Overexpression Of Micrornas-23a Acute Uvb Irradiation-induced Skin Light Injury Intervention And Its Related Target Gene Predictions Verified

Background and objective:Ultraviolet (UV) radiation from the sun, particularly its UVB component (290-320 nm), is the major cause of skin cancer. UVB radiation is regarded as a complete carcinogen, with tumor-initiating as well as tumor-promoting potential. Chemoprevention by naturally occurring agents is gaining attention as a newer dimension in the management of neoplasia, including skin cancer. We recently demonstrated that pre-treatment with baicalin, isolated from baical skullcap root, on Balb/C mice skin significantly decreased the amount of epidermal DNA photo-lesions: cyclobutane pyrimidine dimmers(CPDs) 1, 24 and 48 hours after 180 mJ/cm2 of UVB irradiation as compared to untreated mice. Also, topical application of baicalin, either as a pre-treatment or as a post-treatment, resulted in a significant decrease in UVB mediated increases in skin edema, skin hyperplasia and infiltration of leukocytes. Furthermore, baicalin treatments (pre and post) also resulted in a significant decrease in UVB mediated generation of H2O2 and formation of CPDs. These earlier findings indicated protection of baicalin from single UVB-mediated cutaneous damage in mouse skin. However, whether baicalin can protect against damage due to multiple UVB exposures in mouse skin is not clearly understood. And the mechanism of the chemopreventive effects of baicalin are not known. We did the present research to fill these gaps in our knowledge. Studies have shown that COX-2 plays an important role in hyperproliferative conditions including cancer and has been shown to promote skin tumor development and multiplicity. Furthermore, Ki-67 and PCNA are nuclear proteins that are expressed in proliferating cells and are regarded as marker of proliferation, which if not controlled results in the development of cancer. In this study we used COX-2, Ki-67 and PCNA to assess the protective effect of baicalin on multiple UVB irradiation mediated damages on C57BL/6 mouse epidermis.Methods:According to the experiment design, Female C57BL/6 mice were divided into four groups: control, UVB treatment group, baicalin treatment group and UVB+ baicalin treatment group. 180mJ/cm2 UVB irradiation was conducted on alternate days×10 exposures on C57BL/6 mice and baicalin(1mg/cm2 skin area/mouse/100μL acetone)was pretreated on their dorsal skin 30min before UVB irradiation. Studies were done 24h after the last UVB exposure. The skin samples obtained from mice were stained with hematoxylin and eosin to observe changes in mice skin. The immunohistochemical staining and western blotting were used for COX-2, Ki-67 and PCNA detection.Results:1. Baicalin inhibits multiple UVB exposure mediated hyperplastic responses Multiple exposures of UVB radiation resulted in a significant increase in epidermal thickness compared to control animals. Topical application of baicalin prior to each UVB exposure resulted in significant inhibition of UVB exposure mediated hyperplastic responses;2. Baicalin inhibits multiple UVB exposure mediated increases of COX-2As shown by western blot analysis followed by densitometric analysis of the bands, multiple UVB exposures resulted in an obvious increase in epidermal COX-2 protein. Topical application of baicalin on mouse skin significantly inhibited the UVB mediated increase in COX-2 protein levels. Immunohistochemical analyses further confirmed our western blot data showing a moderate protective effect by baicalin against UVB mediated increases in COX-2. 3. Baicalin inhibits multiple UVB exposure mediated increases of Ki-67As shown by western blot analysis followed by densitometric analysis of the bands, multiple UVB exposures resulted in an obvious increase in epidermal Ki-67 protein. Topical application of baicalin on mouse skin significantly inhibited the UVB mediated increase in Ki-67 protein levels. Immunohistochemical analyses further confirmed our western blot data showing a moderate protective effect by baicalin against UVB mediated increases in Ki-67.4. Baicalin inhibits multiple UVB exposure mediated increases of PCNAAs shown by western blot analysis followed by densitometric analysis of the bands, multiple UVB exposures resulted in an obvious increase in epidermal PCNA protein. Topical application of baicalin on mouse skin significantly inhibited the UVB mediated increase in PCNA protein levels. Immunohistochemical analyses further confirmed our western blot data showing a moderate protective effect by baicalin against UVB mediated increases in PCNA.Conclusions:In summary, our data suggest that multiple exposures of C57BL/6 mice to UVB results in an increase in skin hyperplasia, and that topical application of baicalin prior to UVB radiation significantly inhibits UVB effects, including afford anti-proliferative effect by inhibiting the expression of COX-2, Ki-67 and PCNA. These protective effects of baicalin may even inhibit UVB-induced skin carcinogenesis. Background and objective:MicroRNAs(miRNAs) are about 22-nucleotides, short, noncoding RNAs that are thought to regulate gene expression through sequence-specific base pairing with the 3'-untranslated region (3'-UTR) of target mRNAs. In recent years, several articles have been published showing a possible link between miRNAs and cancers. Differential expression profiles of microRNAs in NIH3T3 cells in response to UVB irradiation has also been reported. However, until now, there is no studies of mechanisms of photoprotective drugs from the aspect of miRNAs. Understanding the mechanisms that contribute to photodamge and find effect targets of photoprotective drugs will be of great value for the development of improved anti-UV strategies. Our group previouely analyzed that baicalin, isolated from baical skullcap root, has a wide range of photoprotective effect, especially our researchs demonstrated that pre-treatment with baicalin on Balb/C mice skin or fibroblast significantly decreases the amount of epidermal cyclobutane pyrimidine dimmers(CPDs) and facilitates its clearance. Furthermore, we compare the profiles of miRNA expression in 3 pairs of untreated mice,UVB irradiated mice and baicalin treated irradiated mice, the results show that 3 miRNAs were down-regulated and 3 miRNAs were up-regulated in UVB irradiated mice compared with untreated counterpart. Differentially expressed miRNAs were predicted to have some relationships with photocarcinogenesis, hypomethylation and apoptosis. 3 miRNAs were down-regulated and 1 miRNA was up-regulated in baicalin treated irradiated mice compared with UVB irradiated mice. miR-23a is the only up-regulated miRNA after baicalin treatment, quantitative PCR also validated the results of mammalian miRNA microarray. Recently miR-23a has been widely studied as tumour suppressors/oncogenes. As the specificly up-regulated miRNA after baicalin treatment, it suggests that maybe miR-23a is the upstream regulatory mechanism which contribute to the photoprotective effect of baicalin. However, whether miR-23a has protective effect on damages mediated by UVB irradiation are still not investigated. In this study, synthetic miR-23a mimics was transfected into HaCaT cells by lipofectamine to assessed the effects of miR-23a on damages mediated by acute UVB irradiation. Then find out meaningful downstream target genes of miR-23a which may be relate to its photoprotective effects by bioinformatics analysis. Furthermore, double-luciferase assay and Wester Blot were used to confirm the effect of miR-23a on its downstream target genes.Methods:According to the experiment design, HaCaT cells were divided into the four groups: control, UVB treatment group, UVB+miR-23a Negative Control transfection treatment group and UVB+miRNA-23a mimics transfection treatment group. 30mJ/cm2 UVB irradiation was conducted and miR-23a mimics /Negative Control was transfected into HaCaT cells with transfection agent before UVB irradiation. Cell proliferation rates were detected by MTT assay after0, 24, 48 and 72 houes after UVB irradiation. The influence of overexpression of miR-23a on apoptosis rate and cell cycle arrest of HaCaT cells after UVB irradiation was detected by flow cytometry. The clearance of CPDs were qualitatively examined by immunofluorescence assay and quantitatively analyzed by immunodotblot assays at 0, 2, 6 and 8 hours after irradiated by UVB. TargetScan and GO-analysis were employed for the prediction the the downstream target genes of miR-23a and their functions. Then find out meaningful downstream target genes of miR-23a which may be relate to its photoprotective effects by bioinformatics analysis. Fuethrermore, doub-luciferase assay and Wester Blot were used to confirm the effect of miR-23a on its downstream target genes.Results: 1. MTT analysis of Cell proliferationCompared with control group, 30mJ/cm2UVB irradiation significantly reduced the proliferating activity of HaCaT cells. Transfection of miR-23a mimics before UVB irradiation can relieve this inhibitive effect, it suggested the possible photoprotective effects and this photoprotective effects is most obvious at 72 hours after UVB irradiation.2. Flow Cytometry analysis of cell cycle and apoptosisUVB irradiation increased apoptosis rate from 0.32% to 6.43%, and induced S-phase arrest of cell cycle. Transfection of miR-23a mimics before UVB irradiation reduced apoptosis rate to 1.46%, and relieve S-phase arrest of cell cycle.3.Qualitative and quantitative analysis of the clearance of CPDsPositive products were observed localizing in the whole nucleus of overall HaCaT cells when detected at 0 hour after UVB irradiation by immunofluorescene microscopy. Meanwhile, the control group showed negative results. Both immunofluorescene microscopy and immunodotblot assay results shows that transfection of miR-23a mimics before UVB irradiation can facilitate the clearance of photoproducts CPDs.4. Bioinformatics analysis of miR-23aBioinformatics analysis showed that the predicted target genes are FUT9,ETNK1,RAB39B,KIAA1467 and TOP1, their functions are closely related with signal transduction, DNA replication, substance metabolism and nucleotide metabolism. TOP1 is the conservation target gene of miR-23a which plays an important role in DNA reparation.5. Doub-luciferase analysis of the luciferase activity of TOP1 3'UTR report geneAs shown by doub-luciferase assay analysis, luciferase activity of Top1 3'UTR were decreased after miR-23a overexpression. The luciferase activity of Top1 3'UTR in miR-23a mimics and Top1 3'UTR -wide type plasmid cotransfected group is Only 45% of the negative control group. It suggested that miR-23a can decrease luciferase activity of Top1 3'UTR by sequence-specific base pairing with the 3'-UTR of Top1.6. Western blot analysis of the protein levels of TOP1As shown by western blot analysis followed by densitometric analysis of the bands, acute UVB exposures resulted in an obvious increase in TOP1 protein. Overexpression of miR-23a significantly inhibited the UVB mediated increase in TOP1 protein levels. It suggested that miR-23a can inhibit UVB mediated increase of TOP1 expression and TOP1 is one of the target genes of miR-23a.Conclusions:TOP1, which plays an important role in DNA reparation, is one of the conservation target genes of miR-23a. Overexpression of miRNA-23a can not only inhibit the cell growh depression, apoptosis and cell cycle arrest of HaCaT cells induced by UVB irradiation, but also facilitate the clearance of photoproducts CPDs by decrease target gene TOP1 expression. It suggested that maybe overexpression of miR-23a can help to treat and prevent UVB-induced dermatoses.