| Objective: Psoriasis is an immune-mediated chronic,inflammatory,immune,and systemic disease induced by the interaction of genetics and environment.The worldwide incidence rate is 2%-3%.Tortured by serious physical and psychological damage,psoriasis patients often face large burden in daily life.At present,the main treatment methods for psoriasis include local therapy,physical therapy,systemic therapy and new biological agents(such as tumor necrosis factor-? [TNF-?] inhibitors,interleukin [IL]-12/23 inhibitors and IL-17 inhibitors etc.),have achieved certain results,but the low cure rate and high recurrence rate are still key issues for clinicians.The main reason is that the etiology of psoriasis is complex,and the internal regulation mechanism has not been clarified.In recent years,it has been found that miRNAs are involved in the occurrence and development of psoriasis,providing a new direction for the study of the pathogenesis of psoriasis,and at the same time exploring new therapeutic target is of great significance.miR-125 a is closely related to the occurrence and development of various immune-associated diseases.The study found that serum miR-125 a in patients with moderate to severe plaque psoriasis treated with etanercept was negatively correlated with the area of psoriasis and the severity index.Multiple logistic regression analysis showed that miR-125 a is an independent predictor of efficacy,so miR-125 a has a good predictive value for the efficacy of etanercept in patients with psoriasis.We speculate that miR-125 a,as a miR closely related to inflammation and immunity,may play a role in the pathogenesis of psoriasis.Studies have shown that IL-23 has a significant effect on the proliferation and differentiation of keratinocytes.T cells and IL-23 receptor(IL-23R)/Janus kinase2(JAK2)/protein kinase B(AKT)/signal in skin tissue Transduction and activation of activator of transcription 3(STAT3)signaling pathway is related to activation.We predicted the miR-125 a target on Target Scan(http://www.targetscan.org/vert?72/)and found that IL-23 R is the target gene of miR-125 a,and studies have shown that keratinocytes express IL-23 R.Therefore,the main purpose of this study is to investigate the expression of miR-125 a in the skin lesions of patients with psoriasis vulgaris and its association with the severity of the disease.And explore whether miR-125 a can regulate the proliferation of keratinocytes through the IL-23 R signaling pathway,and affect the occurrence and development of psoriasis.Methods: This study is divided into two parts.The first part studies the expression of miR-125 a in the skin lesions of patients with psoriasis vulgaris and its relationship with the severity of the disease.The second part uses IL-23 to intervene in human immortalized keratinocytes(HaCaT),and explores the role of miR-125 a targeting IL-23 R signaling pathway in regulating keratinocyte proliferation,differentiation,apoptosis,and inflammation.1.The expression of miR-125 a in the skin lesions of patients with psoriasis vulgaris and its relationship with the severity of the disease.1.1 Clinical materials: 60 psoriasis patients were consecutively recruited,then lesional and non-lesional skin tissue samples were collected.In addition,clinical data of patients were also collected,which consisted of age,gender,disease duration,lesional body surface area(BSA)as well as psoriasis area and severity index(PASI)score was evaluated.1.2 Q-PCR: Extract m RNA from skin tissues of each group,detect the expression level of miR-125 a in psoriasis skin lesions and adjacent normal tissues,and detect IL-1? m RNA,IL-6 m RNA,TNF-? m RNA,IL-17 m RNA expression level.in psoriasis skin lesions.2.The effect of miR-125 a on the proliferation,differentiation,apoptosis and inflammation of keratinocytes through the IL-23 R signaling pathway.2.1 With the immortalized human keratinocyte cell line(HaCaT cells)as the research object,IL-23 was used to interfere with keratinocytes.The plasmid was transfected into miR-125 a,and the following studies were carried out:2.1.1 Plasmid transfection and grouping of HaCaT cells: After 24-hour pretreatment with interleukin(IL)-23,HaCaT cells were divided into miR-125 a overexpression plasmid group(miR-125a(+)group)and overexpression control plasmid group(NC(+)group).2.1.2 Q-PCR: Detect the miR-125 a transfection efficiency of two groups of cells.2.1.3 CCK-8 test: After HaCaT cells were transfected with miR-125 a,the HaCaT cells viability was assessed.2.1.4 Western blot: After plasmid transfection,the protein expression levels of KRT1 and KRT10 and the protein expression levels of IL-23 R,p-AKT,p-JAK2,AKT and JAK2 were detected in the two groups.2.1.5 AV/PI test: After HaCaT cells were transfected with miR-125 a,the apoptosis levels of the two groups were detected.2.1.6 ELISA test: After HaCaT cells were transfected with miR-125 a,extract the supernatant,detect the expression levels of cytokines TNF-?,IL-6,IL-10,and IL-17.2.2 According to the results of the study 2.1,with HaCaT cell line as the research object,IL-23 was used to interfere with keratinocytes.The plasmids were transfected into miR-125 a and miR-125 a plus IL-23 R respectively,and the following studies were carried out:2.2.1 Plasmid transfection and grouping of HaCaT cells: After 24-hour pretreatment with interleukin(IL)-23,HaCaT human immortalized keratinocytes were divided into miR-125 a overexpression plasmid group(miR-125a(+)group)and miR-125 a overexpression plasmid combined with IL-23 R overexpression plasmid group(miR-125a(+)plus IL-23R(+)group).2.2.2 Q-PCR: Detect the miR-125 a transfection efficiency of two groups of cells.2.2.3 CCK-8 test: After HaCaT cells were transfected plasmid,the HaCaT cells viability was assessed.2.2.4 Western blot: After plasmid transfection,the protein expression levels of KRT1 and KRT10 and the protein expression levels of IL-23 R,p-AKT,p-JAK2,AKT and JAK2 were detected in the two groups.2.2.5 AV/PI test: After plasmid transfection,the apoptosis levels of the two groups were detected.2.2.6 ELISA test: After plasmid transfection,extract the supernatant,detect the expression levels of cytokines TNF-?,IL-6,IL-10,and IL-17.2.3 Luciferase reporter assay verified the relationship between miR-125 a and IL-23 R.Construct 4 kinds of plasmids,miR125 a plasmid(A),miR control plasmid(A control),plasmid containing IL-23 R promoter sequence(B),plasmid containing IL-23 R promoter mutation sequence(B mutation).The B plasmid and the B mutant plasmid can express luciferase and are regulated by the A plasmid.Then the plasmids were grouped into 293 T cells.The grouping is as follows: A+B,A control+B,A+B mutation,A control+B mutation.After 48 hours of transfection,the cells were collected and the fluorescence intensity was measured.The effect of miR-125 a on IL-23 R was determined based on the fluorescence intensity.Results: 1.The expression level of miR-125 a is related to the severity of the patient's condition and some inflammatory factors.1.1 miR-125 a was downregulated in lesional skin tissue compared with that in non-lesional skin tissue of psoriasis patients1.2 No correlation of miR-125 a in lesional skin tissue with disease duration was found in psoriasis patients,however,negative association of miR-125 a with lesional BSA and PASI score was discovered in psoriasis patients.1.3 miR-125 a expression in lesional tissue was negatively correlated with TNF-?m RNA,IL-? m RNA and IL-17 m RNA in psoriasis patients.However,no association of miR-125 a with IL-6 m RNA was found.2.miR-125 a negatively regulates IL-23R/JAK2/AKT signaling pathway,inhibits the proliferation and inflammation of keratinocytes,and promotes differentiation and apoptosis.2.1 Q-PCR: Post transfection,miR-125 a was upregulated in miR-125 a group compared with NC(+)group.2.2 CCK-8 test: The cell proliferation was decreased in miR-125 a group compared with NC(+)group at 72 h post transfection.2.3 AV/PI test: The cell apoptosis was enhanced in miR-125 a group compared with NC(+)at 48 h post transfections.2.4 Q-PCR: Post transfection,the expression levels of KRT10 m RNA and KRT1 m RNA in the miR-125 a group increased significantly compared with the NC(+)group.2.5 Western Blot: Post transfection,the expression levels of KRT10 and KRT1 proteins in the miR-125 a group increased significantly compared with the NC(+)group.2.6 Q-PCR: Post transfection,the expression levels of IL-23 R m RNA increased in miR-125 a group compared with NC(+)group.2.7 Western Blot: Post transfection,the protein expression of IL-23 R,JAK2,and p-AKT in the miR-125 a group was significantly reduced compared with the NC(+)group.2.8 ELISA test: Post transfection,the expression levels of TNF-?,IL-6,and IL-17 in the miR-125 a group were significantly reduced compared with the NC(+)group.2.9 Q-PCR: Post transfection,the expression level of miR-125 a in the miR-125a(+)group had no statistical difference compared with the miR-125a(+)plus IL-23R(+)group.2.10 CCK-8 test: The cell proliferation was decreased in miR-125a(+)group compared with miR-125a(+)plus IL-23R(+)group at 48h?72 h post transfection.2.11 AV/PI test: The cell apoptosis was enhanced in miR-125 a group compared with miR-125a(+)plus IL-23R(+)group at 48 h post transfections.2.12 Q-PCR: Post transfection,the expression levels of KRT10 m RNA and KRT1 m RNA in the miR-125a(+)group increased significantly compared with the miR-125a(+)plus IL-23R(+)group.2.13 Western Blot: Post transfection,the protein expression levels of KRT10 and KRT1 in the miR-125a(+)group increased significantly compared with the miR-125a(+)plus IL-23R(+)group.2.14 Q-PCR: Post transfection,the expression levels of IL-23 R m RNA reduced in the miR-125a(+)group compared with miR-125a(+)plus(IL-23R)group.2.15 Western Blot: Post transfection,the protain expression of IL-23 R,JAK2,and p-AKT in the miR-125a(+)group was significantly reduced compared with miR-125a(+)plus IL-23R(+)group.2.16 ELISA test: Post transfection,the expression level of TNF-?,IL-6 and IL-17 in the miR-125a(+)group were significantly reduced compared with miR-125a(+)plus IL-23R(+)group.3.Luciferase reporter assay verified the relationship between miR-125 a and IL-23 R.The dual luciferase report experiment showed that the relative luciferase activity of the WT + miR-125 a group was significantly lower than that of the WT + miR-NC group,while the luciferase activity of the Mut + miR-125 a group was not statistically different from that of the Mut + miR-NC group.conclusion: 1.The relative expression of miR-125 a in psoriasis vulgaris lesion area is lower than that in non-lesion area.2.The expression level of miR-125 a is negatively correlated with the severity of psoriasis vulgaris and is negatively correlated with some inflammatory factors in the skin lesion area.3.miR-125 a can target IL-23R/JAK2/AKT to inhibit keratinocyte proliferation and inflammation,and promote apoptosis and differentiation,and play a protective role in the pathogenesis of psoriasis. |
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