC/PI staining.(4)Western blot was used to detect the changes of the expression of cell cycle proteins related to G1/S transition and apoptosis-related protein after transfection with the SFMBT1-specific siRNA and its negative control into HaCaT cells.(5)Western blot was used to detect the changes of the protein expression of TGF-p 1、P-Smad2 and P-Smad3 after transfection with miR-20a-3p mimics,inhibitor and their respective controls or the SFMBT1-specific siRNA and its negative control in HaCaT cells.Results(1)Results of dual-luciferase reporter assay showed that co-transfection with miR-20a-3p mimics and pmiR-RB-ReportTM-SFMBTl-WT led to an obvious reduction in luciferase activity when compared to the miR-NC control group(P<0.01).However,no statistically significant alteration in luciferase activity was observed in the presence of the mutated 3’-UTR site.In addition,protein levels of SFMBT1 was found to be decreased following miR-20a-3p mimics and increased after transfection with miR-20a-3p inhibitor in HaCaT cells.(2)The results of immunohistochemistry indicated that 65%of psoriasis patients have high expression of SFMBT1,while that in healthy people was only 12.5%(p<0.05)(nuclear staining of epidermis).The protein levels of SFMBT1 was found to be decreased following miR-20a-3p mimics and increased after transfection with miR-20a-3p inhibitor in HaCaT cells.(3)Results of CCK8 assay indicated that knockdown of SFMBT1 inhibited cell proliferation.For EdU assay,the SFMBT1-specific siRNA had less EdU-positive cells at 48 hours after transfection compared with the control group(P<0.05).Flow-cytometric analysis showed that SFMBT1 knockdown increased the proportion of cells at G1 phase(P<0.05)and decreased that of S phase(P<0.01)in HaCaT cells.In addition,results demonstrated that the percentage of early and late apoptotic cells increased compared with the control group after knockdown of SFMBT1(early apoptotic rate:1.45 ± 0.12 vs 0.54 ± 0.02,p<0.001;late apoptotic rate:11.27 ± 1.25 vs 4.16 ± 1.54,p<0.01).(4)Western blot showed that HaCaT cells transfected with the SFMBT1-specific siRNA decreased the expression of cell cycle control protein CDK2,CDK4,Cyclin D and phospho S811-Rb,and increased P21clip1 protein expression.We also found that knockdown of SFMBT1 decreased the expression of apoptosis-related protein survivin.(5)Overexpression of miR-20a-3p promoted the protein expression levels of TGF-p 1 and P-smad2/3 but suppressed that of survivin.Similar results were reported after knockdown of SFMBT1 in HaCaT cells.Conversely,miR-20a-3p knockdown had the opposite results with the protein expression levels of TGF-β1,P-smad2/3 and survivin in HaCaT cells.ConclusionsSFMBT1 is a direct target gene of miR-20a-3p.Interfering with SFMBT1 protein can inhibit cell proliferation,promote cell apoptosis,and arrest cell cycle in G1 phase.Knockdown of SFMBT1 protein affects TGF-β1/Survivin signaling pathway.In conclusion,miR-20a-3p may play a role in the pathogenesis of psoriasis by directly targeting SFMBT1 and TGF-β1/Survivin signaling pathways to affect the proliferation and apoptosis of keratinogenesis,which may be a novel therapeutic target for psoriasis.