The Primary Study On Effect Of MicroRNA In UVB-induced Apoptosis

BackgroundUltraviolet, shorten by UV, is one of the radiation in electromagnetic wave spectrum and has a wavelength between 100 and 400 nm. Solar light and some artificial light sources are the primary sources of UV radiation for human being. Ultraviolet is about 5% in all solar light. According to the International Commission on Illumination, UVB is the ultraviolet which has a wavelength between 280 and 315 nm. Except for UVB, there are UVC and UVA in the solar light. Almost all of the UVC absorbed by the ozone layer efficiently before it arrived at the ground. However, UVA is not absorbed at all and comprises more than 95% of solar radiation that reaches us. It can penetrate deep into the epidermis and dermis of the skin and darkens the melanin in the epidermis. And the melanin can protect the skin from sunburn.And UVB, which we concerned in this study, is a minor but the most active constituent of solar light. It makes up 4% to 5% of coming UV light. It is more genotoxic and about 1000 times more capable of causing sunburn than UVA. It can directly damage DNA by formation of cyclobutane-pyrimidine dimers and pyrimidine-pyrimidone photoproducts and then induces adverse biological effects such as apoptosis and mutation.let-7,miR-21 and miR-24 all are small RNA belonging to microRNA. microRNAs are a large class of small noncoding RNAs consisting of about 22 nucleotides, which regulate target mRNAs on post-transcriptional level. The first microRNAs found by investigators are lin-4 and let-7, which are found in 1993, when investigators looking for how the developmental timing of the nematode called Caenorhabditis elegans is controlled. At present, more than a thousand of microRNAs have been identified. And we have known that they had diverse functions, including the regulation of cellular differentiation, proliferation and apoptosis. That is they play key roles in a wide variety of normal and pathological cellular processes. But, the specific function of most microRNAs still left unkown.Among the solar radiation, as said above, most of UVB (about 95%) is absorbed by the ozone layer efficiently, and it makes up only 4% to 5% of coming UV light. However, the wavelength of UVB is near to the peak value of absorption for DNA and proteins, and it can induce damage to DNA and proteins. Furthermore, the ozone layer was broken seriously these years, more and more UV arrived at ground, and people pay more attention to the effect of UVB on health. But the study of the molecular mechanism of UVB-induced DNA damage, cell cycle arrest and apoptosis is limited by protein-coding genes. There are a few of reports about the roles of many non-coding gene (microRNA gene) in UVB-induced impairment. For this reason, we have made a primary study on effect of let-7, miR-21 and miR-24 on UVB-induced apoptosis.Objective To determine the microRNAs let-7, miR-21 and miR-24 whether participate in UVB-induced apoptosis primarily, and compare their expression levels between cells UVB-exposed and non-UVB-exposed. And then suspect the improvement or inhibition roles of let-7, miR-21 and miR-24 in UVB-induced apoptosis, predict their target genes and the mechanism in cellar signal transduction by online tools.Methods1. Cell CulturesNIH3T3 cells were bought from China Center for Type Culture Collection (CCTCC), HaCaT cells were from our laboratory's storage. The NIH3T3 and HaCaT were cultured in MEM and DMEM medium (Invitrogen, U.S.A.), separately. Both MEM and DMEM medium contained 10% fetal bovine serum (FBS, Si Ji Qing, Hang Zhou.), NaHCO3 30mM, penicillin 100 U/ml and streptomycin 100 mg/ml. Cells were plated at 5×105～6×106 cells/ml in 80mm dishes, and were placed in an incubator at 37℃with 5% carbon dioxide.2. UVB IrradiationThe UV irradiation apparatus used in this study consisted of a bulb and the wavelength peak of the bulb is 305 nm. The approximate irradiance of UVB at the sample level (at a distance of 40cm from the bulb) is 16.5μW/cm2. When the confluent cells were irradiated, the lid of the culture dish was removed and the UVB exposure was performed in PBS after washing the cells with PBS twice.3. Hoechest33342/PI stainingThe method was according to Vybrant(?) Apoptosis Assay Kit #7 (USA, invitrogen, Cat# V-23201, Molecular Probes). The samples were observed on LACAS fluorescence microscope (Germany).4. Cell viability assay and FACSCells were seeded into 96-well plates (about 10 000 cells/well) and were treated in two ways:(1) cells were treated with 0,10,30,50,70, and 100 J/m2 UVB. After irradiation, the cells were cultured in complete culture medium again for a further 12h. (2) the treated group were incubated for 2,4,8,12, and 24h after exposed to 50J/m2 UVB radiation. Subsequently, MTT was added to the culture medium to yield a final MTT concentration of 0.5 mg/ml. Cells were incubated with the MTT for 4 h in a CO2 incubator and then collected and dissolved in DMSO. Colorimetric analysis at 570nm was measured.Cell cycle analysis was performed by flow cytometry for the treated group and the control group. Cells were cultured in 80mm dishes and the treated group was treated in two ways as above. The cells were fixed in chilled 70% ethanol for at least 24h before staining with 100μg/ml propidium iodide (PI) and 100 100μg/ml RNase A (Sigma) and were analyzed using FACScan (Coulter) 30 min after staining.5. RNA Isolation and analysisSmall RNA of NIH3T3 and Total RNA of HaCaT was isolated with the mirVanaTM microRNA Isolation Kit (Cat#1560, Ambion).6.PCR of let-7 and miR-24Conventional RT-PCR was done as described later. Briefly,1μl of small RNA isolated from two teams of cells were reverse transcribed using oligo dT Primer, Random 6 mers and reversed transcriptive enzyme. 7. Real-time PCR of miR-21Conventional RT-PCR and Real-time PCR (or quantitative RT-PCR) of miR-21 were done as described later.1μl of total RNA isolated from each cell were reverse transcribed using TaqMan MicroRNA Reverse Transcription Kit(Ambion, Cat#4366596, USA). The resulting cDNA was amplified by PCR using gene-specific primer pairs; mature miR-21 and U6 hsnRNA were quantified using TaqMan Universal PCR Master Mix Kit (Ambion, Cat#4324018, USA).8. Statistical analysisStatistical analysis was done with the software program SPSS 13.0, quantitative results presented are the mean±standard deviation (SD) (X±S), comparison means was used with One-Way ANOVA, a probability value (P value) of less than 0.05 was considered significant.9. The target genes predict and bioinformatics analysisFor target genes predictions and validations, microRNAs were processed by using miRBase Target (http://www.mirbase.org/), Targetscan (http://www.targetscan.org/), Pictar (http://www.pictar.org/), miRGator (http://genome.ewha.ac.kr/miRGator/). These sources use algorithms such as miRanda to identify potential binding sites for a given microRNA in genomic sequences.The target genes lists were then processed by GO (http://www.geneontology.org/) and miRGator (http://genome.ewha.ac.kr/miRGator/), a web interface that carries out simple data mining using gene ontology. The data mining consists of the assignation of the most characteristic gene ontology term to each cluster of regulated genes.Results1. Hoechest33342/PI stainingWe used Hoechest33342/PI to investigate the changes in the nucleus of cells, and lots of apoptotic bodies containing nuclear fragments were found in UVB-treated cells but a little in untreated cells. At the same time, cytoplasmic shrinkage was observed in cells UVB-treated.2. Cell viability assay and cell cycle analysisUVB irradiation decreased cell viability in a dose-dependent manner. The survival ratio of HaCaT cells decreased gradually after being irradiated by UVB at different dosages and then incubated at the same duration. Our data showed that UVB exposure, at every dose, resulted in decreased cell viability and increased apoptosis. When cells were exposed to UVB (50 J/m2) and then incubated with complete culture medium for different time, the results showed that there was a large change at each point.The effects of UVB irradiation on the progress of HaCaT cells were studied by flow cytometry using PI staining. It showed that UVB induced DNA damage led to cell cycle arrest in the G1 phase.3. PCR of let-7 and miR-24The results of PCR of let-7 and miR-24 visualized in 10% agarose gel showed that both the expression levels of let-7 and miR-24 in NIH3T3 cells treated by UVB were higher than the levels in cells untreated.4. Real-time PCR of miR-21 and Statistical analysis Real-time PCR analysis showed that hsn-miR-21 was highly expressed at 2h and 4h, and the expression levels at 2h was more than six times in comparison with the control. However, the expression level at 8h was decreased by more than 1/2 compared with the control. In addition, the level of hsn-miR-21 was down-regulated at 12h. Taken together, these results suggested that the expression of miR-21 after UVB-exposed was time-dependent.Statistical analysis was done with the software program SPSS 13.0, quantitative results presented are the mean±standard deviation (SD) (X±S), comparison means was used with One-Way ANOVA. The probability value (P value) was less than 0.05, suggesting that the results was considered significant.5. The target genes predict and bioinformatics analysisPredicting microRNA-regulated target genes is a necessary step to understand the functions of microRNA. However, due to the imperfect binding between the majority of human microRNAs and their targets, individual microRNAs may regulate hundreds of target genes. Recently a study indicated that Pictar had an excellent recovery rate in target gene prediction, so we utilized the database Pictar, Targetscan and MicroCosm to obtain predicted target genes of let-7, miR-21 and miR-24 in response to UVB radiation. These microRNA genes, as expected, could potentially regulate several hundred targets.ConclusionsUVB radiation is a hazard factor in sunlight, and can lead to apoptosis of cells. The molecular events in the induction of apoptosis are being actively investigated. However, most of the studies have focused on examining protein-coding genes. Recently, studies have found that miRNAs have been implicated in the regulation of proliferation, differentiation and apoptosis; it seemed possible that miRNAs might contribute to understanding the cellular mechanisms of apoptosis induced by UVB. Our present study involved the miRNAs let-7, miR-21 and miR-24, which were sensitive to UVB.In our study, the results demonstrated that let-7 and miR-24 could be expressed in NIH3T3 cells, and increased after UVB irradiation. And miR-21 could be also expressed in HaCaT cells, and increased after UVB irradiation too. Moreover, we found that the level of miR-21 at 2h and 4h after UVB-treated were significantly higher than that of the control (nearly six-fold). They showed a characteristic of enhancing apoptosis.It is well known that microRNAs have the potential to regulate diverse target genes including cell cycle and apoptosis. We predicted target genes by some famous online tools, and the result showed that the target genes contained pro-apoptotic genes such as Casp3, Bcl2, Bcl212, Cdk5.In summary, the focus of this study was to investigate the relationship between the three microRNA molecules let-7, miR-21, miR-24 and apoptosis of cells in response to UVB irradiation. Although our investigation is very preliminary, we believe that this study will provide a basis for further investigation of their function in signal transduction pathways induced by UVB.