| Objective: To seek the differential expression of micro RNA in epidermal cells of thermal damaged in vitro,then predictive differential expression of miRNAs target gene and its mechanism in the process of wound healing provide new clues for further research on the mechanism of wound healing and targeting therapy.Methods:(1)Isolate,purify and obtain the human primary epidermal cells with enzyme digestion method.(2)Randomly,the cells were divided into two groups as follows: the experimental group will be put into water bath of 51.5 ?temperature and incubated for 35 secends.The controlled group will be put into 37 ?temperature as the same time of experimental group.After thermal injury,the cells will be put on the inverted phase contrast microscope,detection of apoptosis in cell morphology and quantity.Continue to develop in the incubator after 6h samples were collected and extracted total RNAs.(3)Total RNA was isolated from cells/tissues using the Trizol(invitrogen)according to the manufacturer's protocol.RNA purity was assessed using the ND-1000 Nanodrop.Each RNA sample had an A260:A280 ratio above 1.8 and A260:A230 ratio above 2.0.RNA integrity was evaluated using the Agilent 2200 TapeStation(Agilent Technologies,USA)and each sample had the RINe above 7.0.Briefly,RNAs were ligated with 3'RNA adapter,and followed by 5'adapter ligation.Subsequently,the adapter-ligated RNAs were subjected to RT-PCR and amplified with a low-cycle.Then the PCR products were size selected by PAGE gel according to instructions of NEBNext? Multiplex Small RNA Library Prep Set for Illumina?(Illumina,USA).The purified library products were evaluated using the Agilent 2200 TapeStation and diluted to 10 p M for cluster generation in situ on the HiSeq2500 single-end flow cell followed by sequencing(1×50 bp)on Hi Seq 2500.(4)Enrichment analysis was conducted for partially of the targets of differentially expressed microRNAs.Enrichment analysis was conducted for partially of the targets of differentially expressed microRNAs.Results:(1)Under the inverted contrast microscope,the epidermal stem cells are firmly affixed to the wall,small and round shape,strong refraction.After cultivating 2 d,the cell clones grows faster,the sticking wall is firmly,the control group after 37 ? water bath has not seen the apparent cell number decreases,the cell assumes the class circle,the sticking wall firmly.53.5 ? Water bath after the experimental group The number of cells decreased markedly,the cell is irregular shape,the wall is not firm.(2)In the experimental group,33 microRNAs were up-regulated,and 21down-regulated.The most significantly up-regulated microRNAs were hsa-miR-1973,while hsa-miR-4485-3p,hsa-miR-548j-5p,hsa-miR-212-3p,hsa-miR-4461 and so on were significantly up-regulated.Meanwhile,the most significantly down-regulated microRNAs were hsa-mi R-4520-5p,while hsa-miR-4661-5p,hsa-miR-191-3p,hsa-miR-129-5p,hsa-miR-147 b,hsa-mi R-6868-3p and so on were significantly down-regulated.(3)Through the analysis of the mi RNA family of notable differences,the distribution of the miRNA family belonged to the study,and the result showed that the difference miRNA family was mir-4520,mir-548,mir-132,mir-4510,mir-191,mir-129,mir-549,mir-154,mir-7641,mir-1976,mir-147,mir-6511,mir-154,mir-744,mir-1287,mir-3064,mir-1295,mir-1248,mir-181,mir-193,mir-1294,mir-149,mir-515,mir-506,mir-338,mir-17,mir-486,mir-431,mir-4504,mir-29,mir-134,mir-221,mir-194,mir-6511.MiRNAs target gene prediction and functional enrichment analysis hint difference expression miRNAs participates in the biological process of cell proliferation and differentiation,cell growth apoptosis,cell adhesion and migration.Conclusion:(1)There was significant differention of morphology ?quantity and microRNAs profiling in epidermal cells after thermal damaged and normal.(2)There was significant differential mi RNAs expression may be closely related to the mechanisms of wound healing. |
PDF Full Text Request