| BackgroundPsoriasis is a common immune-mediated chronic inflammatory skin disease.Its pathological features are epidermal keratinocyte proliferation,shortened cell division cycle and epidermal cell turnover time.Clinical manifestations were mainly erythema and scales,with long course and repeated attacks.It seriously affects the life quality of patients.The occurrence of this disease is related to various factors such as genetics,environment,immunity,infection,and psychological factors.However,the pathogenesis has not been fully elucidated,and currently there is no fundamental effective cure.Therefore,the study of pathogenesis of psoriasis is the focus and difficulty in the field of dermatology at home and abroad.Recent studies suggest that psoriasis occurs on the basis of genetic factors,and various factors such as infection,mental trauma and trauma induce the misregulation of various immune cells in the body.It leads to the release of inflammatory cytokines,further obstruct the innate and acquired immune function of the body,more inflammatory cytokines are released,leading to infiltration of associated inflammatory cells,eventually leads to characteristic pathological changes in psoriasis:proliferation and abnormal differentiation of keratinocytes,immune inflammatory cells move into the epidermis,dermis.However,the underlying mechanisms leading to epidermal defects and immune dysfunction remain uncertain.With the gradual deepening of research in the field of small molecules at home and abroad,it has been found that a variety of microRNAs(miRNAs)play an extremely important role in the process of skin physiology and pathology.Some miRNAs are abnormally expressed in some skin lesions,such as psoriasis,atopic dermatitis,and skin tumors.In recent years,due to the development of gene chip technology,qT-PCR,dual luciferase reporter experiments test,and new generation of high-throughput sequencing technologies,more and more miRNAs associated with psoriasis have been screened and verified,and the specific relationship with psoriasis can be clarified by targeting specific miRNAs that are endogenous in organisms.Although some miRNAs are closely related to psoriasis,the current research on miRNAs mainly stays at the level of expression.The regulatory mechanism needs to be studied hard.A large amount of miRNA gene expression data needs to be collected in order to find out the relevant mechanism of action in the occurrence and development of psoriasis.The study of prevention,diagnosis and treatment of psoriasis at the molecular biological field has become one of the main directions in the study of psoriasis.Research purposesScreening differentially expressed miRNAs in the epidermal tissue of the lesions of psoriasis patients and normal controls by miRCURY LNATM microRNA Array microarray technology;Using Bioinformatics techniques and molecular biology experiments to investigate the mechanism of miR-320b,which is down-regulated in the epidermis of psoriatic lesions,in the induction of proliferation of normal human epidermal keratinocytes(NHEKs).Part 1 MicroRNA microarry results and validation in epidermis of psoriatic lesionsResearch methods1.The paraffin tissues of the epidermis of 10 cases of psoriasis and 10 epidermis of healthy controls were collected.RNA was extracted from paraffin epidermal tissue by TRIzol method in 8 samples with matching gender,age and sampling location(4 in each psoriasis patient and healthy control group).2.Using miRCURY LNATM microRNA Array(v.18.0)(Exiqon)gene chip technology to analyze the differential expression of miRNAs in the epidermal cells of the skin lesions of psoriasis patients and healthy controls.The chip was scanned using an Axon GenePix 4000B chip scanner,and the probe signal values were read by GenePix Pro 6.0,and the original values obtained were normalized to median values.Differential expression of miRNAs between the two samples was screened using Fold change and P-value.Using U6 as an internal reference,the relative expression level of miRNA was calculated using the 2-AACT method.3.In addition,paraffin tissue from the epidermis of 12 samples(6 from each psoriasis patient and healthy control group)was selected,RNA was extracted,and RT-qPCR was used to detect the expression levels of miR-320b and miR-1246 with obvious differential expression.U6 was used as the internal reference,and the computed data was analyzed by 2-??CT method to further verify the chip results.Result1.The differential expression of miRNAs in paraffin epidermis of 4 psoriasis patients and 4 healthy controls was detected and analyzed by miRCURY LNATM microRNA Array gene chip technology.The results of chip analysis showed that there were 243 miRNA with different expressions between the two groups.Among them,Fold Change,(FC)? 1.5,P-value ?0.05,there are 116 differential expressed miRNAs.Forty-five(35.7%)were up-regulated and 71(56.3%)were down-regulated.2.RT-qPCR was used to detect the expression levels of six obviously differentially expressed miRNAs.Compared with the control group,mir-1246 and let-7d-3p were up-regulated(P<0.05,P<0.01),and mir-30d-5p,let-7g-5p and mir-320b were down-regulated(P<0.05,P<0.01),consistent with the results of the gene chip.There was no statistical difference in the expression levels of miR-4475 between the two groups.3.RT-qPCR was used to detect the expression levels of miR-1246 and miR-320b in the epidermis paraffin tissue of another 12 samples(6 lesions in psoriasis and 6 skins in healthy subjects).The results showed that the expression of miR-1246 was up 1.98-fold(P<0.01)and the expression of miR-320b was down-regulated by 0.26-fold(P<0.01).Part 2 Expression level of microRNA-320b and effect on the proliferation of keratinocytesResearch methods1.The NHEKs were cultured in vitro,and the purified NHEKs were obtained after 2-3 passages.The third generation NHEKs were selected and verified for subsequent experiments.2.RNA was extracted from cell precipitation,and complementary DNA was obtained by reverse transcription.U6 was used as a reference gene to normalize the sample,and RT-PCR detection and 2-??CT method were used to analyze the data.3.Construct the low expression miR-320b lentiviral vector,transfect into NHEKs,down-regulate the expression level of miR-320b in cells,and establish a lentivirus negative control group.4.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(3-(4,5-Dimethylthiazol-2-)after 1,2,3,4 days of transfection Yl)-2,5-Diphenylte-trazoliumBromide(MTT)was used to measure the level of cell prolifer-ation activity.5.Cell proliferation activity was further tested in vitro using Bromodeoxyu-ridine(Brdu).Result1.Using U6 as a standardized internal reference,RT-PCR detected high expression of miR-320b in NHEK.2.MTT test results showed that the cell proliferation activity level of miR-320b low expression group was significantly higher than that of the normal control group on the fourth day of transfection(P<0.001).3.BrdU assay showed that the proliferation of miR-320b low expression group(Down)cells was significantly higher than that of the normal control group(NC)on the fourth day of transfection(P<0.05).Part 3 MicroRNA-320b regulates the phosphorylation levels of STAT3/SAPK/JNK in keratinocytesResearch methods1.Mir-320b target gene was predicted by using miRNA target gene prediction software of Miranda and Targetscan,a biological information database.2.Predicting the binding site of miR-320b to AKT3 via online software(http://www.targetscan.org).The miR-320b binding site mutation of AKT3(pMIR-REPORT Luciferase-AKT3-3'UTR(MT))and the wild type(pMIR-REPORT Luciferase-AKT3-3'UTR(WT))sequence were inserted into the reporter plasmid.The wild type or mutant double reporter vector was co-transfected into 293T cells with miR-320b mimics and a negative control(mimicscontrol),and the luciferase activity of each group was detected under a dual luciferase reporter.3.Using a miR-320b inhibitor(miR-320b inhibitor)and a negative control miR-320b inhibitor control),the lentivirus was transfected into NHEKs,The expression levels of AKT3 protein in the two groups were detected by WB(Western Blot)after 48 hours.GAPDH was used as an endogenous control to verify the regulatory effect of mir320b on the target gene AKT3.4.Cell Signaling Technology(CST)'s PathScan(?)Antibody Array Kit was used to detect and compare the key signaling molecules in the miR-320b low-expression experimental and control groups,and to analyze the possible signaling pathways regulated by miR-320b.Result1.5982 and 1573 potential target genes were obtained by using the microRNA target gene prediction software of Miranda and Targetscan,respectively.The intersection of the two target genes was 553 target genes,including AKT3,PTEN,CMPK1,RUNX1,etc.Further study on AKT3 as it is related to cell proliferation was selected and conducted through literature review.2.The results of the dual luciferase reporter system showed:The Luciferase activity of co-transfected mir-320b mimics and pmir-report Luciferase 3-3'UTR(WT)cells and mirna-320b mimics and pmir-report Luciferase 3-AKT3-3' UTR(MT)cells was significantly reduced compared with the other two groups(P<0.0001).After the mutation of the binding site,there was no significant change in this regulatory relationship(p=0.0507).3.WB(Western blot)analysis showed that the expression level of AKT3 protein was increased in the miR-320b low expression experimental group compared with the normal control group.4.After the downregulation of mirna-320b by NHEKs lentivirus transfection,pathscan detected the related genes in the cell signaling pathway,in which the phosphorylation levels of STAT3 and SAPK/JNK proteins were significantly increased.Conclusion1.miR-320b is low expressed in epidermal tissue of psoriatic lesions2.miR-320b is high expressed in NHEKs,and down-regulation of miR-320b expression promotes keratinocyte proliferation3.AKT3 is a target gene regulated by miR-320b4.Downregulation of miR-320b resulted in upregulated phosphorylation of STAT3/SAPK/JNK proteins5.miR-320b may regulate keratinocyte proliferation by inhibiting AKT3 and regulating STAT3/SAPK/JNK signaling pathway... |
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