Application And Mechanism Of Small Molecular Inhibitor Of Precursor MicroRNA-210 In Psoriasis

Psoriasis is a chronic inflammatory skin disease mainly manifested as scaly erythematous plaques,which is recurrent and persistent,and seriously affects the physical and mental health of patients.At present,topical drugs such as corticosteroid and calcium phosphatase inhibitor ointment are the preferred treatment options for mild to moderate psoriasis,but they can cause a series of adverse reactions such as skin irritation,pruritus,burning,and atrophy,making their clinical application somewhat limited.Biological agents are currently mainly aimed at patients with moderate to severe psoriasis however,and the safety of long-term application of biologics,especially TNF-αinhibitors,is unclear as they may induce malignancy and recurrence of previous tuberculosis and hepatitis virus infections in patients.Therefore,research and development of new topical drugs with better efficacy and fewer adverse effects are urgently needed for psoriasis patients and are of great clinical importance.The previous research of our research group found that micro RNA-210(mi R-210),a micro RNA(mi RNA)closely related to hypoxia induction,was abnormally expressed in psoriasis patients in both lesions and peripheral blood.In addition,mi R-210 expression was also elevated in imiquimod(IMQ)-induced psoriasis-like skin lesions in mice,while intradermal injection of nucleic acids targeting mi R-210 inhibited the differentiation of T helper lymphocytes 1(Th1)and Th17 cells and promoted the differentiation of Th2 cells in mice,thereby reducing the intracutaneous inflammatory response in mice.The drug significantly alleviated IMQ-induced psoriasis-like dermatitis in mice by inhibiting the inflammatory response in the skin.In the nucleus,primary mi RNAs(pri-mi RNAs)are produced by transcription with RNA polymerase and are thousands of nucleotides long.Pri-mi RNA is further sheared by the nucleic acid endonuclease Drosha enzyme to produce a precursor mi RNA(pre-mi RNA)that is about 70nucleotides long and has a hairpin structure.Pre-mi RNA is transported to the cytoplasm where it is sheared by the Dicer enzyme to produce mature mi RNA.therefore,we speculated whether it would be possible to inhibit the expression level of mi R-210 by suppressing mature mi R-210production,thereby regulating the inflammatory response downstream of mi R-210,and acting as a therapeutic agent for psoriasis.It has been reported that Targapremir-210(TGP-210)can specifically bind to the Dicer binding site of pre-mi R-210,blocking the formation of mature mi R-210 by inhibiting shearing of pre-mi R-210 by Dicer.However,the effect of TGP-210 on T lymphocytes and its application and mechanism of action in psoriasis has not been investigated.In this study,we will investigate the application and mechanism of application of TGP-210,a small molecule inhibitor of pre-mi R-210,in the treatment of psoriasis,and further,we will screen a new small-molecule inhibitor with better efficacy than TGP-210by a series of chemical modifications,and test and verify it in vitro and in vivo experiments.In this study,we conducted in vitro experiments using TGP-210,a known pre-mi R-210 inhibitor,and confirmed by flow cytometry,RT-q PCR,and CCK8 cell proliferation assay that TGP-210 can inhibit the expression of mi R-210,inhibit the differentiation of Th1 and Th17 cells,it also inhibited the proliferation of human immortalized keratinocytes(Ha Ca T).Topical application of TGP-210 to IMQ-induced psoriasis-like mouse model significantly improved IMQ-induced psoriasis-like skin lesions in mice,reduced the proportion of Th1 and Th17 cells in skin lesions and spleen,and increased the proportion of Th2 cells.Based on the results of these experiments,our group has developed a series of chemical modifications of TGP-210 and designed a new series of TGP-210-like small molecule compounds,called TD(Targeted drugs).In vitro and in vivo experiments have demonstrated that TD-02,one of a series of TD drugs,can bind to pre-mi R-210 and inhibit the expression of mi R-210,and its effect is better than that of TGP-210.This study is the first to explore the possibility of using pre-mi RNA as a target for the treatment of psoriasis,providing a new option for topical agents in the treatment of mild to moderate psoriasis and a sound basis for the future clinical application of pre-mi R-210 small molecule inhibitors.Part I The effect of TGP-210 in CD4~+T lymphocytes and keratinocytesObjective:To initially explore the effect of TGP-210 in CD4~+T lymphocytes and Ha Ca T cells.Method:1.Collect the peripheral blood of healthy people,density gradient centrifugation combined with immunomagnetic beads adsorption to separate CD4~+T cells,and CD4~+T cells were treated with different concentrations of TGP-210.2.Total RNA of CD4~+T cells was extracted by TRIZOL and real-time fluorescence quantitative polymerase chain reaction(Real-time q PCR,RT-q PCR)was performed to detect the effect of TGP-210 on the expression of mi R-210 in CD4~+T cells.RT-q PCR detection of expression levels of cytokine IFNG,IL4,IL17A and the transcription factors T-bet,GATA3,RORγt,and Foxp3 m RNA in CD4~+T cells by effect of TGP-210.The effect of TGP-210 on the proportion of Th cells as measured by flow cytometry.3.Ha Ca T cells were treated with different concentrations of TGP-210treatment,and RT-q PCR to detect the effect of TGP-210 on the expression level of mi R-210 in Ha Ca T.Effect of TGP-210 on the proliferation of Ha Ca T as detected by the CCK8.Result:1.Treat CD4~+T cells with 2μM TGP-210 significantly reduced the expression level of mi R-210(p<0.05).2.After TGP-210 treatment,IFNG、IL17A m RNA expression levels were trended to downregulated,while those of IL4(p<0.05),and GATA3(p<0.05)m RNA were upregulated in CD4~+T cells.After TGP-210treatment,significantly lowered the proportion of Th1 and Th17 cells(p<0.05)and significantly increased the proportion of Th2 cells(p<0.05).3.TGP-210 treatment significantly inhibited mi R-210 expression levels(p<0.0001)and cell proliferation(p<0.0001)in Ha Ca T cells in a concentration-dependent manner.Conclusions:TGP-210 inhibits the expression level of mi R-210 in CD4~+T cells,and inhibits the differentiation of Th1,Th17 cells in CD4~+T cells,but promotes the differentiation of Th2 in CD4~+T cells,and inhibits mi R-210expression and proliferation of Ha Ca T in a concentration-dependent manner.Part II The effect of TGP-210 on IMQ-induced psoriasis-like dermatitis in miceObjective:To investigate the effects of topical application of TGP-210 on IMQ-induced psoriasis-like dermatitis in mice.Methods:1.Apply to the IMQ-induced mice daily with matrix/calcipotriol/0.5%TGP-210/1%TGP-210 ointment was applied to the depilated areas of each group of mice for 6 consecutive days.2.Record daily changes in the skin lesions of the mice up to day 7,PASI scoring of the skin lesion and execute these mice,and take the spleen and skin lesion tissues from the mice for subsequent experiments.3.The spleen was milled into single cells,and a portion of the spleen was stained with the appropriate antibodies and flow cytometry to detect the proportion of each subpopulation of CD4~+T cells in the spleen;a portion of the spleen was used for sorting CD4~+T cells by immu-nomagnetic bead adsorption and total RNA of CD4~+T cells in the mouse spleen was extracted by TRIZOL.RT-q PCR was performed to detect the expression levels of mi R-210,and IFNG,IL4,IL17A,T-bet,GATA3 and RORγt m RNA in splenic CD4~+T cells from each group of mice.4.The skin lesions of the mice were divided into three parts:one part was made into pathological sections and stained with HE to quantify the acanthosis and the dermal inflammatory cell infiltration.One part was removed the fascial layer and epidermis,and the dermis was preserved and digested into single cells,and the proportion of subpopulation of CD4~+T cells in the skin lesions was detected using the corresponding antibody staining and flow cytometry.One part was preserved in RNA preservation solution,preserved overnight and milled,and total RNA was extracted by TRIZOL.RT-q PCR was performed to detect the expression levels of mi R-210 and IFNG,IL4,IL17A,T-bet,GATA3,and RORγt m RNA in the skin lesion cells of each group of mice.Results:1.In appearance,psoriasis-like lesions were significantly reduced in mice in the calcipotriol or TGP-210 group compared to the vehicle group,with the 1%TGP-210 group being the most significant.2.Pathologically,epidermal hyperkeratosis,acanthosis,and dermal inflammatory cell infiltration were significantly improved in mice in the calcipotriol or TGP-210 group compared to the vehicle group,with the 1%TGP-210 group being the most significant(p<0.0001).3.Mice spleens CD4~+T cells:compared with the vehicle group,mice spleens in the TGP-210 group had decreased proportions of Th1 and Th17cells(p<0.05),and decreased expression levels of mi R-210(p<0.001),and decreased expression levels of IFNG(p<0.0001),IL17A(p<0.05)and RORγt(p<0.001)m RNA.4.CD4~+T cells in dermal tissue of mice with skin lesions:compared with the vehicle group,the proportion of Th1 and Th17 cells were downward trend,the expression level of mi R-210 was decreased(p<0.05),the expression levels of IFNG(p<0.01),RORγt(p<0.05)and T-bet(p<0.05)m RNA were decreased,and the expression levels of GATA(p<0.01)m RNA were increased in the TGP-210 group。Conclusion:1.Topical application of TGP-210 significantly improved the skin lesions of IMQ-induced psoriasis-like dermatitis mice,and reduced IMQ-induced acanthosis and infiltration of dermal inflammatory cells in mice.2.Topical application of TGP-210 inhibited mi R-210 expression in mouse spleen CD4~+T cells,suppressed the differentiation of Th17 cells,and inhibited the expression of IFNG,IL17A and RORγt m RNA.3.Topical application of TGP-210 had a tendency to inhibit inflam-matory infiltration of Th1 and Th17 cells in the mice dermis,significantly inhibited mi R-210 expression in skin lesion tissues,inhibited IFNG,T-bet,and RORγt m RNA expression,significantly promoted GATA3 m RNA expression.Part III Screening for novel effective pre-mi R-210 small molecule inhibitorsObjective:To investigate the best small-molecule inhibitor of mi R-210expression among the TD series drugs and to further explore its role in CD4~+T cells and Ha Ca T cells.Methods:1.TGP-210 was used as a positive control,the affinity between TD series drugs and pre-mi R-210 was detected by surface plasmon resonance(SPR).2.Peripheral blood was collected from healthy individuals and CD4~+T cells were isolated by density gradient centrifugation combined with immunomagnetic bead adsorption.CD4~+T cells were treated with TD series drugs,and total RNA of CD4~+T cells was extracted by TRIZOL,and the expression level of mi R-210 in each group of CD4~+T cells was detected by RT-q PCR,and to screen for the small molecule inhibitor with the best inhibition effect on mi R-210 expression.3.After screening the best small molecule inhibitor,TD-02,CD4~+T cells were treat with different concentrations of TD-02,and collect the cells.and detect the proportion of Th1,Th2 and Th17 subpopulations in each group of CD4~+T cells by flow cytometry.Total RNA of CD4~+T cells was extracted by TRIZOL,and the expression levels of mi R-210,IFNG,IL4and IL17A m RNA in each group of CD4~+T cells were detected by RT-q PCR.4.The effect of different concentrations of TD-02 on the expression level of mi R-210 in Ha Ca T cells was detected by RT-q PCR;the effect of different concentrations of TD-02 on the proliferation of Ha Ca T cells was detected by the CCK8 kit.Results:1.The affinities of TD-02,TD-04 and TD-06 with pre-mi R-210 were found to be higher than that of TGP-210 after SPR assay.And TD-02 had the best inhibiting effect on expression level of mi R-210 among TD series drugs(p<0.001).2.The proportion of Th1 and Th17 cells decreased(p<0.05)and the proportion of Th2 cells increased(p<0.01)in CD4~+T cells after treatment with TD-02.The expression levels of IFNG(p<0.05)and IL17A(p<0.01)m RNA decreased in CD4~+T cells after TD-02 treatment.3.TD-02 treatment significantly inhibited mi R-210 expression levels(p<0.0001)and cell proliferation(p<0.0001)in Ha Ca T cells in a concentration-dependent manner.Conclusion:1.TD-02 has a higher affinity with pre-mi R-210 than TGP-210,and TD-02 had the best inhibitory effect on mi R-210 in CD4~+T cells among the TD series drugs.2.TD-02 and TGP-210 have similar effects in CD4~+T cells:inhibiting mi R-210 expression in CD4~+T cells,thereby inhibiting Th1 and Th17 cell differentiation and promoting Th2 cell differentiation.3.TD-02 significantly inhibits mi R-210 expression in Ha Ca T cells,thereby suppressing cell proliferation in a concentration-dependent manner.Part IV The effect of TD-02 on IMQ-induced psoriasis-like dermatitis in miceObjective:To investigate the effects of topical application of TD-02 on IMQ-induced psoriasis-like dermatitis in mice.Methods:1.Apply to the IMQ-induced mice with matrix/calcipotriol/0.008%TD-02/0.024%TD-02/0.080%TD-02 ointment for 6 consecutive days.2.PASI scoring of the skin lesion and execute these mice,and take the spleen and skin lesion tissues from the mice for subsequent experiments.3.The spleens were ground into single cells,and a portion of the spleen cells was stained with the appropriate antibodies in combination with flow cytometry to detect the proportion of each subpopulation of CD4~+T cells in the mouse spleen;a portion cells was used for sorting CD4~+T cells by immunomagnetic bead adsorption,and the total RNA of the mouse spleen CD4~+T cells was extracted by the TRIZOL.The expression levels of mi R-210,and cytokines IFNG,IL4 and IL17A m RNA in CD4~+T cells of each group of mice were detected by RT-q PCR.4.The mouse skin lesions were divided into three parts:one part was made into pathological sections analyzed by HE staining to quantify the acanthosis and dermal inflammatory cell infiltration.One part was removed the fascial layer and epidermis from the mouse skin lesions,and the dermis was preserved and digested into single cells,and the proportion of each subpopulation of CD4~+T cells in the skin lesions was detected using the corresponding antibody staining and flow cytometry.The other part was preserved in RNA preservation solution,preserved overnight and then ground.Total RNA was extracted by TRIZOL.RT-q PCR was performed to detect the expression levels of mi R-210 and cytokines IFNG,IL4 and IL17A m RNA in the lesions of each group of mice.Results:1.In appearance,psoriasis-like lesions were significantly reduced in mice in mice in the calcipotriol or TD-02 group compared to the matrix control group,with the most pronounced in the 0.080%TD-02 group(p<0.0001).2.pathologically,epidermal hyperkeratosis,acanthosis(p<0.01)and dermal inflammatory cell infiltration(p<0.0001)were significantly improved in mice in the 0.080%TD-02 group compared to the matrix control group.3.Mouse spleen CD4~+T cells:Compared with the matrix control group,the proportion of Th17 cells in the spleen CD4~+T cells decreased(p<0.05)and and the proportion of Th2 cells increased(p<0.01)in the TD-02 group.mi R-210 expression levels decreased(p<0.01),IFNG(p<0.05)and IL17A(p<0.01)m RNA expression levels decreased,while IL4 m RNA expression level had increased trend.4.Mouse dermal CD4~+T cells:Compared with the matrix control group,the percentage of Th1(p<0.01)and Th17(p<0.0001)cells in the dermis of mice in the TD-02 group was significantly reduced.Expression level of mi R-210 was decreased(p<0.0001),IFNG(p<0.0001)and IL17A(p<0.0001)m RNA expression levels were decreased,while IL4m RNA expression level was increased(p<0.001).Conclusion:1.Topical application of TD-02 significantly improved the appear-ance and pathological changes of IMQ-induced psoriasis-like mice.2.Topical application of high concentrations of 0.080%TD-02ointment topically inhibited the differentiation of splenic CD4~+T cells to Th17 cells,and promoted their differentiation to Th2 cells by suppressing the expression of mi R-210 in splenic CD4~+T cells and inhibiting the expression of IFNG and IL17A m RNA,and had a tendency to promoting the expression of IL4 m RNA in mice.3.Topical application of high concentrations of 0.080%TD-02significantly inhibited the inflammatory infiltration of Th1 and Th17 cells in the dermis,inhibited the expression of IFNG and IL17A m RNA,and promoted the expression of IL4 m RNA in skin lesion by inhibiting the expression of mi R-210 in mouse skin lesions.Summary:In this study,we found that TGP-210,a known small molecule inhibitor of pre-mi R-210,inhibited the differentiation of CD4~+T cells to Th1 and Th17 cells and promoted the differentiation of CD4~+T cells to Th2 cells in vitro and in vivo,and had some immunomodulatory effects;and that topical application of TGP-210 could improve IMQ-induced psoriasis-like dermatitis mice.Based on the above,we found that TD-02,a small molecule inhibitor of pre-mi R-210 with better affinity than TGP-210,has similar immunomodulatory effects,and can improve IMQ-induced psoriasis-like dermatitis mice by topical application of TD-02.This thesis contains 50 graphs,50 tables and 100 references.