MicroRNAs Expression And Its Function Research In Chronic Actinic Dermatitis

Chronic actinic dermatitis(CAD)is a kind common idiopathic photosensitive dermatitis.In recent years,with the deterioration of the environment and expansion of the ozone layer holes,the incidence of CAD increased in Asian.Because of chronic actinic dermatitis occurred mainly in the surface the neck,limbs and other exposed parts,it may affect the patient’s mental health.CAD patients accompanied by itching,it brings heavy burden to the patients.Although a large number of studies suggest that immune dysfunction induced by delayed type hypersensitivity may be associated with CAD,so far,the pathogenesis of CAD is not clear.Therefore,the occurrence and development of CAD is a complex disease process,and other mechanisms may also be present.MicroRNA(miRNA)is a kind of conservative,19-24 nucleotides in length non-encoding RNA,which widely exists in eukaryotic organisms.miRNA promotes the degradation of mRNA by combining the target gene sequence with the target gene mRNA 3 ’untranslated region(31 UTR)completely or incompletely.Athough these miRNA accounted for only 1%of the human genome,it regulates the expression,modification,transcription and translation processes of more than 1/3 of the human genes.These target genes play an important role in cell proliferation,differentiation and apoptosis,and other biological functions.More and more studies have confirmed that abnormal expression of miRNA in many kinds of skin diseases.The abnormal expression of miRNA may play a role in regulation of apoptosis or proliferation in keratinocytes,fibroblasts and melanocytes.The discovery of miRNA may become a molecular marker with high application value,provide a new direction for the early diagnosis and prognosis of various skin diseases.But so far,the research on chronic actinic dermatitis which focused on screening and analysing the expression of miRNA is still few.Part 1:Screening of differentially expressed miRNAs in chronic actinic dermatitisObjective:To screen differentially expressed miRNAs in chronic actinic dermatitis,acquire miRNA expression profile of chronic actinic dermatitis.Methods:Total RNA was exracted from skin lesions of 3 patients with chronic actinic dermatitis and 3 normal skin tissues.Agilent miRNA chips were used to screen the differential miRNA expression of chronic actinic dermatitis.Differential miRNAs were further validated using quantitative real-time PCR(qRT-PCR)in the lesions of 10 patients with chronic actinic dermatitis and 10 skin tissue samples of the control group.Through Cytoscape,DAVID and other related software,we finaly carried out the prediction and functional analysis of target genes in differentially expressed miRNA.Result:1.The results of total RNA extraction and quality detection showed the 28S and 18S bands were clear,and the ratio of A260/A280 was between 1.8-2.2.Therefore the results of RNA extraction met the requirements of the chip experiment.2.miRNA chip results showed that there was a significant difference in the miRNA expression of chronic actinic dermatitis.We found 18 miRNAs expression were up-regulated,and the expression of 16 miRNAs was down regulated.3.qRT-PCR method was used to verify the 5 differentially miRNA,and the results were consistent with the results of the chips.4.The target genes of differentially expressed miRNAs may be involved in PI3K/AKT and other signaling pathways,and are associated with cell proliferation.Conclusion:MiRNA microarray can be used for the analysis of miRNA expression in chronic actinic dermatitis.Through Agilent miRNA chip screening and the qRT-PCR verification,there are 18 up-regulated miRNAs,16 miRNAs were down regulated.This study provides experimental basis for the further study of chronic actinic dermatitis.Part 2:biological function of miRNA-21 in HaCaT cellObjective:To investigate the biological function of miRNA-21 in HaCaT cell.Methods:The synthetic miRNA-21 mimic and inhibitor were transfected into HaCaT cells.The transfection efficiency was detected by PCR.Cell Counting(CCK-8)method and EdU test were used to compare the proliferation ability of the cells after transfection,and the apoptosis of the cells after transfection was detected using flow cytometry.Results:1.The results of PCR showed that miRNA-21 mimic could upregulate the expression of miRNA-21 in the HaCaT cells.Meanwhile,miRNA-21 inhibitor could down regulate the expression of miRNA-21 in the HaCaT cells by more than 15 folds.2.Effects of miRNA-21 on the biological behavior of HaCaT cells,(1)Detection of cell proliferation using CCK8 assay:respectively detected the OD value of the cells in 12 hours,24 hours,36 hours,48 hours and 72 hours.The results showed the proliferation activity of HaCaT cells transfected with miRNA-21 mimic increased compared with the control group(P<0.05).(2)EdU assay was further used to detect the proliferation of cells.The results of EdU showed that the proliferation activity of HaCaT cells transfected with miRNA-21 mimic increased compared with the control cells(P<0.05).(3)Apoptosis was detected by flow cytometry(FCM).The apoptosis of HaCaT cells transfected with miRNA-21 after 48h was analyzed by flow cytometry(FCM).The results showed that the apoptosis rate of miRNA-21 mimic group was significantly lower than that of NC group(P<0.05)Conclusion:MiRNA-21 can increase the cell proliferation of chronic actinic dermatitis,inhibit cell apoptosis,suggested that miRNA-21 may be involved in the development of chronic actinic dermatitis.Part 3:miRNA-21 regulates biological behavior of HaCaT cells in chronic actinic dermatitis via PTENObjective:To predict and verify the target gene of miRNA-21,and to explore the biological mechanism of miRNA-21 in regulating cell proliferation and apoptosis of HaCaT cell.Methods:by miRNA target gene prediction database,such as TargetScan,picTar,RNA22,PITA and miRanda,we carried out online detection or screening of target genes of miRNA-21.At the cellular level and tissue level,we further detected the mechanism and signaling pathway using Western blotting,immunohistochemistry and rescue experiment.Result:1.Prediction of target genes of miRNA-21:Using target gene prediction database,three and above the intersection of the software of miRNA-21 target genes were 150.The final choice of 5 target genes was further study as a possible target gene of miRNA-21.2.Validation of 5 target genes:3 related protein expression of miRNA-21 inhibitor group in HaCaT cells was higher than that of NC group,the difference was statistically significant(P<0.05).3.The level of organization to further verify the expression of the 3 proteins.The results showed that the expression of PTEN in chronic actinic dermatitis lesions reduced,which were consistent with the cell level.4.The different combinations of miRNA-21 inhibitor and PTEN_siRNA were transfected into HaCaT cells.The expression of PTEN was observed in order to verify the target gene.We co-transfected miRNA-21 inhibitor and PTEN siRNA into HaCaT cells and found the expression of PTEN decreased significantly in HaCaT cells.Therefore,miRNA-21 can target and regulate expression of PTEN.5.Compared with untransfected group,PTEN expression increased in HaCaT cells which transfected miRNA-21 inhibitor,however p-AKT expression decreased.Conclusion:This study suggests that miRNA-21 may activate the PI3K/Akt signaling pathway by directly regulating key molecules PTEN and Akt in the PI3K/Akt signaling pathway of HaCaT cells.